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1.  Novel Avian-Origin Influenza A (H7N9) Virus Attachment to the Respiratory Tract of Five Animal Models 
Journal of Virology  2014;88(8):4595-4599.
We determined the pattern of attachment of the avian-origin H7N9 influenza viruses A/Anhui/1/2013 and A/Shanghai/1/2013 to the respiratory tract in ferrets, macaques, mice, pigs, and guinea pigs and compared it to that in humans. The H7N9 attachment pattern in macaques, mice, and to a lesser extent pigs and guinea pigs resembled that in humans more closely than the attachment pattern in ferrets. This information contributes to our knowledge of the different animal models for influenza.
doi:10.1128/JVI.03190-13
PMCID: PMC3993775  PMID: 24478425
2.  Novel H7N9 Influenza Virus Shows Low Infectious Dose, High Growth Rate, and Efficient Contact Transmission in the Guinea Pig Model 
Journal of Virology  2014;88(3):1502-1512.
The zoonotic outbreak of H7N9 subtype avian influenza virus that occurred in eastern China in the spring of 2013 resulted in 135 confirmed human cases, 44 of which were lethal. Sequencing of the viral genome revealed a number of molecular signatures associated with virulence or transmission in mammals. We report here that, in the guinea pig model, a human isolate of novel H7N9 influenza virus, A/Anhui/1/2013 (An/13), is highly dissimilar to an H7N1 avian isolate and instead behaves similarly to a human seasonal strain in several respects. An/13 was found to have a low 50% infectious dose, grow to high titers in the upper respiratory tract, and transmit efficiently among cocaged guinea pigs. The pH of fusion of the hemagglutinin (HA) and the binding of virus to fixed guinea pig tissues were also examined. The An/13 HA displayed a relatively elevated pH of fusion characteristic of many avian strains, and An/13 resembled avian viruses in terms of attachment to tissues. One important difference was seen between An/13 and both the H3N2 human and the H7N1 avian viruses: when inoculated intranasally at a high dose, only the An/13 virus led to productive infection of the lower respiratory tract of guinea pigs. In sum, An/13 was found to retain fusion and attachment properties of an avian influenza virus but displayed robust growth and contact transmission in the guinea pig model atypical of avian strains and indicative of mammalian adaptation.
doi:10.1128/JVI.02959-13
PMCID: PMC3911619  PMID: 24227867
3.  Evidence for Novel Hepaciviruses in Rodents 
PLoS Pathogens  2013;9(6):e1003438.
Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.
Author Summary
The hepatitis C virus (HCV) is one of the most relevant causes of liver disease and cancer in humans. The lack of a small animal models represents an important hurdle on our way to understanding, treating, and preventing hepatitis C. The investigation of small mammals could identify virus infections similar to hepatitis C in animals that can be kept in laboratories, such as rodents, and can also yield insights into the evolution of those ancestral virus lineages out of which HCV developed. Here, we investigated a worldwide sample of 4,770 rodents, 2,939 bats, 210 horses and 858 cats and dogs for HCV-related viruses. New viruses were discovered in European bank voles (Myodes glareolus) and South African four-striped mice (Rhabdomys pumilio). The disease in bank voles was studied in more detail, suggesting that infection of the liver occurs with similar symptoms to those caused by HCV in humans. These rodents might thus enable the development of new laboratory models of hepatitis C. Moreover, the phylogenetic history of those viruses provides fascinating new ideas regarding the evolution of HCV ancestors.
doi:10.1371/journal.ppat.1003438
PMCID: PMC3688547  PMID: 23818848
4.  European H16N3 Gull Influenza Virus Attaches to the Human Respiratory Tract and Eye 
PLoS ONE  2013;8(4):e60757.
We explored the attachment of an H16N3 influenza virus to human, mallard, and gull tissues using virus histochemistry applied to tissue microarrays and employing human and mallard viruses as references. Of the viruses tested, the H16N3 gull virus most readily attached to the human respiratory tract and eye. These results underscore the need to assess the potential for gull influenza viruses to replicate in human tissues and further investigate the role of gulls in influenza virus ecology.
doi:10.1371/journal.pone.0060757
PMCID: PMC3620227  PMID: 23593303
5.  The Multibasic Cleavage Site in H5N1 Virus Is Critical for Systemic Spread along the Olfactory and Hematogenous Routes in Ferrets 
Journal of Virology  2012;86(7):3975-3984.
The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space—including along the olfactory route—and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.
doi:10.1128/JVI.06828-11
PMCID: PMC3302532  PMID: 22278228
6.  Influenza A and B Virus Attachment to Respiratory Tract in Marine Mammals 
Emerging Infectious Diseases  2012;18(5):817-820.
Patterns of virus attachment to the respiratory tract of 4 marine mammal species were determined for avian and human influenza viruses. Attachment of avian influenza A viruses (H4N5) and (H7N7) and human influenza B viruses to trachea and bronchi of harbor seals is consistent with reported influenza outbreaks in this species.
doi:10.3201/eid1805.111828
PMCID: PMC3358047  PMID: 22516350
influenza; viruses; virus attachment; virus receptors; influenza A virus; influenza B virus; human influenza virus; avian influenza virus; marine mammals; respiratory tract
7.  Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species 
Veterinary Research  2012;43(1):28.
This study assessed the presence of sialic acid α-2,3 and α-2,6 linked glycan receptors in seven avian species. The respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, golden pheasant, ostrich, and mallard were tested by means of lectin histochemistry, using the lectins Maackia amurensis agglutinin II and Sambucus nigra agglutinin, which show affinity for α-2,3 and α-2,6 receptors, respectively. Additionally, the pattern of virus attachment (PVA) was evaluated with virus histochemistry, using an avian-origin H4N5 virus and a human-origin seasonal H1N1 virus. There was a great variation of receptor distribution among the tissues and avian species studied. Both α-2,3 and α-2,6 receptors were present in the respiratory and intestinal tracts of the chicken, common quail, red-legged partridge, turkey, and golden pheasant. In ostriches, the expression of the receptor was basically restricted to α-2,3 in both the respiratory and intestinal tracts and in mallards the α-2,6 receptors were absent from the intestinal tract. The results obtained with the lectin histochemistry were, in general, in agreement with the PVA. The differential expression and distribution of α-2,3 and α-2,6 receptors among various avian species might reflect a potentially decisive factor in the emergence of new viral strains.
doi:10.1186/1297-9716-43-28
PMCID: PMC3368784  PMID: 22489675
8.  Assessment of the Antiviral Properties of Recombinant Porcine SP-D against Various Influenza A Viruses In Vitro 
PLoS ONE  2011;6(9):e25005.
The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.
doi:10.1371/journal.pone.0025005
PMCID: PMC3173486  PMID: 21935489
9.  The Pattern of Influenza Virus Attachment Varies among Wild Bird Species 
PLoS ONE  2011;6(9):e24155.
The ability to attach to host cells is one of the main determinants of the host range of influenza A viruses. By using virus histochemistry, we investigate the pattern of virus attachment of both a human and an avian influenza virus in colon and trachea sections from 12 wild bird species. We show that significant variations exist, even between closely related avian species, which suggests that the ability of wild birds to serve as hosts for influenza viruses strongly varies among species. These results will prove valuable to assess the possibilities of interspecies transmission of influenza viruses in natural environments and better understand the ecology of influenza.
doi:10.1371/journal.pone.0024155
PMCID: PMC3164702  PMID: 21909418
10.  Highly Pathogenic Avian Influenza Virus H5N1 Infects Alveolar Macrophages without Virus Production or Excessive TNF-Alpha Induction 
PLoS Pathogens  2011;7(6):e1002099.
Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine. In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.
Author Summary
Alveolar macrophages (AM), which reside in the alveolar lumen, usually dampen down the host immune response to incoming pathogens. However, they are thought to increase inflammation during highly pathogenic avian influenza virus (HPAIV) H5N1 infections, which cause severe and often fatal disease in humans. This is based on experiments with human macrophages cultured from monocytes rather than with human AM. Here we show that human AM, collected via broncho-alveolar lavage from healthy volunteers, can become infected with HPAIV H5N1. However, this results in neither induction of the pro-inflammatory cytokine TNF-alpha nor virus production. Therefore, AM are most likely not responsible for the excessive cytokine response or high viral load during human HPAIV H5N1 infections as assumed previously. These data significantly changes our insight into the pathogenesis of HPAIV H5N1 pneumonia in humans, indicating that other cells than AM must be responsible for the excessive pro-inflammatory cytokine profile observed during HPAIV H5N1 infections.
doi:10.1371/journal.ppat.1002099
PMCID: PMC3121882  PMID: 21731493
11.  Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding▿ ‡  
Journal of Virology  2010;84(22):11802-11813.
The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.
doi:10.1128/JVI.01136-10
PMCID: PMC2977876  PMID: 20844044
12.  In Vitro Assessment of Attachment Pattern and Replication Efficiency of H5N1 Influenza A Viruses with Altered Receptor Specificity▿  
Journal of Virology  2010;84(13):6825-6833.
The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.
doi:10.1128/JVI.02737-09
PMCID: PMC2903244  PMID: 20392847
13.  Molecular Determinants of Adaptation of Highly Pathogenic Avian Influenza H7N7 Viruses to Efficient Replication in the Human Host ▿  
Journal of Virology  2009;84(3):1597-1606.
Two viruses isolated during the highly pathogenic avian influenza (HPAI) H7N7 virus outbreak in The Netherlands in 2003, one isolated from a person with conjunctivitis and one from a person who died as the result of infection, were used for an in vitro study of influenza A virus pathogenicity factors. The two HPAI H7N7 viruses differed in 15 amino acid positions in five gene segments. Assays were designed to investigate the role of each of these substitutions in attachment and entry, transcription and genome replication, and virus production and release as determined by hemagglutinin (HA), polymerase proteins, nonstructural protein 1 (NS1), and neuraminidase (NA). These in vitro studies confirmed the roles of the E627K substitution in basic polymerase 2 (PB2) and the A143T substitution in HA in pathogenicity observed in a mouse model previously. However, the in vitro studies identified a contribution of acidic polymerase (PA) and NA to the efficient replication in human cells of the fatal case virus, despite the fact that these are rarely marked as determinants of pathogenicity in in vivo studies. With the exception of PB2 E627K, all substitutions contributing to enhanced replication of the fatal case virus in vitro were present in poultry viruses prior to transmission to the human fatal case, indicating that viruses with enhanced replication efficiency in the mammalian host can be generated in poultry. Thus, detailed in vitro analyses of mutations facilitating replication of avian influenza viruses in mammalian cells are important to assess the zoonotic risk posed by these viruses and, in addition, highlight the value of in vitro studies to complement animal models.
doi:10.1128/JVI.01783-09
PMCID: PMC2812334  PMID: 19939933
14.  Wild Ducks as Long-Distance Vectors of Highly Pathogenic Avian Influenza Virus (H5N1) 
Emerging Infectious Diseases  2008;14(4):600-607.
Some duck species are potential long-distance vectors; others are more likely to function as sentinels.
Wild birds have been implicated in the expansion of highly pathogenic avian influenza virus (H5N1) outbreaks across Asia, the Middle East, Europe, and Africa (in addition to traditional transmission by infected poultry, contaminated equipment, and people). Such a role would require wild birds to excrete virus in the absence of debilitating disease. By experimentally infecting wild ducks, we found that tufted ducks, Eurasian pochards, and mallards excreted significantly more virus than common teals, Eurasian wigeons, and gadwalls; yet only tufted ducks and, to a lesser degree, pochards became ill or died. These findings suggest that some wild duck species, particularly mallards, can potentially be long-distance vectors of highly pathogenic avian influenza virus (H5N1) and that others, particularly tufted ducks, are more likely to act as sentinels.
doi:10.3201/eid1404.071016
PMCID: PMC2570914  PMID: 18394278
disease vectors; ducks; epidemiology; immunohistochemistry; influenza A virus; H5N1 subtype; influenza in birds; pathology; reverse transcriptase polymerase chain reaction; virology; research
15.  Antiviral Treatment with Alpha Interferon Up-Regulates CD14 on Liver Macrophages and Its Soluble Form in Patients with Chronic Hepatitis B 
To investigate whether therapy with alpha interferon (IFN-α) induces changes in intrahepatic antigen-presenting cells (APCs), we obtained liver biopsy specimens before, during, and after therapy with IFN-α from chronic hepatitis B patients whose viral load had already been reduced by at least 8 weeks of treatment with lamivudine. HLA-DR, CD1a, and CD83 were not modified by the therapy. The intralobular expression of CD68 on Kupffer cells remained stable, denoting no changes in the number of resident macrophages during IFN-α treatment. In contrast, CD14 was weakly expressed in the absence of IFN-α and was significantly up-regulated during therapy. At the same time, the levels of soluble CD14 and interleukin-10 in plasma increased significantly. In vitro, monocytes maintained in the presence of IFN-α differentiated into macrophages or dendritic cells with higher levels of expression of CD14 than that for the control cultures. During therapy with IFN-α, T-cell infiltration in the portal spaces was reduced, mainly due to a significant decrease in the number of CD8+ T cells. These findings show that IFN-α is biologically active on APCs in vivo and in vitro and suggest that this newly described regulatory function, together with the already known inhibitory effects on lymphocytes, may cooperate to reduce inflammation and consequent tissue damage in patients with chronic viral hepatitis.
doi:10.1128/AAC.49.2.590-599.2005
PMCID: PMC547278  PMID: 15673738

Results 1-15 (15)