European Biosafety Association

PLoSPathogens
27 March 2009
Overview
Gram-negative bacteria-producing extended-spectrum β-lactamases (ESBLs) are found to be truly multiresistant pathogens causing severe clinical problems.
The consensus view about antimicrobial resistance is that severe clinical problems arise from the emergence of antibiotic resistance in Gram-negative pathogens causing nosocomial infections, and from the lack of new antimicrobial agents to challenge the threat. There are four disturbing trends (extending substrate spectra) in the increasing antimicrobial resistance of Gram-negative pathogens: (i) class B β-lactamases (metallo-β-lactamases) conferring resistance to almost all β-lactam antibiotics; (ii) a bifunctional aminoglycoside-modifying enzyme; (iii) the evolution of a fluoroquinolone-modifying enzyme from an aminoglycoside acetyltransferase; and (iv) a new plasmid-borne fluoroquinolone efflux determinant. These disturbing trends indicate that options for the treatment of health-care–associated Gram-negative infections are perilously limited as the organisms expand their ability to evade existing antimicrobial agents. Here we wish to draw attention to a new disturbing trend (the recently emerging class C extended-spectrum β-lactamases [ESBLs]), and to the antimicrobial drug development for class C ESBLs. We suggest also that the category of ESBLs has to be expanded.

Animal infectious disease

PloS One
5 February 2010
Abstract
Since the advent of highly pathogenic variants of avian influenza virus (HPAIV), the main focus of avian influenza research has been the characterization and detection of HPAIV hemagglutinin (HA) from H5 and H7 subtypes. However, due to the high mutation and reassortation rate of influenza viruses, in theory any influenza strain may acquire increased pathogenicity irrespective of its subtype. A comprehensive antigenic characterization of influenza viruses encompassing all 16 HA and 9 neuraminidase subtypes will provide information useful for the design of differential diagnostic tools, and possibly, vaccines. We have expressed recombinant HA proteins from 3 different influenza virus HA subtypes in the baculovirus system. These proteins were used to generate polyclonal rabbit antisera, which were subsequently employed in epitope scanning analysis using peptide libraries spanning the entire HA. Here, we report the identification and characterization of linear, HA subtype-specific as well as inter subtype-conserved epitopes along the HA proteins. Selected subtype-specific epitopes were shown to be suitable for the differentiation of anti-HA antibodies in an ELISA.
 

PLoS Pathogens
22 January 2010
Author Summary
Prions are infectious proteins that cause heritable, sporadic, and transmissible diseases in humans and other mammals. These infectious proteins arise when the normal form of the prion protein (PrP) adopts a self-perpetuating conformation. This disease-causing PrP form is frequently distinguished from normal PrP by its resistance to digestion by proteases although considerable evidence shows that protease-sensitive prions occur naturally in humans and sheep. Here we describe the generation of novel protease-sensitive synthetic prions. After producing recombinant PrP of the wild-type mouse sequence in Escherichia coli, we polymerized the protein into an amyloid fiber conformation. Mice inoculated with these amyloid fibers developed extensive neurodegeneration characteristic of prion disease, but did not generate protease-resistant PrP. Prions from sick animals were transmitted to healthy animals, which likewise developed neurodegeneration but not protease-resistant prions. These novel synthetic prions demonstrate that truncated wild-type PrP can undergo a conformational change that becomes infectious yet the protein remains protease sensitive.
 

PloS One
9 December 2009
Abstract
A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrPC) into a disease related, alternatively folded isoform (PrPSc). The accumulation of PrPSc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.

Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrPC from the surface of prion-infected neuronal cells. The cell surface is a site where PrPC molecules may be converted to PrPSc and glimepiride treatment reduced PrPSc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A2 (cPLA2) and the production of prostaglandin E2 that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrPC expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.

Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrPSc formation and neuronal damage in prion diseases.
 

CDC, EID
December 2009
Abstract
To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrPd) to initiate conversion of the human normal cellular form of prion protein of the 3 major PRNP polymorphic variants in vitro. Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility.
 

PloS Pathogens
26 November 2009
Author Summary
Prion diseases belong to the class of neurodegenerative disorders that include Alzheimer, Parkinson and Huntington diseases. In each of these disorders, a specific protein in the brain changes shape and accumulates, leading to neuronal loss and damage. These diseases are uniformly fatal after a period of neurodegeneration, dementia and motor dysfunction. Quinacrine, an antimalarial drug, is able to eliminate prions from dividing cells in culture, yet is ineffective in diseased mice and human patients. Here, we provide an explanation for this failure. Our data indicate that the administration of quinacrine results in the proliferation of drug-resistant prions. This insight will enable us to develop more effective antiprion therapeutics in the future.
 

PLoS One
12 November 2009
Abstract
Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrPC) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrPSc, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrPC molecules. A great deal of effort has been devoted to developing protocols for purifying PrPSc for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrPSc. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.

We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrPSc and mutant PrP aggregates at electrophoretic homogeneity. PrPSc purified from prion-infected mice was able to seed misfolding of PrPC in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.

The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.
 

PLoS One
5 November 2009
Abstract
Recently much attention has been given to developing national-scale micro-simulation models for livestock diseases that can be used to predict spread and assess the impact of control measures. The focus of these models has been on directly transmitted infections with little attention given to vector-borne diseases such as bluetongue, a viral disease of ruminants transmitted by Culicoides biting midges. Yet BT has emerged over the past decade as one of the most important diseases of livestock.

We developed a stochastic, spatially-explicit, farm-level model to describe the spread of bluetongue virus (BTV) within and between farms. Transmission between farms was modeled by a generic kernel, which includes both animal and vector movements. Once a farm acquired infection, the within-farm dynamics were simulated based on the number of cattle and sheep kept on the farm and on local temperatures. Parameter estimates were derived from the published literature and using data from the outbreak of bluetongue in northern Europe in 2006. The model was validated using data on the spread of BTV in Great Britain during 2007. The sensitivity of model predictions to the shape of the transmission kernel was assessed.

The model is able to replicate the dynamics of BTV in Great Britain. Although uncertainty remains over the precise shape of the transmission kernel and certain aspects of the vector, the modeling approach we develop constitutes an ideal framework in which to incorporate these aspects as more and better data become available. Moreover, the model provides a tool with which to examine scenarios for the spread and control of BTV in Great Britain.
 

BMC Virology Journal
28 October 2009
Abstract
The European swine influenza viruses (SIVs) show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008- 2009. The majority of the European H1N2 swine influenza viruses described so far possess haemagglutinin (HA) of the humanlike H1N2 SIV viruses and the neuraminidase (NA) of either the European H1N2 or H3N2 SIV-like viruses. The Swedish isolate has an avian-like SIV HA and a H3N2 SIV-like NA, which is phylogenetically more closely related to H3N2 SIV NAs from isolates collected in the early ´80s than to the NA of H3N2 origin of the H1N2 viruses isolated during the last decade, as depicted by some German strains, indicative of independent acquisition of the NA genes for these two types of reassortants. The internal genes proved to be entirely of avian-like SIV H1N1 origin. The prevalence of this SIV variant in pig populations needs to be determined, as well as the suitability of the routinely used laboratory reagents to analyze this strain.
The description of this H1N2 SIV adds further information to influenza epidemiology and supports the necessity of surveillance for influenza viruses in pigs.
 

PLoS One
6 October 2009
Abstract
Atypical scrapie or Nor98 has been identified as a transmissible spongiform encephalopathy (TSE) that is clearly distinguishable from classical scrapie and BSE, notably regarding the biochemical features of the protease-resistant prion protein PrPres and the genetic factors involved in susceptibility to the disease. In this study we transmitted the disease from a series of 12 French atypical scrapie isolates in a transgenic mouse model (TgOvPrP4) overexpressing in the brain ~0.25, 1.5 or 6× the levels of the PrPARQ ovine prion protein under the control of the neuron-specific enolase promoter. We used an approach based on serum PrPc measurements that appeared to reflect the different PrPc expression levels in the central nervous system. We found that transmission of atypical scrapie, much more than in classical scrapie or BSE, was strongly influenced by the PrPc expression levels of TgOvPrP4 inoculated mice. Whereas TgOvPrP4 mice overexpressing ~6× the normal PrPc level died after a survival periods of 400 days, those with ~1.5× the normal PrPc level died at around 700 days. The transmission of atypical scrapie in TgOvPrP4 mouse line was also strongly influenced by the prnp genotypes of the animal source of atypical scrapie. Isolates carrying the AF141RQ or AHQ alleles, associated with increased disease susceptibility in the natural host, showed a higher transmissibility in TgOvPrP4 mice. The biochemical analysis of PrPres in TgOvPrP4 mouse brains showed a fully conserved pattern, compared to that in the natural host, with three distinct PrPres products. Our results throw light on the transmission features of atypical scrapie and suggest that the risk of transmission is intrinsically lower than that of classical scrapie or BSE, especially in relation to the expression level of the prion protein.


 

PLoS One
7 September 2009
Abstract
The prion consists essentially of PrP ^Sc , a misfolded and aggregated conformer of the cellular protein PrP ^C . Whereas PrP ^C deficient mice are clinically healthy, expression of PrP ^C variants lacking its central domain (PrP _ΔCD ), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrPC (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrP _ΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrP _ΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrPΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants.
 

PLoS One
3 September 2009
Abstract
In prion disease, the peripheral expression of PrPC is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrPSc accumulation, localisation of nerve fibres and PrPC expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep.
Laser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrPC and PrPSc in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrPSc in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrPSc and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrPSc and nerves. Some nerve fibres were observed to accompany blood vessels into the PrPSc-laden germinal centres. However, the close association between nerves and PrPSc was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres.
The findings suggest that the degree of PrPSc accumulation does not depend on the expression level of PrPC. Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrPSc.


 

PLos One
28 July 2009
Abstract
In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). While the precise mechanism of the PrPC to PrPSc conversion is not understood, it is clear that host PrPC expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrPSc, or of a prion disease developing in farmed fish is alarming and requires further evaluation.

Medicalnewstoday
27 August 2009
Overview
For the first time, Whitehead Institute researchers have shown definitively that mutations associated with prion diseases are sufficient to cause a transmissible neurodegenerative disease.
The discovery is reported in the August 27 edition of the journal Neuron.
 

CDC, EID
August 2009 (epub ahead of print)
Abstract
How susceptible pigs are to infection with sheep prions is unknown. We show, through transmission experiments in transgenic mice expressing porcine prion protein (PrP), that the susceptibility of this mouse model to bovine spongiform encephalopathy (BSE) can be enhanced after its passage in ARQ sheep, indicating that the pathogenicity of the BSE agent is modified after passage in sheep. Transgenic mice expressing porcine PrP were, nevertheless, completely resistant to infection with a broad panel of classical scrapie isolates from different sheep PrP genotypes and with different biochemical characteristics. The atypical (Nor98 like) isolate (SC-PS152) was the only scrapie isolate capable of transmission in these mice, although with a marked transmission barrier. Unexpectedly, the atypical scrapie agent appeared to undergo a strain phenotype shift upon transmission to porcine-PrP transgenic mice and acquired new strain properties, suggesting that atypical scrapie agent may exhibit different phenotypes depending on the host cellular PrP or other genetic factors.
 

PLoS One
15 July 2009
Abstract
Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood.
The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, ~40% of the ~2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three ~90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors.
The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.

PLoS One
1 July 2009
Abstract
Despite overwhelming evidence implicating the prion protein (PrP) in prion disease pathogenesis, the normal function of this cell surface glycoprotein remains unclear. In previous reports we demonstrated that PrP mediates cellular iron uptake and transport, and aggregation of PrP to the disease causing PrP-scrapie (PrPSc) form results in imbalance of iron homeostasis in prion disease affected human and animal brains. Here, we show that selective deletion of PrP in transgenic mice (PrPKO) alters systemic iron homeostasis as reflected in hematological parameters and levels of total iron and iron regulatory proteins in the plasma, liver, spleen, and brain of PrPKO mice relative to matched wild type controls. Introduction of radiolabeled iron (59FeCl3) to Wt and PrPKO mice by gastric gavage reveals inefficient transport of 59Fe from the duodenum to the blood stream, an early abortive spike of erythropoiesis in the long bones and spleen, and eventual decreased 59Fe content in red blood cells and all major organs of PrPKO mice relative to Wt controls. The iron deficient phenotype of PrPKO mice is reversed by expressing Wt PrP in the PrPKO background, demonstrating a functional role for PrP in iron uptake and transport. Since iron is required for essential metabolic processes and is also potentially toxic if mismanaged, these results suggest that loss of normal function of PrP due to aggregation to the PrPSc form induces imbalance of brain iron homeostasis, resulting in disease associated neurotoxicity.

PLoS Pathogens
19 June 2009
Author Summary
Prion diseases are transmissible fatal neurodegenerative diseases caused by aberrant metabolism of the cellular prion protein (PrPC). The transmissible agent is PrPSc, a misfolded version (conformer) of PrP capable of converting PrPC into PrPSc. PrPSc can be generated de novo in inherited prion diseases due to synthesis of aberrant PrP forms from a mutated PrP gene. Such mutant PrP forms, analogous to other aberrant proteins, should typically be destroyed by various cellular ‘quality control’ (QC) pathways; however, several human diseases result from an eventual breakdown in these QC systems, often due to prolonged bombardment by mutant proteins. We have therefore sought to identify the specific pathways that normally cope with disease-causing misfolded PrPs. By carefully following the generation and turnover of these mutant PrPs in cells, we have discovered an intracellular QC pathway that selectively routes biochemically aberrant PrP species to lysosomes. As the lysosomal system has been implicated as a site for conversion of PrPC to PrPSc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrPSc in familial prion disease. Importantly, these findings suggest that eventual changes or breakdown of this QC pathway may contribute to disease progression.

PLoS One
18 June 2009
Abstract
Doppel protein (Dpl) is a paralog of the cellular form of the prion protein (PrPC), together sharing common structural and biochemical properties. Unlike PrPC, which is abundantly expressed throughout the central nervous system (CNS), Dpl protein expression is not detectable in the CNS. Interestingly, its ectopic expression in the brain elicits neurodegeneration in transgenic mice. Here, by combining native isoelectric focusing plus non-denaturing polyacrylamide gel electrophoresis and mass spectrometry analysis, we identified two Dpl binding partners: rat alpha-1-inhibitor-3 (α1I3) and, by sequence homology, alpha-2-macroglobulin (α2M), two known plasma metalloproteinase inhibitors. Biochemical investigations excluded the direct interaction of PrPC with either α1I3 or α2M. Nevertheless, enzyme-linked immunosorbent assays and surface plasmon resonance experiments revealed a high affinity binding occurring between PrPC and Dpl. In light of these findings, we suggest a mechanism for Dpl-induced neurodegeneration in mice expressing Dpl ectopically in the brain, linked to a withdrawal of natural inhibitors of metalloproteinase such as α2M. Interestingly, α2M has been proven to be a susceptibility factor in Alzheimer's disease, and as our findings imply, it may also play a relevant role in other neurodegenerative disorders, including prion diseases.
 

PLoS One
16 June 2009
Abstract
Key to understanding the epidemiology and pathogenesis of prion diseases, including chronic wasting disease (CWD) of cervids, is determining the mode of transmission from one individual to another. We have previously reported that saliva and blood from CWD-infected deer contain sufficient infectious prions to transmit disease upon passage into naïve deer. Here we again use bioassays in deer to show that blood and saliva of pre-symptomatic deer contain infectious prions capable of infecting naïve deer and that naïve deer exposed only to environmental fomites from the suites of CWD-infected deer acquired CWD infection after a period of 15 months post initial exposure. These results help to further explain the basis for the facile transmission of CWD, highlight the complexities associated with CWD transmission among cervids in their natural environment, emphasize the potential utility of blood-based testing to detect pre-clinical CWD infection, and could augur similar transmission dynamics in other prion infections.

PLoSOne
11 June 2009
Abstract
The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

PLoS One
29 May 2009
Abstract
Prion strain identification has been hitherto achieved using time-consuming incubation time determinations in one or more mouse lines and elaborate neuropathological assessment. In the present work, we make a detailed study of the properties of PrP-overproducing Tga20 mice. We show that in these mice the four prion strains examined are rapidly and faithfully amplified and can subsequently be discriminated by a cell-based procedure, the Cell Panel Assay.

Virology journal
26 May 2009
Abstract
The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland.
To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland.
It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated.
Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

PLoS Pathogens
15 May 2009
Author Summary
Prions are the unprecedented infectious agent associated with prion diseases, which are composed exclusively of a misfolded protein. In this study we report the de novo formation of infectious prion protein in vitro, which when inoculated into wild type animals is able to induce a new disease phenotype with unique clinical, neuropathological and biochemical characteristics. The findings described in this article are very important, because they support the prion hypothesis and provide a simple model for studying the mechanism responsible for the appearance of sporadic prion disease. The data also suggest that the universe of possible prions is not restricted to those currently known, but that likely many new forms of infectious protein foldings may be produced. This is worrisome, because it raises the possibility that novel and perhaps more aggressive infectious prion foldings may spontaneously originate in diverse species, leading to the emergence of new and unpredictable forms of transmissible diseases.

PLoS Pathogens
8 May 2009
Abstract
Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrPC), denoted PrPSc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrPC into PrPSc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrPC and PrPSc and measured PrPSc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.

CDC
24 April 2009
Overview
Swine Influenza (swine flu) is a respiratory disease of pigs caused by type A influenza virus that regularly causes outbreaks of influenza in pigs. Swine flu viruses cause high levels of illness and low death rates in pigs. Swine influenza viruses may circulate among swine throughout the year, but most outbreaks occur during the late fall and winter months similar to outbreaks in humans. The classical swine flu virus (an influenza type A H1N1 virus) was first isolated from a pig in 1930.

Eurosurveillance
26 March 2009
Overview
Arboviruses are arthropod-borne viruses, which include West Nile fever virus (WNFV), a mosquito-borne virus, Rift Valley fever virus (RVFV), a mosquito-borne virus, and Crimean-Congo haemorrhagic fever virus (CCHFV), a tick-borne virus. These arthropod-borne viruses can cause disease in different domestic and wild animals and in humans, posing a threat to public health because of their epidemic and zoonotic potential. In recent decades, the geographical distribution of these diseases has expanded. Outbreaks of WNF have already occurred in Europe, especially in the Mediterranean basin. Moreover, CCHF is endemic in many European countries and serious outbreaks have occurred, particularly in the Balkans, Turkey and Southern Federal Districts of Russia. In 2000, RVF was reported for the first time outside the African continent, with cases being confirmed in Saudi Arabia and Yemen. This spread was probably caused by ruminant trade and highlights that there is a threat of expansion of the virus into other parts of Asia and Europe.
In the light of global warming and globalisation of trade and travel, public interest in emerging zoonotic diseases has increased. This is especially evident regarding the geographical spread of vectorborne diseases. A multi-disciplinary approach is now imperative, and groups need to collaborate in an integrated manner that includes vector control, vaccination programmes, improved therapy strategies, diagnostic tools and surveillance, public awareness, capacity building and improvement of infrastructure in endemic regions.

PLoSOne
18 March 2009
Abstract
Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids (e.g. deer, elk, and moose). The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD). Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrPCWD was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA). The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrPCWD levels by traditional assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP) mice (373±3days in 2 of 9 urine-inoculated mice and 342±109 days in 8 of 9 saliva-inoculated mice). These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.

PLoS Pathogens
13 March 2009
Abstract
Neurotoxicity in all prion disorders is believed to result from the accumulation of PrP-scrapie (PrPSc), a β-sheet rich isoform of a normal cell-surface glycoprotein, the prion protein (PrPC). Limited reports suggest imbalance of brain iron homeostasis as a significant associated cause of neurotoxicity in prion-infected cell and mouse models. However, systematic studies on the generality of this phenomenon and the underlying mechanism(s) leading to iron dyshomeostasis in diseased brains are lacking. In this report, we demonstrate that prion disease–affected human, hamster, and mouse brains show increased total and redox-active Fe (II) iron, and a paradoxical increase in major iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) at the end stage of disease. Furthermore, examination of scrapie-inoculated hamster brains at different timepoints following infection shows increased levels of Tf with time, suggesting increasing iron deficiency with disease progression. Sporadic Creutzfeldt-Jakob disease (sCJD)–affected human brains show a similar increase in total iron and a direct correlation between PrP and Tf levels, implicating PrPSc as the underlying cause of iron deficiency. Increased binding of Tf to the cerebellar Purkinje cell neurons of sCJD brains further indicates upregulation of TfR and a phenotype of neuronal iron deficiency in diseased brains despite increased iron levels. The likely cause of this phenotype is sequestration of iron in brain ferritin that becomes detergent-insoluble in PrPSc-infected cell lines and sCJD brain homogenates. These results suggest that sequestration of iron in PrPSc–ferritin complexes induces a state of iron bio-insufficiency in prion disease–affected brains, resulting in increased uptake and a state of iron dyshomeostasis. An additional unexpected observation is the resistance of Tf to digestion by proteinase-K, providing a reliable marker for iron levels in postmortem human brains. These data implicate redox-iron in prion disease–associated neurotoxicity, a novel observation with significant implications for prion disease pathogenesis.


 

Emerging Infectious Disease

Nature
13 July 2009
Abstract
Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses1. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

CDC, EID
April 2009
Overview
The international course Emerging Viruses: Global Approaches and Specificities of the Amazon Region was held in Porto Velho, Rondonia, Brazil, November 17–December 7, 2007. Organized as part of the Amsud-Pasteur research collaboration program (sponsored by Institut Pasteur), the course was held there primarily because Rondonia State is located in the Amazon region, a particular environment in which new viruses could emerge as a consequence of ecologic changes brought about by exploration and other human activities.

Emerging Infectious Disease - Avian Influenza

PLoS One
3 February 2010
Abstract
For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents.
 

PLoS Pathogens
24 December 2009
Author Summary
H5N1 influenza viruses have caused over 400 human infections in 15 countries and continue to circulate in poultry and wild birds. Most human infections resulted from direct contact with virus-contaminated poultry or poultry products. It would be disastrous if H5N1 viruses acquired the ability to efficiently transmit among humans, because the mortality rate may exceed 60%. However, the genetic basis for transmission of H5N1 influenza viruses is largely unknown. Here, we demonstrate that the amino acid residue at 701 of PB2 is a prerequisite for transmission of H5N1 viruses in a mammalian guinea pig model. Interestingly, we found that the absence of glycosylation at residues 158–160 of the HA gene is pivotal for the H5N1 virus to bind to human-like receptors and to transmit in a mammal host. These findings are important for assessing the pandemic potential of H5N1 field isolates.
 

PLoS One
22 October 2009
Abstract
Several species of wild raptors have been found in Eurasia infected with highly pathogenic avian influenza virus (HPAIV) subtype H5N1. Should HPAIV (H5N1) reach North America in migratory birds, species of raptors are at risk not only from environmental exposure, but also from consuming infected birds and carcasses. In this study we used American kestrels as a representative species of a North American raptor to examine the effects of HPAIV (H5N1) infection in terms of dose response, viral shedding, pathology, and survival. Our data showed that kestrels are highly susceptible to HPAIV (H5N1). All birds typically died or were euthanized due to severe neurologic disease within 4–5 days of inoculation and shed significant amounts of virus both orally and cloacally, regardless of dose administered. The most consistent microscopic lesions were necrosis in the brain and pancreas. This is the first experimental study of HPAIV infection in a North American raptor and highlights the potential risks to birds of prey if HPAIV (H5N1) is introduced into North America.
 

Virology Journal
7 October 2009
Abstract
Avian influenza viruses, belonging to the family Orthomyxoviridae, possess distinct combinations of hemagglutinin (H) and the neuraminidase (N) surface glycoproteins. Typing of both H and N antigens is essential for the epidemiological and surveillance studies. Therefore, a rapid, sensitive, and specific method for their assay is desirable and an enzyme-linked immunosorbent assay (ELISA) can be useful for this purpose, using recombinant proteins.
The nucleoprotein (NP) and truncated neuraminidase subtype 3 and 7 of avian influenza virus (AIV) were expressed in Saccharomyces cerevisiae (S. cerevisiae) and used to develop an indirect ELISA for antibody detection. The developed assays were evaluated with a panel of 61 chicken serum samples. The performance of NP-ELISA was compared with the commercially available ProFlok(R) AIV ELISA kit. The results showed comparable agreement and sensitivity between the two tests indicating that NP-ELISA assay can be used for screening the influenza type A antibody in AIV-infected birds. The N3- and N7- ELISAs also reacted specifically to their type-specific sera and did not exhibit any cross-reaction with heterologous neuraminidase subtype specific sera.
The study demonstrates the expression of the NP, N3, and N7 proteins of AIV in S. cerevisiae and their application in developing an indirect ELISA for detecting NP, N3 and N7 antibodies from AIV-infected chicken sera. The described indirect ELISAs are rapid, sensitive, specific and can be used as promising tests during serological surveillance.

PLoS One 20 August 2009
Abstract
The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×102 vs. 2.3×104 vs. 8.7×104; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections.
 

PLoS One
5 August 2009
Abstract
Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.
Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.
Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.

CDC, EID
August 2009
Abstract
Hemagglutination-inhibition (HI) and neutralization are used to evaluate vaccines against influenza virus A (H5N1); however, poor standardization leads to interlaboratory variation of results. A candidate antibody standard (07/150) was prepared from pooled plasma of persons given clade 1 A/Vietnam/1194/2004 vaccine. To test human and sheep antiserum, 15 laboratories used HI and neutralization and reassortant A/Vietnam/1194/2004, A/turkey/Turkey/1/2005 (clade 2.2), and A/Anhui/1/2005 (clade 2.3.4) viruses. Interlaboratory variation was observed for both assays, but when titers were expressed relative to 07/150, overall percentage geometric coefficient of variation for A/Vietnam/1194/2004 was reduced from 125% to 61% for HI and from 183% to 81% for neutralization. Lack of reduced variability to clade 2 antigens suggested the need for clade-specific standards. Sheep antiserum as a standard did not reliably reduce variability. The World Health Organization has established 07/150 as an international standard for antibody to clade 1 subtype H5 and has an assigned potency of 1,000 IU/ampoule.

PLoS One
17 July 2009
Abstract
The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA) stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt), WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49th to 68th positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.
 

PLoS Medicine
23 June 2009
Background
In a 2007 article in PLoS Medicine, Holger J. Schünemann and colleagues described a new process used by the World Health Organization for rapidly developing clinical management guidelines in emergency situations. These situations include outbreaks of emerging infectious diseases. The authors discussed how they developed such a “rapid advice” guideline for the pharmacological management of avian influenza A (H5N1) virus infection. The guideline recommends giving the antiviral drug oseltamivir at a dose of 75 mg twice daily for five days. In this Debate, Nicholas White argues that such dosing is inadequate, Robert Webster and Elena Govorkova say that combination antiviral therapy should be used, and Tim Uyeki reminds us that clinical care of patients with H5N1 entails much more than antiviral treatment. These issues may also apply to therapy of patients hospitalized with severe disease due to novel swine-origin influenza A (H1N1) virus infection.

PLoS One
19 June 2009
Abstract
We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses.
We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5:3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log10 TCID50/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains.
Our findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.

WHO
May 2009
Overview
A new H5N1 recombinant vaccine virus has been developed by the WHO Collaborating Center for the Surveillance, Epidemiology and Control of Influenza at the Centers for Disease Control and Prevention (WHO CC), Atlanta, USA from A/Egypt/2321-NAMRU3/2007 (H5N1; Clade 2.2.1), thanks to the Ministry of Health & Population of Egypt for providing the virus specimens. This recombinant vaccine virus is available for distribution, under a Material Transfer Agreement (MTA).

PLoSOne
14 May 2009
Abstract
Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.
Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.
The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.
 

PLOS One
7 May 2009
Abstract
The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.
We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.
Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.
 

Medicalnewstoday 2 May 2009
Overview
A study published this week in the Proceedings of the National Academy of Sciences of the United States of America shows that Aflunov®, the Novartis investigational pre-pandemic avian influenza vaccine formulated with Novartis' proprietary MF59® adjuvant, can elicit a broadly cross-reactive immune response covering all known H5N1 antigenic variants, even when that booster dose is administered six years after the initial priming dose.
 

PLoSOne
29 April 2009
Abstract
Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that γ-ray inactivated flu virus can induce cross-reactive Tc cell responses.
Here, we report that intranasal administration of purified γ-ray inactivated human influenza A virus preparations (γ-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of γ-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections.
Intranasal γ-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.

PLoSOne
29 April 2009
Abstract
Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that γ-ray inactivated flu virus can induce cross-reactive Tc cell responses.
Here, we report that intranasal administration of purified γ-ray inactivated human influenza A virus preparations (γ-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of γ-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections.
Intranasal γ-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.

CIDRAP
23 April 2009

PLoS Medicine
21 April 2009
Abstract
Transmission of highly pathogenic avian H5N1 viruses from poultry to humans have raised fears of an impending influenza pandemic. Concerted efforts are underway to prepare effective vaccines and therapies including polyclonal or monoclonal antibodies against H5N1. Current efforts are hampered by the paucity of information on protective immune responses against avian influenza. Characterizing the B cell responses in convalescent individuals could help in the design of future vaccines and therapeutics.
To address this need, we generated whole-genome–fragment phage display libraries (GFPDL) expressing fragments of 15–350 amino acids covering all the proteins of A/Vietnam/1203/2004 (H5N1). These GFPDL were used to analyze neutralizing human monoclonal antibodies and sera of five individuals who had recovered from H5N1 infection. This approach led to the mapping of two broadly neutralizing human monoclonal antibodies with conformation-dependent epitopes. In H5N1 convalescent sera, we have identified several potentially protective H5N1-specific human antibody epitopes in H5 HA[(-10)-223], neuraminidase catalytic site, and M2 ectodomain. In addition, for the first time to our knowledge in humans, we identified strong reactivity against PB1-F2, a putative virulence factor, following H5N1 infection. Importantly, novel epitopes were identified, which were recognized by H5N1-convalescent sera but did not react with sera from control individuals (H5N1 naïve, H1N1 or H3N2 seropositive).
This is the first study, to our knowledge, describing the complete antibody repertoire following H5N1 infection. Collectively, these data will contribute to rational vaccine design and new H5N1-specific serodiagnostic surveillance tools.

Virology Journal
2 April 2009
Abstract
Avian influenza virus H5N1 is a major concern as a potential global pandemic. It is thought that multiple key events must take place before efficient human-to-human transmission of the virus occurs. The first step in overcoming host restriction is viral entry which is mediated by HA, responsible for both viral attachment and viral/host membrane fusion. HA binds to glycans-containing receptors with terminal sialic acid (SA). It has been shown that avian influenza viruses preferentially bind to _2,3-linked SAs, while human influenza A viruses exhibit a preference for _2,6-linked SAs. Thus it is believed the precise linkage of SAs on the target cells dictate host tropism of the viruses.
We demonstrate that H5N1 HA/HIV pseudovirus can efficiently transduce several human cell lines including human lung cells. Interestingly, using a lectin binding assay we show that the presence of both _2,6-linked and _2,3-linked SAs on the target cells does not always correlate with efficient transduction. Further, HA substitutions of the residues implicated in switching SA-binding between avian and human species did not drastically affect HA-mediated transduction of the target cells or target cell binding.
Our results suggest that a host factor(s), which is yet to be identified, is required for H5N1 entry in the host cells.
 

PLoSOne
18 March 2009
Abstract
Although vaccination can be a useful tool for control of avian influenza epidemics, it might engender emergence of a vaccine-resistant strain. Field and experimental studies show that some avian influenza strains acquire resistance ability against vaccination. We investigated, in the context of the emergence of a vaccine-resistant strain, whether a vaccination program can prevent the spread of infectious disease. We also investigated how losses from immunization by vaccination imposed by the resistant strain affect the spread of the disease.
We designed and analyzed a deterministic compartment model illustrating transmission of vaccine-sensitive and vaccine-resistant strains during a vaccination program. We investigated how the loss of protection effectiveness impacts the program. Results show that a vaccination to prevent the spread of disease can instead spread the disease when the resistant strain is less virulent than the sensitive strain. If the loss is high, the program does not prevent the spread of the resistant strain despite a large prevalence rate of the program. The epidemic's final size can be larger than that before the vaccination program. We propose how to use poor vaccines, which have a large loss, to maximize program effects and describe various program risks, which can be estimated using available epidemiological data.
We presented clear and simple concepts to elucidate vaccination program guidelines to avoid negative program effects. Using our theory, monitoring the virulence of the resistant strain and investigating the loss caused by the resistant strain better development of vaccination strategies is possible.


 

PLoSOne
17 March 2009
Abstract
Highly pathogenic avian influenza virus A/H5N1 was first officially reported in Africa in early 2006. Since the first outbreak in Nigeria, this virus spread rapidly to other African countries. From its emergence to early 2008, 11 African countries experienced A/H5N1 outbreaks in poultry and human cases were also reported in three of these countries. At present, little is known of the epidemiology and molecular evolution of A/H5N1 viruses in Africa. We have generated 494 full gene sequences from 67 African isolates and applied molecular analysis tools to a total of 1,152 A/H5N1 sequences obtained from viruses isolated in Africa, Europe and the Middle East between 2006 and early 2008. Detailed phylogenetic analyses of the 8 gene viral segments confirmed that 3 distinct sublineages were introduced, which have persisted and spread across the continent over this 2-year period. Additionally, our molecular epidemiological studies highlighted the association between genetic clustering and area of origin in a majority of cases. Molecular signatures unique to strains isolated in selected areas also gave us a clearer picture of the spread of A/H5N1 viruses across the continent. Mutations described as typical of human influenza viruses in the genes coding for internal proteins or associated with host adaptation and increased resistance to antiviral drugs have also been detected in the genes coding for transmembrane proteins. These findings raise concern for the possible human health risk presented by viruses with these genetic properties and highlight the need for increased efforts to monitor the evolution of A/H5N1 viruses across the African continent. They further stress how imperative it is to implement sustainable control strategies to improve animal and public health at a global level.
 

WHO
Overview
The development of representative H5N1 candidate vaccine viruses by the WHO Global Influenza Programme is being conducted as one compnent of the overall global strategy for pandemic preparedness. This summary provides an update on the characterization of H5N1 viruses circulating in birds, those that have caused human infections and the current status of the development of candidaze H5N1 vaccine viruses. This information may be used to guide national authorities as regards decisions on procurement of H5N1 vaccines.



 

Emerging Infectious Disease - SARS

PLoS Pathogens
5 February 2010
Author Summary
Severe acute respiratory syndrome coronavirus (SARS-CoV) infection causes acute lung injury that may develop into the life-threatening acute respiratory distress syndrome (ARDS) in mostly elderly individuals. Although SARS-CoV infection can be fatal, most patients recover, suggesting that protective host responses are operational to combat the viral infection. Therefore, we used age as predisposing factor to obtain insight into the pathogenesis of SARS-CoV. In this study, we show that SARS-CoV-infected aged macaques developed significantly more pathology than young adult animals, which could not be contributed to differences in viral replication. Using comparative microarray analyses, it was shown that although the nature of the host response to SARS-CoV infection was similar in aged and young adult macaques, the severity was significantly different, with aged macaques displaying an increase in differential expression of genes associated with inflammation. Interestingly, type I IFN-β mRNA levels correlated negatively with gross pathology. Therapeutic treatment of aged macaques with type I IFN reduced pathology without affecting virus replication. However, pro-inflammatory gene expression was significantly diminished. Thus, modulation of the host response by type I IFNs provides a promising outlook for novel intervention strategies.
 

PLoS One
17 December 2009
Abstract
The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) and inhibitory effects of a dominant-negative form of eIF2α on GRP78 promoter activity, (2) increased translation of activating transcription factor 4 (ATF4) mRNA, and (3) ATF4-dependent activation of the C/EBP homologous protein (CHOP) gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN) signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1) degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity.
 

PLoS One
17 November 2009
Abstract
Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases.

Purified triSpike proteins were readily cleaved in vitro by three different airway proteases: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC) and amino acid sequencing analyses identified two arginine residues (R667 and R797) as potential protease cleavage site(s). The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A). Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment.

These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.
 

PLoS One
13 November 2009
Abstract
Severe acute respiratory syndrome (SARS), caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA)–mediated host–virus interactions in bronchoalveolar stem cells (BASCs) at the onset of infection by correlating the “BASC–microRNome” with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA–mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-), a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. Overall, this investigation reveals another mode of exploitation of cellular miRNA machinery by virus to their own advantage.
 

PLoS Pathogens
15 May 2009
Author Summary
The genome of the SARS coronavirus codes for 16 non-structural proteins that are involved in replicating this huge RNA (approximately 29 kilobases). The roles of many of these in replication (and/or transcription) are unknown. We attempt to derive conclusions concerning the possible functions of these proteins from their three-dimensional structures, which we determine by X-ray crystallography. Non-structural protein 3 contains at least seven different functional modules within its 1922-amino-acid polypeptide chain. One of these is the so-called SARS-unique domain, a stretch of about 338 residues that is completely absent from any other coronavirus. It may thus be responsible for the extraordinarily high pathogenicity of the SARS coronavirus, compared to other viruses of this family. We describe here the three-dimensional structure of the SARS-unique domain and show that it consists of two modules with a known fold, the so-called macrodomain. Furthermore, we demonstrate that these domains bind unusual nucleic-acid structures formed by consecutive guanosine nucleotides, where four strands of nucleic acid are forming a superhelix (so-called G-quadruplexes). SUD may be involved in binding to viral or host-cell RNA bearing this peculiar structure and thereby regulate viral replication or fight the immune response of the infected host cell.
 

Emerging Infectious Disease - New Influenza A (H1N1)

CDC, EID
March 2010, Epub ahead of print
To the Editor
In April 2009, the outbreak of influenza A pandemic (H1N1) 2009 virus was reported. Subsequently, the disease spread throughout the world, and the pandemic alert level was raised to level 6 in June by the World Health Organization. Pandemic (H1N1) 2009 virus infection spread to Thailand and is now found throughout Thailand. Similar to the effects of other viruses, pandemic (H1N1) 2009 virus may cause neurologic complications. Associated neurologic symptoms were first reported from Dallas, Texas, USA: 4 children experienced unexplained seizures or had an alteration of consciousness level that was associated with this virus (1). We report an adult patient with pandemic (H1N1) 2009 infection who had neurologic complications.
 

PLoS One
11 February 2010
Abstract
Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies.

The hemagglutinin (HA) and neuraminidase (NA) matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains) and a highly pathogenic avian influenza A virus (H5N1) were studied using a pseudotyped particle (pp) system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005) could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern.

Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.
 

PLoS One
11 February 2010
Abstract
The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.
 

PLoS One
5 February 2010
Abstract
The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic.
 

PLoS One
18 January 2010


Abstract
The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated.
HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007–2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes.
Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.

PLoS One
15 January 2010


Abstract
Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of α2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

CDC, EID
March 2010
Abstract
In 2009, a new H1N1 influenza virus (pandemic [H1N1] 2009) emerged in Mexico, spread to the United States (1), and subsequently caused the first influenza pandemic of the 21st century (2). The emergence of pandemic (H1N1) 2009 virus is imperfectly understood, but an early switch in hemagglutinin (HA) receptor specificity is essential to allow interspecies transmission (3–5).

Pandemic (H1N1) 2009 virus strains were recently reported to be reassortants of the North American and European swine lineages (6). Phylogenetic evidence suggests that this reassortment event occurred 10–17 years ago (7). These data suggest that the current pandemic (H1N1) 2009 virus strains should have receptor specificity typically found in the HA of mammalian viruses (Neu5Acα2,6Gal). In addition, they may have lost the ability to replicate in avian hosts, the natural reservoir species. To test these hypotheses, we examined the biological properties of pandemic (H1N1) 2009 virus, including receptor specificity, erythrocyte binding, and ability to replicate in avian species.

BMC Infectious Diseases
7 January 2010
Abstract
A pandemic novel H1N1 swine-origin influenza virus has emerged. Most recently the World Health Organization has announced that in a country-dependent fashion, up to 15 % of cases may require hospitalization, often including respiratory support. It is now clear that healthy children and young adults are disproportionately affected, most unusually among those with severe respiratory disease without underlying conditions. One possible explanation for this case age distribution is the doctrine of Original Antigenic Sin, i.e., novel H1N1 may be antigenically similar to H1N1 viruses that circulated at an earlier time. Persons whose first exposure to influenza viruses was to such similar viruses would be relatively immune. However, this principle is not sufficient to explain the graded susceptibility between ages 20 and 60, the reduced susceptibility in children below age 10, and the unusual toxicity observed.

We collected case data from 11 countries, about 60% of all cases reported through mid-July 2009. We compared sequence data for the hemagglutinin of novel H1N1 with sequences of H1N1 viruses from 1918 to the present. We searched for sequence differences that imply loss of antigenicity either directly through amino acid substitution or by the appearance of sites for potential glycosylation proximal to sites known to be antigenic in humans. We also considered T-cell epitopes. In our composite, over 75% of confirmed cases of novel H1N1 occurred in persons [less than or equal to] 30 years old, with peak incidence in the age range 10-19 years. Less than 3% of cases occurred in persons over 65, with a gradation in incidence between ages 20 and 60 years. The sequence data indicates that novel H1N1 is most similar to H1N1 viruses that circulated before 1943. Novel H1N1 lacks glycosylation sites on the globular head of hemagglutinin (HA1) near antigenic regions, a pattern shared with the 1918 pandemic strain and H1N1 viruses that circulated until the early 1940s. Later H1N1 viruses progressively added new glycosylation sites likely to shield antigenic epitopes, while T-cell epitopes were relatively unchanged.

In this evolutionary context, Original Antigenic Sin exposure should produce an immune response increasingly mismatched to novel H1N1 in progressively younger persons. We suggest that it is this mismatch that produces both the gradation in susceptibility and the unusual toxicity. Several murine studies suggest specific cell types as a likely basis of the unusual toxicity. These studies also point to widely available pharmaceutical agents as plausible candidates for mitigating the toxic effects. The principle of Original Antigenic Sin modified by glycosylation appears to explain both the case age distribution and the unusual toxicity pattern of the novel H1N1 pandemic. In addition, it suggests pharmaceutical agents for immediate investigation for mitigation potential, and provides strategic guidance for the distribution of pandemic mitigation resources of all types.
 

CDC, EID (Epub ahead of print)
January 2010
Overview
We previously reported detection of double resistance to oseltamivir and amantadine of influenza virus A (H1N1) in Hong Kong during the first half of 2008 (1). Three different strains of A/Hong Kong/2652/2006-like (clade 2C) viruses that carried the S31N mutation in the matrix (M2) gene associated with amantadine resistance acquired a neuraminidase (NA) gene with CAT→TAT change at position 274 through either reassortment with an oseltamivir-resistant A/Brisbane/59/2007-like (clade 2B) virus or spontaneous H274Y mutation in the NA gene. A clade 2C strain resistant to both oseltamivir and amantadine also was detected in Cambodia in 2007 (2).
 

The Lancet
4 January 2010
Summary
With the ongoing 2009 pandemic of influenza A H1N1, development of pandemic influenza vaccines has generated much interest. We investigated the safety and immunogenicity of a whole-virion, inactivated, adjuvanted pandemic H1N1 vaccine in adult and elderly volunteers, given without or simultaneously with the 2009—10 seasonal trivalent influenza vaccine.

This prospective, randomised study was undertaken in two centres in Hungary. 355 participants, including 203 adults (18—60 years) and 152 elderly people (>60 years), were assigned by stratified randomisation to either 0·5 mL of the pandemic vaccine (Fluval P, a monovalent vaccine with 6 μg haemagglutinin per 0·5 mL content and aluminium phosphate gel adjuvant; n=178) or 0·5 mL of the pandemic vaccine and 0·5 mL of the seasonal trivalent vaccine (Fluval AB, a trivalent inactivated whole-virion influenza vaccine; n=177). All vaccinations were done by specific study personnel, who did not take part in the assessment of safety or immunogenicity. Co-primary objectives were safety and immunogenicity by haemagglutinin inhibition testing. All analyses were done according to a pre-established analysis plan. This study is registered with ClinicalTrials.gov, number NCT01010893.

Two participants receiving the pandemic vaccine only (group 1) and one receiving pandemic and seasonal vaccines (group 2) were lost to follow-up. Participants in both groups developed antibody responses against the pandemic influenza A H1N1 virus (group 1: seroconversion for adults 74·3%, 95% CI 64—6—82·4 and for elderly people 61·3%, 49·1—72·4; group 2: 76·8%, 67·2—84·7 and 81·8%, 71·4—89·7, respectively). Single doses of 6 μg fulfilled European Union and US licensing criteria for interpandemic and pandemic influenza vaccines. Simultaneously, participants in group 2 developed the immune responses needed for licensing for all three seasonal strains in the seasonal vaccine for the 2009—10 season. All adverse events were rare, mild, and transient; the most frequent were pain at injection site (eight cases in group 1 vs 18 in group 2) and fatigue for 1—2 days after vaccination (three vs five cases).

The present pandemic vaccine is safe and immunogenic in healthy adult and elderly patients, and needs low doses and only one injection to trigger immune responses to comply with licensing criteria. It can be safely co-administered with the 2009—10 seasonal influenza vaccine.


 

The Lancet
4 January 2010
Summary
Data are needed from large clinical trials of paediatric, adult, and elderly people to fi nd the appropriate antigen dose and vaccination schedule for the 2009 pandemic influenza A

H1N1. We therefore report preliminarysafety and immunogenicity results after one injection of a licensed monovalent pandemic H1N1 vaccine in the USA.

We randomly assigned healthy children (aged 6–35 months and 3–9 years) and adults (18–64 years and ≥65 years) to vaccine containing per dose 7·5 μg (children and adults), 15 μg (children and adults), or 30 μg (adultsonly) haemagglutinin in two placebo-controlled, observer-masked, multicentre phase 2 studies done in the USA. Participants were allocated with an interactive voice-response system or computer-generated randomisation lists with opaque scratchable patches. Primary outcome was haemagglutination inhibition antibody response 21 days after the fi rst of two planned vaccinations (interim analysis of studies in progress). Analyses were by full-analysis set. The trials are registered with ClinicalTrials.gov as NCT00953524 and NCT00952419.

410 of 423 children and 724 of 750 adults given an active vaccine, and 50 of 51 children and 95 of 99 adults given placebo were assessed for immunogenicity on day 21. After active vaccination, 45 of 101 (45%; 95% CI 35–55) to 47 of 94 (50%; 40–61) infants aged 6–35 months, 75 of 109 (69%; 59–77) to 80 of 106 (75%; 66–83) 3–9-year-old children, 134 of 141 (95%; 90–98) to 144 of 144 (100%; 98–100) of 18–64-year-old adults, and 93 of 100 (93%; 86–96) to 93 of 98 (95%; 89–98) elderly adults were seroprotected (proportion with titres ≥1:40). No vaccine-related serious adverse events occurred. Injection-site and systemic reactions were reported by up to about 50% of every age and vaccine group, with no noticeable diff erences between vaccine and placebo groups.

One dose of vaccine was highly immunogenic in adults, suggesting that it aff orded suffi cient protection against this pandemic infl uenza A H1N1 virus. Two doses of vaccine will probably be needed in children younger than 9 years. Safety and reactogenicity of the vaccine were acceptable and similar to those of seasonal vaccine.
 

PLoS One One
1 January 2010
Abstract
The pandemic influenza virus (2009 H1N1) was recently introduced into the human population. The hemagglutinin (HA) gene of 2009 H1N1 is derived from “classical swine H1N1” virus, which likely shares a common ancestor with the human H1N1 virus that caused the pandemic in 1918, whose descendant viruses are still circulating in the human population with highly altered antigenicity of HA. However, information on the structural basis to compare the HA antigenicity among 2009 H1N1, the 1918 pandemic, and seasonal human H1N1 viruses has been lacking. By homology modeling of the HA structure, here we show that HAs of 2009 H1N1 and the 1918 pandemic virus share a significant number of amino acid residues in known antigenic sites, suggesting the existence of common epitopes for neutralizing antibodies cross-reactive to both HAs. It was noted that the early human H1N1 viruses isolated in the 1930s–1940s still harbored some of the original epitopes that are also found in 2009 H1N1. Interestingly, while 2009 H1N1 HA lacks the multiple N-glycosylations that have been found to be associated with an antigenic change of the human H1N1 virus during the early epidemic of this virus, 2009 H1N1 HA still retains unique three-codon motifs, some of which became N-glycosylation sites via a single nucleotide mutation in the human H1N1 virus. We thus hypothesize that the 2009 H1N1 HA antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in humans. Indeed, amino acid substitutions predicted here are occurring in the recent 2009 H1N1 variants. The present study suggests that antibodies elicited by natural infection with the 1918 pandemic or its early descendant viruses play a role in specific immunity against 2009 H1N1, and provides an insight into future likely antigenic changes in the evolutionary process of 2009 H1N1 in the human population.

PLoS One
31 December 2010
Abstract
Initial reports in May 2009 of the novel influenza strain H1N1pdm estimated a case fatality rate (CFR) of 0.6%, similar to that of seasonal influenza. In July 2009, however, Argentina reported 3056 cases with 137 deaths, representing a CFR of 4.5%. Potential explanations for increased CFR included virus reassortment or genetic drift, or infection of a more vulnerable population. Virus genomic sequencing of 26 Argentinian samples representing both severe and mild disease indicated no evidence of reassortment, mutations associated with resistance to antiviral drugs, or genetic drift that might contribute to virulence. Furthermore, no evidence was found for increased frequency of risk factors for H1N1pdm disease.

We examined nasopharyngeal swab samples (NPS) from 199 cases of H1N1pdm infection from Argentina with MassTag PCR, testing for 33 additional microbial agents. The study population consisted of 199 H1N1pdm-infected subjects sampled between 23 June and 4 July 2009. Thirty-nine had severe disease defined as death (n = 20) or hospitalization (n = 19); 160 had mild disease. At least one additional agent of potential pathogenic importance was identified in 152 samples (76%), including Streptococcus pneumoniae (n = 62); Haemophilus influenzae (n = 104); human respiratory syncytial virus A (n = 11) and B (n = 1); human rhinovirus A (n = 1) and B (n = 4); human coronaviruses 229E (n = 1) and OC43 (n = 2); Klebsiella pneumoniae (n = 2); Acinetobacter baumannii (n = 2); Serratia marcescens (n = 1); and Staphylococcus aureus (n = 35) and methicillin-resistant S. aureus (MRSA, n = 6). The presence of S. pneumoniae was strongly correlated with severe disease. S. pneumoniae was present in 56.4% of severe cases versus 25% of mild cases; more than one-third of H1N1pdm NPS with S. pneumoniae were from subjects with severe disease (22 of 62 S. pneumoniae-positive NPS, p = 0.0004). In subjects 6 to 55 years of age, the adjusted odds ratio (OR) of severe disease in the presence of S. pneumoniae was 125.5 (95% confidence interval [CI], 16.95, 928.72; p<0.0001).

The association of S. pneumoniae with morbidity and mortality is established in the current and previous influenza pandemics. However, this study is the first to demonstrate the prognostic significance of non-invasive antemortem diagnosis of S. pneumoniae infection and may provide insights into clinical management.
 

PLoS One
23 December 2009
Abstract
The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host.
Blood was sampled within the first two days of the presentation of signs of infection from 10 healthy volunteers; from 18 cases of flu-like syndrome; and from 31 cases of infection by H1N1 confirmed by reverse RT-PCR. Absolute counts of subtypes of monocytes and of lymphocytes were determined after staining with monoclonal antibodies and analysis by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and stimulated with various bacterial stimuli. Concentrations of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-18, interferon (FN)-alpha and of IFN-gamma were estimated in supernatants by an enzyme immunoassay. Infection by H1N1 was accompanied by an increase of monocytes. PBMCs of patients evoked strong cytokine production after stimulation with most of bacterial stimuli. Defective cytokine responses were shown in response to stimulation with phytohemagglutin and with heat-killed Streptococcus pneumoniae. Adaptive immune responses of H1N1-infected patients were characterized by decreases of CD4-lymphocytes and of B-lymphocytes and by increase of T-regulatory lymphocytes (Tregs).

Infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses characterized by defective cytokine responses to S.pneumoniae. Alterations of the adaptive immune responses are predominated by increase of Tregs. These findings signify a predisposition for pneumococcal infections after infection by H1N1 influenza.
 

PLoS One
17 December 2009
Abstract
Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies.

We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus.

Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride.
 

WHO
30 November 2009

Please be aware that the updated and revised guidance document "Laboratory biorisk management for laboratories handling human specimens suspected or confirmed to contain influenza A (H1N1) causing the current international epidemics" from 30 November 2009 is now available on the web.

CDC,EID
December 2009, Epub ahead of print
Overview
As the world struggles with the challenges of influenza A pandemic (H1N1) 2009, it is clear that treatment options for critically ill infected patients are suboptimal because deaths continue to be reported in otherwise young and healthy patients. Extracorporeal membrane oxygenation (ECMO) is an established therapeutic option for patients with medically refractory cardiogenic or respiratory failure. We describe the successful use of ECMO in a patient with complicated pneumonia and influenza A pandemic (H1N1) 2009 virus infection.
 

CIDRAP
30 November 2009
Overview
This is an CIDRAP-authored overview updated with new research.
 

PLoS One
17 November 2009
Abstract
The Pandemic (H1N1) 2009 is spreading to numerous countries and causing many human deaths. Although the symptoms in humans are mild at present, fears are that further mutations in the virus could lead to a potentially more dangerous outbreak in subsequent months. As the primary immunity-eliciting antigen, hemagglutinin (HA) is the major agent for host-driven antigenic drift in A(H3N2) virus. However, whether and how the evolution of HA is influenced by existing immunity is poorly understood for A(H1N1). Here, by analyzing hundreds of A(H1N1) HA sequences since 1918, we show the first evidence that host selections are indeed present in A(H1N1) HAs. Among a subgroup of human A(H1N1) HAs between 1918~2008, we found strong diversifying (positive) selection at HA1 156 and 190. We also analyzed the evolutionary trends at HA1 190 and 225 that are critical determinants for receptor-binding specificity of A(H1N1) HA. Different A(H1N1) viruses appeared to favor one of these two sites in host-driven antigenic drift: epidemic A(H1N1) HAs favor HA1 190 while the 1918 pandemic and swine HAs favor HA1 225. Thus, our results highlight the urgency to understand the interplay between antigenic drift and receptor binding in HA evolution, and provide molecular signatures for monitoring future antigenically drifted 2009 pandemic and seasonal A(H1N1) influenza viruses.

PLoS One
6 November 2009
Abstract
Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase®), a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI) strain (H5N1). Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.
 

PLOS One
6 November 2009
Abstract
The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.



 

CIDRAP
30 October 2009

CDC, Taiwan
20 October 2009
Overview
On October 20, 2009, the Central Epidemic Command Center (CECC) announced the discovery of the first Oseltamivir-resistant pandemic influenza A (H1N1) virus strain in Taiwan. According to statistics announced by the World Health Organization (WHO), a total of ten countries have discovered a total of 35 strains of antiviral-reisistant pandemic influenza A (H1N1) virus. According to the epidemiological investigation conducted by the Taiwan Centers for Disease Control (Taiwan CDC), the first Oseltamivir-resistant pandemic influenza A (H1N1) virus strain in Taiwan was discovered in an individual case and no signs of cluster outbreak or further transmission have been detected. In addition, CECC has reported the case to WHO.
 

CIDRAP
9 October 2009

CDC, EID October 2009
Abstract
During June 2–8, 2009, an outbreak of influenza A pandemic (H1N1) 2009 occurred among 30 members of a tour group in China. To identify the mode of transmission and risk factors, we conducted a retrospective cohort investigation. The index case-patient was a female tourist from the United States. Secondary cases developed in 9 (30%) tour group members who had talked with the index case-patient and in 1 airline passenger (not a tour group member) who had sat within 2 rows of her. None of the 14 tour group members who had not talked with the index case-patient became ill. This outbreak was apparently caused by droplet transmission during coughing or talking. That airborne transmission was not a factor is supported by lack of secondary cases among fellow bus and air travelers. Our findings highlight the need to prevent transmission by droplets and fomites during a pandemic.
 

ECDC, daily updated

PLoS One
17 August 2009
Abstract
Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.

WHO Guidance documents

Information on Laboratory biorisk management for laboratories handling human specimens suspected or confirmed to contain influenza A (H1N1) causing the current international epidemics 

CIDRAP
31 August 2009

WHO
20 August 2009
Executive summary
The purpose of this document is to provide a basis for advice to clinicians on the use of the currently available antivirals for patients presenting with illness due to influenza virus infection as well the potential use of the medicines for chemoprophylais. The document addresses specifically the two neuraminidase inhibitors oseltamivir and zanamivir, and the two M2 inhibitors amantadine and rimantadine. It includes recommendations on the use of some other potential pharmacological treatments. While the focus of the document is on management of patients with pandemic influenza (H1N1) 2009 virus infection, the document includes guidance on the use of the antivirals for other seasonal influenza virus strains, and for infections due to novel influenza A virus strains. WHO recommends that country and local public health authorities issue local guidance for clinicians from time to time that places these recommendations in the context of epidemiological and antiviral susceptibility data on the locally circulating influeza strains.
This guidance expands on the recommendations published in May 2009 titled ʺClinical management of human infection with new influenza A (H1N1) virus: Initial guidanceʺ. The recommendations are based on a review of data obtained with previously circulating strains, and treatment of human H5N1 influenza virus infections. It is anticipated that as the prevalence and severity of the current epidemic changes, further information will become available that may warrant revision of the recommendations.



 

CDC
30 July 2009
Overview
With the new H1N1 virus continuing to cause illness, hospitalizations and deaths in the US during the normally flu-free summer months and some uncertainty about what the upcoming flu season might bring, CDC's Advisory Committee on Immunization Practices has taken an important step in preparations for a voluntary novel H1N1 vaccination effort to counter a possibly severe upcoming flu season. On July 29, ACIP met to consider who should receive novel H1N1 vaccine when it becomes available.
 

PLoS One
28 July 2009
Abstract
In April 2009, novel swine-origin influenza viruses (S-OIV) were identified in patients from Mexico and the United States. The viruses were genetically characterized as a novel influenza A (H1N1) strain originating in swine, and within a very short time the S-OIV strain spread across the globe via human-to-human contact.
We conducted a comprehensive computational search of all available sequences of the surface proteins of H1N1 swine influenza isolates and found that a similar strain to S-OIV appeared in Thailand in 2000. The earlier isolates caused infections in pigs but only one sequenced human case, A/Thailand/271/2005 (H1N1).
Differences between the Thai cases and S-OIV may help shed light on the ability of the current outbreak strain to spread rapidly among humans.

CIDRAP
27 July 2009

ECDC
20 July 2009
Executive summary
The interim ECDC risk assessment for Europe is that the 2009 pandemic influenza A(H1N1) virus will continue to spread, but many uncertainties remain. Though it seems that most of those infected in the US and in Europe experience a mild and self-limiting infection, this picture is still unclear as there has not been enough transmission to judge the effects, especially in those more at risk. The indications from Europe and North America are that there are significant differences between the pandemic and the seasonal influenzas as regards more severe disease: there seems to be an underrepresentation of older people and a prominent representation of adults under the age of 60 with chronic ill health (including very obese persons), pregnant women, and very young children. If confirmed this will have important implications for early treatment and vaccination policies.

ECDC
19 May 2009
Overview
The ECDC intends to produce a series of interim guidance documents on suggested procedures to be put in place in order to reduce the risk of transmission of the new influenza A (H1N1) virus.
This guidance only applies to a situation when there are relatively few persons under investigation, and will be revised if there seems to be a wider spread.
This particular document specifically relates to the management of persons defined below and their close contacts from a public health perspective. It does not cover the detailed medical management of patients, especially those with severe infection.
 

WHO 13 July 2009
Overview
On 7 July 2009, the Strategic Advisory Group of Experts (SAGE) on Immunization held an extraordinary meeting in Geneva to discuss issues and make recommendations related to vaccine for the pandemic (H1N1) 2009.
SAGE reviewed the current pandemic situation, the current status of seasonal vaccine production and potential A(H1N1) vaccine production capacity, and considered potential options for vaccine use.

WHO
10 July 2009
Background
This document updates the interim WHO guidance on global surveillance of pandemic (H1N1) 2009 virus infection in humans. The guidance has been revised to make it applicable to current global pandemic phase 6. It will be further reviewed and modified as the pandemic (H1N1) 2009 evolves.
Standardized and coordinated international information sharing is crucial for the management of the pandemic at global and national levels. National authorities need to know how the pandemic is evolving, not only in their own country, but also in neighbouring countries and continents. The continual flow and analysis of information provided by individual countries contributes to the development of a global picure that:

• results in a better understanding of critical clinical, epidemiological and virological features of the (H1N1) 2009 pandemic
• guides global prevention and control activities
• allows health care providers and public health authorities to modify their own strategies for case management, community mitigation, and health resource allocation
• reduces the impact of inaccurate and unconfirmed rumours.

CIDRAP
2 July 2009
Overview
The novel H1N1 (swine) influenza now circling the globe causes more serious lung disease than seasonal flu strains and sheds from the lung and throat tissue where it reproduces at higher rates, according to two animal studies published today—findings that could explain autopsies and case reports of severe pneumonia as well as the virus's rapid spread.
 

HPA 19 June 2009
Overview
Swine flu is a respiratory illness caused by a virus that usually infects pigs. People do not normally get swine flu but human infection can happen. The latest outbreaks in countries around the world have been caused by a new version of the swine flu virus called influenza A/H1N1v.
This is an updated compilation of the currently most important questions concerning swine flu for the public.
 

ScienceDaily
12 June 2009
Overview
A new, easy-to-perform method for detecting both seasonal influenza A virus and the emerging H1N1 swine-derived influenza A virus in human clinical samples offers a fast, sensitive, and cost-effective diagnostic test that runs on standard laboratory equipment. This timely and broadly applicable molecular technique is described in a new article.

CIDRAP
10 June 2009
Overview
This is a CIDRAP-authored update on the current state of knowledge on swine flu.
 

CDC
9 June 2009
Overview
The objective is to provide interim guidance on which groups should be vaccinated with the 23-valent pneumococcal polysaccharide vaccine (PPSV23) to prevent pneumococcal infections during the outbreak of novel influenza A(H1N1).

WHO
26 May 2009
Overview
The novel influenza A (H1N1) virus which is causing infection among humans, was first identified in the United States on 15 April 2009, and shown to be genetically related to recent swine influenza viruses, but to have a genetic make‐up not previously detected among viruses infecting either swine or human populations1. It was later shown that outbreaks of respiratory illness experienced in several parts of Mexico in March and April 2009 were due to these novel influenza A (H1N1) viruses2. Subsequently the novel A (H1N1) virus has caused outbreaks of disease in several countries, initially in North America, and has now spread to 46 countries worldwide. The influenza A (H1N1) virus is thus considered to pose a pandemic threat and on 29 April 2009 WHO raised its influenza pandemic alert level to phase 5.
The emergence and spread of the novel influenza A (H1N1) virus has been of great concern globally. The selection, development and availability of A (H1N1) vaccine viruses are essential components of the global strategy for pandemic preparedness and response. This summary provides an update on the characterization of available novel influenza A (H1N1) viruses affecting humans and makes a recommendation on suitable viruses for development of vaccines against this emergent influenza A (H1N1) infection. WHO, through the Global Influenza Surveillance Network, will continue to monitor the evolution of the influenza A (H1N1) virus, and will update recommendations of vaccine viruses whenever needed.
This recommendation does not replace the annual WHO recommendations of vaccine viruses for seasonal influenza vaccines. WHO will update the recommendations of vaccine viruses for inclusion in seasonal influenza vaccines in September 2009 for the southern hemisphere and in February 2010 for the northern hemisphere, as usual.
 

CIDRAP
22 May 2009
Information
This is a CIDRAP-authored overview, updated with new research.

ECDC
18 May 2009
Overview
Within the last 24 hours, 14 new confirmed cases were reported in the EU and EFTA countries, reaching a total of 263 confirmed cases reported from 16 countries. The table of figures and the maps will be updated regularly. The ECDC report gives an update of confirmed cases as of 18 May.
 

ECDC
15 May 2009
Overview
This is an update on current questions concering swine flu.

CDC
13 May 2009
Overview
These interim recommendations are subject to change as more information becomes available.
This guidance is for all individuals who may be processing or performing diagnostic testing on clinical specimens from patients with suspected novel influenza A (H1N1) virus infection, or performing viral isolation.

WHO
13 May 2009
Overview
The WHO Collaborating Centre for Influenza at CDC Atlanta, United States of America, has made available the M2 detailed pyrosequencing protocol, attached, for antiviral suceptibility testing of influenza A (H1N1). This document updates and replaces the protocol for antiviral suceptibility testing by pyrosequencing, published on 29 April 2009.
 

Virology Journal
7 May 2009
Abstract (provisional)
Beginning in March 2009, an outbreak of influenza in North America was found to be caused by a new strain of influenza virus, designated Influenza H1N1 2009, which is a reassortant of swine, avian and human influenza viruses. Over a thousand total cases were identified with the first month, chiefly in the United States and Mexico, but also involving several European countries. Actions concerning Influenza H1N1 2009 need to be based on fact and science, following recommendations of public health officials, and not fueled by political, legal or other interests. Every influenza outbreak or pandemic is unique, so the facts of each one must be studied before an appropriate response can be developed. While reports are preliminary, through the first 4 weeks of the outbreak it does not appear to be severe either in terms of the attack rate in communities or in the virulence of the virus itself. However, there are significant changes in both the hemagglutinin and neuraminidase proteins of the new virus, 27.2% and 18.2% of the amino acid sequence, from prior H1N1 isolates in 2008 and the current vaccine. Such a degree of change qualifies as an "antigenic shift", even while the virus remains in the H1N1 family of influenza viruses, and may give influenza H1N1 2009 significant pandemic potential. Perhaps balancing this shift, the novel virus retains more of the core influenza proteins from animal strains than successful human influenza viruses, and may be inhibited from its maximum potential until further reassortment or mutation better adapts it to multiplication in humans. While contact and respiratory precautions such as frequent handwashing will slow the virus through the human population, it is likely that development of a new influenza vaccine tailored to this novel Influenza H1N1 2009 strain will be essential to blunt its ultimate pandemic impact.
 

Eurosurveillance
7 May 2009
Overview
The recent detection of a novel influenza A(H1N1) virus has led to the first WHO declaration of a Public Health Event of International Concern under the International Health Regulations (IHR 2005). Here we review the early epidemiological findings of confirmed cases in Mexico, the United States, Canada and EU/EFTA countries. Strengthened surveillance and continued, transparent communication across public health agencies globally will be necessary in coming months.

CIDRAP
7 May 2009
Information
This ia a CIDRAP-authored overview updated with new research.
 

WHO
5 May 2009
Overview
So far most people who have contracted the new A (H1N1) virus have experienced influenza-like symptoms (such as sore throat, cough, runny nose, fever, malaise, headache, joint/muscle pain) and recovered without antiviral treatment.
Antiviral drugs may reduce the symptoms and duration of illness, just as they do for seasonal influenza. They also may contribute to preventing severe disease and death. Influenza A (H1N1) is a new virus and only a small number of people with the infection have been treated for it with antiviral drugs. WHO is in touch with public health authorities and clinicians in affected countries and is gathering information about how effective the drugs are.

ECDC
4 May 2009
Overview
In the absence of a specific vaccine, there are a number of personal measures that people may take to reduce their risk of acquiring influenza. Influenza is a viral infection that spreads from person to person principally when people cough or sneeze, or by direct or indirect contact with respiratory secretions from infectious persons that are on their hands or on surfaces. The following recommendations are based in part on evidence from studies and in part on judgement based on public health experience.

ECDC
4 May 2009
Overview
The influenza A(H1N1) virus that has recently been found is a new virus subtype of influenza affecting humans, which contains genes from pig, bird and human influenza viruses, in a combination that has never been observed before anywhere in the world. In addition there is every indication that this virus has been transmitting from human to human and the resulting illnesses have been severe in a substantial number of cases in Mexico. Outside Mexico the disease has been mild, and there has only been one severe case reported in the EU.
 

CDC
2 May 2009
Background
Rapid influenza diagnostic tests can help in the diagnosis and management of patients who present with signs and symptoms compatible with influenza. Such tests detect seasonal influenza A and B viral nucleoprotein antigens in respiratory specimens. The currently circulating novel H1N1 influenza virus (also referred to as swine flu) is an influenza A virus. Data are not yet available to inform recommendations on the use of rapid influenza diagnostic tests in patients with novel H1N1 virus infection. It is reasonable to assume that rapid diagnostic tests that detect influenza A viral nucleoprotein antigen can detect novel H1N1 flu infection in respiratory specimens as these nucleoprotein antigens are highly conserved across influenza A viruses. However, the sensitivity and specificity of the different rapid tests is not yet known for this novel virus. CDC has received anecdotal reports of false positive and false negative results. Clinicians may consider using rapid diagnostic tests as part of their evaluation of patients with signs and symptoms compatible with influenza, but results should be interpreted with caution. Confirmation of novel H1N1 flu infection can only be made by reverse-transcription polymerase chain reaction (RT-PCR) or viral culture.

WHO
2 May 2009
Overview
This document provides questions and answers to vaccine use released by WHO.

CIDRAP
1 May 2009

HPA
1 May 2009


Overview

Symptoms of swine influenza are similar to those of seasonal influenza, usually a feverish illness accompanied by one or more or more of cough, sore throat, headache and muscle aches. For most people this illness appears to be mild. Infection with this virus is treatable with the anti-viral drugs oseltamivir (Tamiflu®) and zamanivir (Relenza®).

CIDRAP
1 May 2009

HPA
30 April 2009
Overview
Swine influenza is a respiratory disease normally found in pigs but human cases can and do happen.
The current human cases were first reported in Southern California and Texas. Cases of swine influenza have now been confirmed in several countries including Mexico, US, Canada, Spain and the United Kingdom.
This strain of swine influenza contains a combination of genetic material typical to avian, swine and human flu viruses. Transmission of this new swine influenza virus is thought to occur in the same way as seasonal flu. Antiviral drugs are available which can effectively prevent and treat the infection, most reported cases of this infection outside of Mexico have recovered fully without the need for medical attention or antivirals.
 

HPA
30 April 2009
Overview
Swine influenza is a respiratory disease normally found in pigs but human cases can and do happen.
The current human cases were first reported in Southern California and Texas. Cases of swine influenza have now been confirmed in several countries including Mexico, US, Canada, Spain and the United Kingdom.
This strain of swine influenza contains a combination of genetic material typical to avian, swine and human flu viruses. Transmission of this new swine influenza virus is thought to occur in the same way as seasonal flu. Antiviral drugs are available which can effectively prevent and treat the infection, most reported cases of this infection outside of Mexico have recovered fully without the need for medical attention or antivirals.

HPA
29 April 2009
Overview
Post exposure prophylaxis is indicated for close contacts who were exposed to a probable or confirmed case during the period when the latter was symptomatic AND last exposure occurred no more than 7 days ago. Any probable or confirmed human case of swine influenza A/H1N1 should be notified to the local Health Protection Unit (HPU) as soon as possible.

CDC
29 April 2009
Objective
To provide interim guidance on appropriate specimen collection, storage, processing, and testing for patients with suspected swine-origin influenza A (H1N1) virus infection.

HPA
29 April 2009
Overview
Post exposure prophylaxis is indicated for close contacts who were exposed to a probable or confirmed case during the period when the latter was symptomatic AND last exposure occurred no more than 7 days ago. Any probable or confirmed human case of swine influenza A/H1N1 should be notified to the local Health Protection Unit (HPU) as soon as possible.

CDC
29 April 2009
Objective
To provide interim guidance on appropriate specimen collection, storage, processing, and testing for patients with suspected swine-origin influenza A (H1N1) virus infection.
 

CDC
28 April 2009 (update)
Objective
To provide interim guidance on the use of antiviral agents for treatment and chemoprophylaxis of swine influenza A (H1N1) virus infection. This includes patients with confirmed, probable or suspected swine influenza A (H1N1) virus infection and their close contacts.

CDC
28 April 2009 (update)
Objective
To provide interim guidance on the use of antiviral agents for treatment and chemoprophylaxis of swine influenza A (H1N1) virus infection. This includes patients with confirmed, probable or suspected swine influenza A (H1N1) virus infection and their close contacts.

 

WHO
27 April 2009
Introduction
The audiences for this guidance document are the National Focal Points for the International Health Regulations (IHR(2005)) and competent national public health authorities. The primaryfocus of this guidance document is global surveillance. It also gives some suggestions on the types of signals that Member States and IHR States Parties can capture in their event based
surveillance. These signals can aid identification of individuals for whom investigation of swine influenza A(H1N1) virus infection is warranted.
This is an interim WHO guidance on the global surveillance of the emerging swine influenza A(H1N1) virus infection. This is a living document that will be reviewed on a weekly basis and modified in accordance with changes in the epidemiology of this virus. As the event evolves, there will be a need to switch surveillance activities to the longer term monitoring of the disease. WHO will alert countries when a change in surveillance objectives and methods occurs.
WHO’s data requirements will remain as flexible as possible to accommodate different surveillance systems and reporting capacity around the world.
This document will form part of a suite of guidance documents being produced by WHO in response to this public health emergency of international concern as determined by the Director General of WHO on 25 April 2009. New influenza virus sub‐types and clusters of unknown and unusual disease are notifiable to WHO in accordance with the Annex 2 decision instrument of the IHR (2005).
At this early stage of the outbreak of swine influenza A(H1N1) virus, the main aims of surveillance are the early warning of virus spread and laboratory confirmation of virus circulating in new geographical areas and countries. Accordingly, WHO encourages all Member States and IHR States Parties to enhance their surveillance and diagnostic capacity for influenza and other acute respiratory infections, building on exiting surveillance structure and resources.

WHO
27 April 2009
Introduction
The audiences for this guidance document are the National Focal Points for the International Health Regulations (IHR(2005)) and competent national public health authorities. The primaryfocus of this guidance document is global surveillance. It also gives some suggestions on the types of signals that Member States and IHR States Parties can capture in their event based
surveillance. These signals can aid identification of individuals for whom investigation of swine influenza A(H1N1) virus infection is warranted.
This is an interim WHO guidance on the global surveillance of the emerging swine influenza A(H1N1) virus infection. This is a living document that will be reviewed on a weekly basis and modified in accordance with changes in the epidemiology of this virus. As the event evolves, there will be a need to switch surveillance activities to the longer term monitoring of the disease. WHO will alert countries when a change in surveillance objectives and methods occurs.
WHO’s data requirements will remain as flexible as possible to accommodate different surveillance systems and reporting capacity around the world.
This document will form part of a suite of guidance documents being produced by WHO in response to this public health emergency of international concern as determined by the Director General of WHO on 25 April 2009. New influenza virus sub‐types and clusters of unknown and unusual disease are notifiable to WHO in accordance with the Annex 2 decision instrument of the IHR (2005).
At this early stage of the outbreak of swine influenza A(H1N1) virus, the main aims of surveillance are the early warning of virus spread and laboratory confirmation of virus circulating in new geographical areas and countries. Accordingly, WHO encourages all Member States and IHR States Parties to enhance their surveillance and diagnostic capacity for influenza and other acute respiratory infections, building on exiting surveillance structure and resources.
 

WHO
25 April 2009
Overview
This is a guidance document for influenza laboratories providing information on sample collection and handling and laboratory tests.
 

WHO
25 April 2009
Overview
This is a guidance document for influenza laboratories providing information on sample collection and handling and laboratory tests.

General

Medicalnewstoday
25 January 2010
Overview
Current research suggests that the flu may predispose to secondary bacterial infections, which account for a significant proportion of mortality during flu pandemics. The related report by Lee et al, "A mouse model of lethal synergism between influenza virus and Haemophilus influenzae ," appears in the February 2010 issue of The American Journal of Pathology .
 

PloS Pathogens
22 January 2010
Author Summary
Antimicrobial resistance in bacterial organisms is a worldwide problem widely discussed from clinical, economical and social points of view. The number of new resistance mechanisms and microorganisms resistant to new drugs is increasing all over the world. The development and spread of antibiotic resistance in bacterial communities represents an excellent model for testing the predictive potential of evolutionary principles at short time-scales. A number of studies have tried to predict the selection of resistant variants when a new drug is commercialized. However, in many cases there is no correlation between in vitro predictions and in-field observations. For this reason, it can be suspected that the ability to predict the emergence of new resistant variants might be incomplete unless we know the evolutionary forces acting on the genetic diversification processes. Using the CTX-M β-lactamases as a model, a combination of molecular phylogenetic approaches and experimental site-specific mutagenesis has allowed us to establish evolutionary trajectories. We have demonstrated that two synthetic antibiotics, cefotaxime and ceftazidime, were the selective forces driving the diversification of CTX-M enzymes, but only when both antibiotics were simultaneously present in the environment. We also predict that, if the current selective landscape is not modified, variants carrying the mutation D240G will be more prevalent and diverse in the future.
 

PLoS One
1 January 2010
Abstract
Prion diseases are a family of rare, progressive, neurodegenerative disorders that affect humans and animals. The most common form of human prion disease, Creutzfeldt-Jakob disease (CJD), occurs worldwide. Variant CJD (vCJD), a recently emerged human prion disease, is a zoonotic foodborne disorder that occurs almost exclusively in countries with outbreaks of bovine spongiform encephalopathy.

This study describes the occurrence and epidemiology of CJD and vCJD in the United States.

Analysis of CJD and vCJD deaths using death certificates of US residents for 1979–2006, and those identified through other surveillance mechanisms during 1996–2008. Since CJD is invariably fatal and illness duration is usually less than one year, the CJD incidence is estimated as the death rate. During 1979 through 2006, an estimated 6,917 deaths with CJD as a cause of death were reported in the United States, an annual average of approximately 247 deaths (range 172–304 deaths). The average annual age-adjusted incidence for CJD was 0.97 per 1,000,000 persons. Most (61.8%) of the CJD deaths occurred among persons ≥65 years of age for an average annual incidence of 4.8 per 1,000,000 persons in this population. Most deaths were among whites (94.6%); the age-adjusted incidence for whites was 2.7 times higher than that for blacks (1.04 and 0.40, respectively). Three patients who died since 2004 were reported with vCJD; epidemiologic evidence indicated that their infection was acquired outside of the United States.

Surveillance continues to show an annual CJD incidence rate of about 1 case per 1,000,000 persons and marked differences in CJD rates by age and race in the United States. Ongoing surveillance remains important for monitoring the stability of the CJD incidence rates, and detecting occurrences of vCJD and possibly other novel prion diseases in the United States.
 

PLoS One
23 December 2009
Abstract
With the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive.

We thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA.

This work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.
 

Medicalnewstoday
5 December 2009
Overview
Most scientists believe that staph infections are caused by many bacterial cells that signal each other to emit toxins. The signaling process is called quorum sensing because many bacteria must be present to start the process. But the Jeff Brinker research group has determined that the very first stage of staph infection, when bacteria switch from a harmless to a virulent form, occurs in a single cell and that this individual process can be stopped by the application of a simple protein.
 

PLoS Pathogens
26 October 2009
Abstract
Interspecies transmission of pathogens may result in the emergence of new infectious diseases in humans as well as in domestic and wild animals. Genomics tools such as high-throughput sequencing, mRNA expression profiling, and microarray-based analysis of single nucleotide polymorphisms are providing unprecedented ways to analyze the diversity of the genomes of emerging pathogens as well as the molecular basis of the host response to them. By comparing and contrasting the outcomes of an emerging infection with those of closely related pathogens in different but related host species, we can further delineate the various host pathways determining the outcome of zoonotic transmission and adaptation to the newly invaded species. The ultimate challenge is to link pathogen and host genomics data with biological outcomes of zoonotic transmission and to translate the integrated data into novel intervention strategies that eventually will allow the effective control of newly emerging infectious diseases.
 

PloS Pathogens
26 October 2009
Abstract
Influenza A virus causes annual epidemics and occasional pandemics of short-term respiratory infections associated with considerable morbidity and mortality. The pandemics occur when new human-transmissible viruses that have the major surface protein of influenza A viruses from other host species are introduced into the human population. Between such rare events, the evolution of influenza is shaped by antigenic drift: the accumulation of mutations that result in changes in exposed regions of the viral surface proteins. Antigenic drift makes the virus less susceptible to immediate neutralization by the immune system in individuals who have had a previous influenza infection or vaccination. A biannual reevaluation of the vaccine composition is essential to maintain its effectiveness due to this immune escape. The study of influenza genomes is key to this endeavor, increasing our understanding of antigenic drift and enhancing the accuracy of vaccine strain selection. Recent large-scale genome sequencing and antigenic typing has considerably improved our understanding of influenza evolution: epidemics around the globe are seeded from a reservoir in East-Southeast Asia with year-round prevalence of influenza viruses; antigenically similar strains predominate in epidemics worldwide for several years before being replaced by a new antigenic cluster of strains. Future in-depth studies of the influenza reservoir, along with large-scale data mining of genomic resources and the integration of epidemiological, genomic, and antigenic data, should enhance our understanding of antigenic drift and improve the detection and control of antigenically novel emerging strains.
 

PLoS One
21 October 2009
Abstract
Prion diseases are considered to be transmissible. The existence of sporadic forms of prion diseases such as scrapie implies an environmental source for the infectious agent. This would suggest that under certain conditions the prion protein, the accepted agent of transmission, can survive in the environment. We have developed a novel technique to extract the prion protein from soil matrices. Previous studies have suggested that environmental manganese is a possible risk factor for prion diseases. We have shown that exposure to manganese is a soil matrix causes a dramatic increase in prion protein survival (~10 fold) over a two year period. We have also shown that manganese increases infectivity of mouse passaged scrapie to culture cells by 2 logs. These results clearly verify that manganese is a risk factor for both the survival of the infectious agent in the environment and its transmissibility.
 

PLoS One
20 October 2009
Abstract
There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.
 

J of Royal society
22 September 2009
Abstract
Over the past few years, prompted by pandemic preparedness initiatives, the debate over the modes of transmission of influenza has been rekindled and several reviews have appeared. Arguments supporting an important role for aerosol transmission that were reviewed included prolonged survival of the virus in aerosol suspensions, demonstration of the low infectious dose required for aerosol transmission in human volunteers, and clinical and epidemiological observations were disentanglements of large droplets and aerosol transmission was possible. Since these reviews were published, several new studies have been done and generated new data. These include direct demonstration of the presence of influenza viruses in aerosolized droplets from the tidal breathing of infected persons and in the air of an emergency department; the establishment of the guinea pig model for influenza transmission, where it was shown that aerosol transmission is important and probably modulated by temperature and humidity; the demonstration of some genetic determinants of airborne transmission of influenza viruses as assessed using the ferret model; and mathematical modelling studies that strongly support the aerosol route. These recent results and their implication for infection control of influenza are discussed in this review.
 

Global influenza Programme, WHO
August 2009
Overview
This is an online repository of standardized training material gathered from multiple international partners involved in the delivery of influenza and pandemic preparedness training. This library constitutes of influenza related training material and technical background documents to provide easy access to these materials and ensure that the latest developments in knowledge are used. This material is regularly updated through peer-review to ensure that changes resulting from the experiences gained using the material as well as changes due to the development of knowledge of influenza are incorporated in a more timely and efficient manner.

PLoS Neglected Tropical Diseases
28 July 2009
Author Summary
Vector-borne diseases constitute a largely neglected and enormous burden on public health in many resource-challenged environments, demanding efficient control strategies that could be developed through improved understanding of pathogen transmission. Human movement—which determines exposure to vectors—is a key behavioral component of vector-borne disease epidemiology that is poorly understood. We develop a conceptual framework to organize past studies by the scale of movement and then examine movements at fine-scale—i.e., people going through their regular, daily routine—that determine exposure to insect vectors for their role in the dynamics of pathogen transmission. We develop a model to quantify risk of vector contact across locations people visit, with emphasis on mosquito-borne dengue virus in the Amazonian city of Iquitos, Peru. An example scenario illustrates how movement generates variation in exposure risk across individuals, how transmission rates within sites can be increased, and that risk within sites is not solely determined by vector density, as is commonly assumed. Our analysis illustrates the importance of human movement for pathogen transmission, yet little is known—especially for populations most at risk to vector-borne diseases (e.g., dengue, leishmaniasis, etc.). We outline several important considerations for designing epidemiological studies to encourage investigation of individual human movement, based on experience studying dengue.

PLos One
17 July 2009
Abstract
A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA) moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2β2–2β3 loops of the heptameric precursor to generate a 14-strand transmembrane β barrel.
We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the β barrel, and a third one, F324, that lies part way up the barrel.
Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus.

PloS Pathogens
10 July 2009
Abstract
Coronaviruses are viral pathogens that cause a variety of lethal diseases in birds and mammals, and common colds in humans. In 2003, however, an animal coronavirus was able to infect humans and produced severe acute respiratory syndrome (SARS), causing a near pandemic. Such events are likely to reoccur in the future, and new antiviral strategies are necessary. A small coronavirus protein called ‘envelope’ is important for pathogenesis, affecting the formation of the viral envelope and the distribution of the virus in the body.
In vitro studies have shown that synthetic coronavirus envelope proteins have channel activity that in some cases has been inhibited by the drug hexamethylene amiloride, but not by amiloride. In the present paper, we have characterized the structure responsible for this channel activity. We have also determined the binding site of the drug hexamethylene amiloride in the channel, and shown that amiloride has only a mild effect on the NMR signals from the protein. The validity of these results is supported using mammalian cells expressing full length SARS-CoV E, where channel activity was inhibited by hexamethylene amiloride, but only mildly by amiloride. The structural model described for this channel provides a valuable insight into coronavirus envelope protein ion channel activity, and could serve as a platform for the development of novel anti-viral drugs.
 

PloS Pathogens
26 June 2009
Overview
West Nile virus (WNV) is a small, enveloped, mosquito-transmitted, positive-stranded RNA virus of the Flaviviridae family. This virus is related to other arthropod-borne viruses that cause human disease globally, including dengue, yellow fever, and Japanese encephalitis viruses. WNV cycles in nature primarily between Culex mosquitoes and birds, but also infects human, horses, and other vertebrates. Over the latter half of the 20th century, outbreaks of WNV infection have been reported in Europe, Asia, and Australia. In 1999, WNV was introduced into the Western Hemisphere in New York City.

This article gives information on the virus, pathogenesis and the genetic basis of human infection.

CDC, EID
June 2009
Abstract
Influenza A subtype H7 viruses have resulted in >100 cases of human infection since 2002 in the Netherlands, Italy, Canada, the United States, and the United Kingdom. Clinical illness from subtype H7 infection ranges from conjunctivitis to mild upper respiratory illness to pneumonia. Although subtype H7 infections have resulted in a smaller proportion of hospitalizations and deaths in humans than those caused by subtype H5N1, some subtype H7 strains appear more adapted for human infection on the basis of their virus-binding properties and illness rates among exposed persons. Moreover, increased isolation of subtype H7 influenza viruses from poultry and the ability of this subtype to cause severe human disease underscore the need for continued surveillance and characterization of these viruses. We review the history of human infection caused by subtype H7. In addition, we discuss recently identified molecular correlates of subtype H7 virus pathogenesis and assess current measures to prevent future subtype H7 virus infection.

ECDC
17 June 2009
Overview
ECDC has created a new series of publications: a yearly compilation of executive summaries from its most important documents. The new series is targeted at policymakers and will be translated into all official EU languages, plus Icelandic and Norwegian. The first edition, Summary of key publications 2008, is a compilation of summaries from nine selected ECDC documents published in 2008. Highlights include short versions of the Framework action plan to fight tuberculosis in the European Union, the Annual epidemiological report on communicable diseases in Europe 2008 and the HIV/AIDS surveillance in Europe – 2007.

PLoS Pathogens
29 May 2009
Author Summary
Injection of pure anthrax lethal toxin (LT) at the levels found in terminally-ill, anthrax-infected animals induces an atypical vascular collapse in several experimental animals without the associated hemorrhagic manifestations and disseminated intravascular coagulopathies seen in bacteria-induced shock states. Nevertheless, LT alone produces the pleural edema that is a classic manifestation of anthrax. All the histopathological changes previously observed in LT-treated mice can be explained as predictable sequelae of vascular insufficiency. In this report, electron microscopic analysis was used to identify rapid and striking pathological changes in endothelial cells, myocytes, mitochondria, and the sarcoplasmic reticulum cisternae in the hearts of LT-challenged mice. These morphological changes were paralleled by release of very high levels of multiple cardiac injury biomarkers. LT-induced pathological changes occurred earlier and with markedly higher severity in nNOS knockout mice compared to wild-type or iNOS or eNOS knockout mice, reflecting the well-established role of nNOS in maintenance of cardiac function, and suggesting that nitric oxide produced by nNOS counteracts the damage induced by LT.
 

Medicalnewstoday
25 May 2009
Overview
It appears that some superbugs have evolved to develop the ability to manipulate the immune system to everyone's advantage.
A team of researchers at The University of Western Ontario, led by Joaquin (Quim) Madrenas of the Robarts Research Institute, has discovered some processes that reduce the lethal effects of toxins from superbugs, allowing humans and microbes to co-evolve. This discovery may lead to novel alternatives to antibiotics that specifically target the toxic effects of these superbugs. The findings are being published in the journal Nature Medicine and are available online today.

PLoSOne
22 May 2009
Abstract
RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.
 

CDC, EID
June 2009 (Ahead of print)
Abstract
A high rate of oseltamivir resistance in seasonal influenza virus A (H1N1) infection was reported in Europe in the winter of 2007–08 (1). Of subtype H1N1 isolates tested in 18 European countries, 59 (13.5%) of 437 were resistant to oseltamivir and carried the substitution of histidine by tyrosine at residue 274 (H274Y) of the neuraminidase (NA) gene. Genetic analysis showed that all oseltamivir-resistant strains of subtype H1N1 virus remained susceptible to amantadine.
In Hong Kong Special Administrative Region, People’s Republic of China, amantadine and oseltamivir are not commonly used to treat patients with influenza. Surveillance of amantadine and oseltamivir resistance among influenza viruses was begun in 2006 after a high rate of amantadine resistance was reported among subtype H3N2 viruses (2) and a stockpile of oseltamivir was purchased for pandemic preparedness. Oseltamivir resistance was first detected in January 2008. We report detection of subtype H1N1 virus isolates that became resistant to amantadine and oseltamivir because of genetic reassortment and spontaneous mutation.

Medicalnewstoday
24 April 2009
Overview
Doctors have no specific drugs to treat dengue fever, a viral illness spread by mosquitoes that sickens 50 million to 100 million people worldwide each year. Instead, the only treatments they can recommend for this painful and sometimes fatal illness (20,000 deaths globally each year) are fluids, rest and non-aspirin pain and fever reducers. Now, researchers have identified cellular components in mosquitoes and in humans that dengue virus uses to multiply inside these hosts after infecting them. Their findings could lead to the development of anti-dengue drugs that would inhibit one or more of these host factors, thus curtailing infection and the development of disease. The National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, funded the research, which was led by Mariano Garcia-Blanco, M.D., Ph.D., of Duke University Medical Center. The research appears in the current issue of the journal Nature.
 

PLosPathogens
3 April 2009
Author Summary
Bacillus anthracis, the etiological agent of anthrax, can infect mammals either accidentally or as a potential consequence of a terrorism threat. Pulmonary infection is a life-threatening form of the disease, causing a near 100% mortality rate in the absence of appropriate therapy. Thus, it is important to understand the mechanisms of host defense against B. anthracis. We examined the effects of various B. anthracis strains on lung inflammation in a mouse model of pulmonary anthrax and on human lung epithelial cells, the first barrier of lung against invading pathogens. We showed that a B. anthracis strain expressing lethal toxin inhibits inflammation. In contrast, a strain in which this toxin has been inactivated induces lung inflammation. We next examined the mechanisms involved in the inhibitory effect of lethal toxin. We showed that B. anthracis injects lethal toxin into epithelial cells, blocks the molecules associated on the chromosome, and thus represses production of mediators involved in inflammation. As the latter is a key process in host defense, its alteration by lethal toxin predisposes the host to infection by B. anthracis. This effect on the chromosomal machinery may represent an efficient strategy used by B. anthracis for invading the host.

PLoSOne
1 April 2009
Abstract
Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion.
 

PLoS Neglected Tropical Diseases
31 March 2009
Introduction
The Disease Vector Database is a global, free, public, Web-accessible resource presenting data on the geographical distribution of infectious disease vectors and reservoirs. At present, the Database contains records for dengue and malaria vectors and Chagas disease and leishmaniasis vectors and reservoirs. Future versions of the Database will include parasite data. These data can be used for a variety of purposes including the construction of ecological niche models and disease risk maps. The Database permits downloading data in formats designed to facilitate such use, for instance, as input files for popular niche modeling software packages such as Maxent and GARP.

PLoSOne
16 March 2009
Abstract
Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene (bont/B) occurs in strains of C. botulinum type B, Ab, and A(B), and whether plasmid carriage is bont/B subtype-related.
PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont/B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont/B subtype genes. All five known bont/B subtype genes were detected among the strains; the most frequently detected subtype genes were bont/B1 and /B2. Surprisingly, the bont/B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont/B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont/B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont/B1 subtype genes were located on plasmids, whereas all but one of the bont/B2 subtypes were chromosomally-located). Three bivalent isolates carried both bont/A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from ~55 kb to ~245 kb.
The unexpected finding of the widespread distribution of plasmids harboring the bont/B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety.

PLosOne
13 March 2009
Abstract
Disease introduction into the New World during colonial expansion is well documented and had a major impact on indigenous populations; however, few diseases have been associated with early human migrations into North America. During the late Pleistocene epoch, Asia and North America were joined by the Beringian Steppe ecosystem which allowed animals and humans to freely cross what would become a water barrier in the Holocene. Anthrax has clearly been shown to be dispersed by human commerce and trade in animal products contaminated with Bacillus anthracis spores. Humans appear to have brought B. anthracis to this area from Asia and then moved it further south as an ice-free corridor opened in central Canada ~13,000 ybp. In this study, we have defined the evolutionary history of Western North American (WNA) anthrax using 2,850 single nucleotide polymorphisms (SNPs) and 285 geographically diverse B. anthracis isolates. Phylogeography of the major WNA B. anthracis clone reveals ancestral populations in northern Canada with progressively derived populations to the south; the most recent ancestor of this clonal lineage is in Eurasia. Our phylogeographic patterns are consistent with B. anthracis arriving with humans via the Bering Land Bridge. This northern-origin hypothesis is highly consistent with our phylogeographic patterns and rates of SNP accumulation observed in current day B. anthracis isolates.
Continent-wide dispersal of WNA B. anthracis likely required movement by later European colonizers, but the continent's first inhabitants may have seeded the initial North American populations.
 

Polio

Medicalnewstoday
24 April 2009
Overview
Emergency measures have been launched by the Government of Southern Sudan to stop a polio outbreak spreading across the Horn of Africa. Previously restricted to Southern Sudan and western Ethiopia, the outbreak has this year spread to Kenya, Uganda and northern Sudan.
To urgently address the spread of disease - and recognizing that the epicentre of the outbreak is Southern Sudan - the President of the Government of Southern Sudan, His Excellency General Salva Kiir Mayardit, has launched a 'President Action Plan for Polio Eradication in Southern Sudan', forming an Inter-Ministerial Coordination Committee to specifically address the crisis. The President of the Government of Southern Sudan's office has urged all state governors to give full and active support to the outbreak response activities.
 

Surveillance

PLoS One
31 August 2009
Abstract
The case fatality ratio (CFR), the ratio of deaths from an infectious disease to the number of cases, provides an assessment of virulence. Calculation of the ratio of the cumulative number of deaths to cases during the course of an epidemic tends to result in a biased CFR. The present study develops a simple method to obtain an unbiased estimate of confirmed CFR (cCFR), using only the confirmed cases as the denominator, at an early stage of epidemic, even when there have been only a few deaths.

Our method adjusts the biased cCFR by a factor of underestimation which is informed by the time from symptom onset to death. We first examine the approach by analyzing an outbreak of severe acute respiratory syndrome in Hong Kong (2003) with known unbiased cCFR estimate, and then investigate published epidemiological datasets of novel swine-origin influenza A (H1N1) virus infection in the USA and Canada (2009). Because observation of a few deaths alone does not permit estimating the distribution of the time from onset to death, the uncertainty is addressed by means of sensitivity analysis. The maximum likelihood estimate of the unbiased cCFR for influenza may lie in the range of 0.16–4.48% within the assumed parameter space for a factor of underestimation. The estimates for influenza suggest that the virulence is comparable to the early estimate in Mexico. Even when there have been no deaths, our model permits estimating a conservative upper bound of the cCFR.

Although one has to keep in mind that the cCFR for an entire population is vulnerable to its variations among sub-populations and underdiagnosis, our method is useful for assessing virulence at the early stage of an epidemic and for informing policy makers and the public.
 

CDC,EID
April 2009
Abstract
BioSense is a US national system that uses data from health information systems for automated disease surveillance. We studied 4 time-series algorithm modifications designed to improve sensitivity for detecting artificially added data. To test these modified algorithms, we used reports of daily syndrome visits from 308 Department of Defense (DoD) facilities and 340 hospital emergency departments (EDs). At a constant alert rate of 1%, sensitivity was improved for both datasets by using a minimum standard deviation (SD) of 1.0, a 14–28 day baseline duration for calculating mean and SD, and an adjustment for total clinic visits as a surrogate denominator. Stratifying baseline days into weekdays versus weekends to account for day-of-week effects increased sensitivity for the DoD data but not for the ED data. These enhanced methods may increase sensitivity without increasing the alert rate and may improve the ability to detect outbreaks by using automated surveillance system data.