H5N1 Avian Virus Research Articles

Immunomodulatory Effect of Purified Exotoxins of <i>Staphylococcus aureus</i> in Association with Bird Flu Virus Vaccine in Broilers

Immunization is the most effective method still used against infectious agents. Although not always, vaccines ineffectiveness is reported enormously against many of the pathogens throughout the world in poultry, particularly in case of killed or sub unit vaccine. The current project is, therefore, carried out as a preliminary study on broiler chickens to investigate the modulation of immune system against avian influenza virus in association with purified Staphylococcus aureus toxoid. After isolation of Gram positive cocci bacteria on mannitol salt agar from raw milk, yogurt and chicken meat were subsequently biochemically characterized by using rapid diagnostic kit. Pure culture of S. aureus was inoculated into digitally controlled bio-fermentor containing mannitol salt broth for production of toxins. Enormous production of bacteria was passed through sequence of filtration system based on 0.45 μm followed by 0.22 μm size. The centrifugation of the filtrate was made at 10,000 rpm for 60 minutes at 5℃ followed by 56,100 rpm for 20 minutes and clear supernatant containing Staphylococcus enterotoxin (SEs) was obtained. Bradford estimation of proteins further provided 305 μg/ml of SEs toxoid. Four types of oil adjuvant avian influenza type H9N2 virus vaccines (without toxoid, 91.5 μg/0.3ml, 22.8 μg/0.3ml and 11.43 μg/0.3ml) were injected into healthy AI H9N2 susceptible broilers and anti-H9N2 HI antibody titers were measured in terms of hemagglutination inhibition test. It was observed that on the 8th day, post vaccination cumulative mean anti AIH9 HI antibody titer of G-1, G-2, G-3, G-4 and G-5 was 3.13 ± 0.406, 5.13 ± 0.246, 3.96 ± 0.159, 3.25 ± 0.237 and 0.78 ± 0.467 respectively. It was found that all the vaccines induced protective titers 18 days’ post vaccination, but vaccine containing 91.5 μg/dose of SEs toxoid showed significantly higher (P , particularly in inactivated vaccines, could be the better choice for modulation of immediate and better immune response in future vaccines technologies.

Interaction between avian influenza subtype H9N2 and Newcastle disease virus vaccine strain (LaSota) in chickens

BackgroundH9N2 avian influenza virus is endemic in Egyptian poultry flocks. The role of the live viral vaccines such as LaSota in exaggeration of the clinical picture of H9N2 infection under field conditions is significantly important leading to severe economic losses due to higher mortality and lower growth performance. This experiment was designed to identify the possible interaction between experimental infection with H9N2 virus and NDV live vaccine (LaSota strain) in broiler chickens. Six groups each of 20 broiler chicks were used. Three groups (G1–3) were infected with H9N2 and vaccinated with LaSota, 3 days before, at the same day or 3 days post vaccination (dpv), while the remaining groups (G4–6) were non-vaccinated infected, vaccinated non-infected and non-vaccinated non-infected.ResultsThe highest mortality rate (37.5%) was noticed in chickens of G1 (H9N2 infected 3 days prior LaSota vaccination). Also, this bird group had the most severe clinical signs, histopathological lesions and the longest viral shedding for 9 days post infection (dpi). In the 2nd and 3rd groups, the mortality rate was the similar (31.2%) with less pronounced clinical signs, histopathological lesions and H9N2 shedding was for only 6 dpi with the least shedding quantity in chickens of G3. The control non-vaccinated infected chickens (G4) had 18.7% mortality with the least degree of clinical signs, lesions and the highest viral shedding quantity but only for 6 dpi. At 35 days of age, there was a statistical significant decrease (P < 0.05) in chicken’s body weight of all H9N2 infected groups from G1 to G4 compared to non-infected control groups, G5 and G6 respectively.ConclusionIt was clear that laSota vaccination significantly affect H9N2 infection in broiler chickens regarding clinical signs, mortality rate, lesions, performance and viral shedding.

Severe human infection with a novel avian-origin influenza A(H7N4) virus

Human infections with influenza H7 subtypes, such as H7N9, have raised concerns worldwide. Here, we report a human infection with a novel influenza A(H7N4) virus. A 68 years-old woman with cardiovascular and cholecystic comorbidities developed rapidly progressed pneumonia with influenza-like-illness as initial symptom, recovered after 23 days-hospitalization including 8 days in ICU. Laboratory indicators for liver and blood coagulation dysfunction were observed. Oseltamivir phosphate, glucocorticoids and antibiotics were jointly implemented, with nasal catheterization of oxygen inhalation for this patient. We obtained the medical records and collected serial respiratory and blood specimens from her. We collected throat, cloacal and/or feces samples of poultry and wild birds from the patient’s backyard, neighborhood, local live poultry markets (LPMs) and the nearest lake. All close contacts of the patient were followed up and sampled with throat swabs and sera. Influenza viruses and other respiratory pathogens were tested by real-time RT-PCR, viral culturing and/or sequencing for human respiratory and bird samples. Micro-neutralizing assay was performed for sera. A novel reassortant wild bird-origin H7N4 virus is identified from the patient and her backyard poultry (chickens and ducks) by sequencing, which is distinct from previously-reported avian H7N4 and H7N9 viruses. At least four folds increase of neutralizing antibodies to H7N4 was detected in her convalescent sera. No samples from close contacts, wild birds or other poultry were tested positive for H7N4 by real-time RT-PCR.

Avian influenza A H7N9 virus infects human astrocytes and neuronal cells and induces inflammatory immune responses

Seasonal, pandemic, and avian influenza virus infections may be associated with central nervous system pathology, albeit with varying frequency and different mechanisms. Here, we demonstrate that differentiated human astrocytic (T98G) and neuronal (SH-SY5Y) cells can be infected by avian H7N9 and pandemic H1N1 viruses. However, infectious progeny viruses can only be detected in H7N9 virus infected human neuronal cells. Neither of these viral strains can generate infectious progeny virus in human astrocytes despite replication of viral genome was observed. Furthermore, H7N9 virus triggered high pro-inflammatory cytokine expression, while pandemic H1N1 virus induced only low cytokine expression in either brain cell type. The experimental finding here is the first data to demonstrate that avian H7N9 virus can infect, transcribe, and replicate its viral genome; induce cytokine upregulation; and cause cytopathic effects in human brain cells, which may potentially lead to profound central nervous system injury. Observation for neurological problems due to H7N9 virus infection deserves further attention when managing these patients.

PA-X: a key regulator of influenza A virus pathogenicity and host immune responses.

PA-X, a fusion protein belonging to influenza A viruses (IAVs), integrating the N-terminal 191 amino acids of PA gene and the ribosomal frame-shifting product that lengthens out to 41 or 61 amino acids. Since its discovery in 2012, multiple functions have been attributed to this small protein, including a process, where wide-spread protein synthesis in infected host cells is shut down (called host shutoff), and viral replication, polymerase activity, viral-induced cell apoptosis, PA nuclear localization, and virulence are modulated. However, many of its proposed functions may be specific to strain, subtype, host, or cell line. In this review, we start by describing the well-defined global host-shutoff ability of PA-X and the potential mechanisms underlying it. We move on to the role played by PA-X in modulating innate and acquired immune responses in the host. We then systematically discuss the role played by PA-X in modulating the virulence of influenza viruses of different subtypes and host origins, and finish with a general overview of the research advances made in identifying the host cell partners that interact with PA-X. To uncover possible clues about the differential effects of PA-X in modulating viral virulence, we focus on systemically evaluating polymorphisms in PA-X from various viral subtypes and hosts, including avian and human H5N1, H5N6, H9N2, and H7N9 viruses. Finally, we conclude with a proposition regarding the possible future research directions for this important protein.

A Multiplex Asymmetric Reverse Transcription-PCR Assay Combined With an Electrochemical DNA Sensor for Simultaneously Detecting and Subtyping Influenza A Viruses.

The reliable and rapid detection of viral pathogens that cause respiratory infections provide physicians several advantages in treating patients and managing outbreaks. The Luminex respiratory virus panel (RVP) assay has been shown to be comparable to or superior to culture/direct fluorescent-antibody assays (DFAs) and nucleic acid tests that are used to diagnose respiratory viral infections. We developed a multiplex asymmetric reverse transcription (RT)-PCR assay that can simultaneously differentiate all influenza A virus epidemic subtypes. The amplified products were hybridized with an electrochemical DNA sensor, and the results were automatically acquired. The limits of detection (LoDs) of both the Luminex RVP assay and the multiplex RT-PCR-electrochemical DNA sensor were 101 TCID50 for H1N1 virus and 102 TCID50 for H3N2 virus. The specificity assessment of the multiplex RT-PCR-electrochemical DNA sensor showed no cross-reactivity among different influenza A subtypes or with other non-influenza respiratory viruses. In total, 3098 respiratory tract specimens collected from padiatric patients diagnosed with pneumonia were tested. More than half (43, 53.75%) of the specimens positive for influenza A viruses could not be further subtyped using the Luminex RVP assay. Among the remaining 15 specimens that were not subtyped, not degraded, and in sufficient amounts for the multiplex RT-PCR-electrochemical DNA sensor test, all (100%) were H3N2 positive. Therefore, the sensitivity of the Luminex RVP assay for influenza A virus was 46.25%, whereas the sensitivity of the multiplex RT-PCR-electrochemical DNA sensor for the clinical H1N1 and H3N2 specimens was 100%. The sensitivities of the multiplex RT-PCR-electrochemical DNA sensor for the avian H5N1, H5N6, H9N2, and H10N8 viruses were 100%, whereas that for H7N9 virus was 85.19%. We conclude that the multiplex RT-PCR-electrochemical DNA sensor is a reliable method for the rapid and accurate detection of highly variable influenza A viruses in respiratory infections with greater detection sensitivity than that of the Luminex xTAG assay. The high mutation rate of influenza A viruses, particularly H3N2 during the 2014 to 2016 epidemic seasons, has a strong impact on diagnosis. A study involving more positive specimens from all influenza A virus epidemic subtypes is required to fully assess the performance of the assay.

Evaluation of the potential defensive strategy against Influenza A in cell line models.

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 and H7N9 viruses have a potential pandemic threat for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialylation. Tissue specific expression of the sialidases could protect domestic birds from infection.

Evaluation of the potential defensive strategy against Influenza A in cell line models

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 and H7N9 viruses have a potential pandemic threat for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialylation. Tissue specific expression of the sialidases could protect domestic birds from infection.

Development and evaluation of a real-time RT-PCR assay for detection of a novel avian influenza A (H5N6) virus

As of Aug 25, 2017, 17 incidences of human infection and 6 deaths due to the novel H5N6 virus have been reported in China. Genetic analysis of the viral genome revealed that this reassortant virus is highly pathogenic to poultry, and that the virus has a risk of transmission to humans. Accordingly, the development of a rapid, sensitive, and specific molecular diagnostic assay is critical for public health. In this study, a real-time reverse-transcription PCR (RT-PCR) assay was developed to specifically detect the novel H5N6 virus, with primer pairs targeting the hemagglutinin and neuraminidase gene sequences of this virus. RNA was extracted from throat swab specimens from patients with influenza-like illness (ILIs), and environmental samples were collected from live poultry markets (LPMs) for H5N6 virus detection by real-time RT-PCR. The method was demonstrated to enable specific detection of the avian H5N6 virus, with no cross-reactivity with seasonal influenza viruses (H1N1, H1N1 pdm09, H3N2 or B); H5N1, H7N9, H9N2 viruses; or other human respiratory viruses. The detection limit of the assay was 1.0 × 101 copies per reaction for N6 and 1.0 × 102 copies per reaction for H5 assays. The assay is a powerful tool for rapid, sensitive, and specific detection of H5N6 virus infection in specimens derived from humans, animals, and the environment.

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses.IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk.

Influenza Infection in Humans Induces Broadly Cross-Reactive and Protective Neuraminidase-Reactive Antibodies

Antibodies to the hemagglutinin (HA) and neuraminidase (NA) glycoproteins are the major mediators ofprotection against influenza virus infection. Here, we report that current influenza vaccines poorly display key NA epitopes and rarely induce NA-reactive B cells. Conversely, influenza virus infection induces NA-reactive B cells at a frequency that approaches (H1N1) or exceeds (H3N2) that of HA-reactive B cells. NA-reactive antibodies display broad binding activity spanning the entire history of influenza A virus circulation in humans, including the original pandemic strains of both H1N1 and H3N2 subtypes. The antibodies robustly inhibit the enzymatic activity of NA, including oseltamivir-resistant variants, and provide robust prophylactic protection, including against avian H5N1 viruses, invivo. When used therapeutically, NA-reactive antibodies protected mice from lethal influenza virus challenge even 48hr post infection. These findings strongly suggest that influenza vaccines should be optimized to improve targeting of NA for durable and broad protection against divergent influenza strains.

Signal Immune Reactions of Macrophages Differentiated from THP-1 Monocytes to Infection with Pandemic H1N1PDM09 Virus and H5N2 and H9N2 Avian Influenza A Virus.

In culture of THP-1 cells differentiated into macrophages with PMA (THP-PMA macrophages) infected with influenza viruses of subtypes H1, H5 and H9, we measured the expression of TLR7 and RIG1 receptor genes, sensors of viral RNA and ribonucleoprotein, and the levels of production of inflammatory cytokines IL-1β, TNFα, IL-10, and IFNα. The sensitivity and inflammatory response of THP-PMA macrophages to pandemic influenza A virus H1N1pdm09 and avian influenza H5N2 and H9N2 viruses correlate with the intracellular level of their viral RNA and activation of the RIG1 gene. Abortive infection is accompanied by intensive macrophage secretion of TNFα, IL-1β, and toxic factors inducing cell death. Activity of endosomal TLR7 receptor gene changed insignificantly in 24 h after infection and significantly decreased in 48 and 72 h under the action of H5N2 and H9N2, which correlated with manifestation of the cytopathogenic effect of these viruses. H5N2 and H9N2 avian viruses in THP-PMA macrophages are strong activators of the expression of the gene of the cytoplasmic RIG1 receptor 24 and 48 h after infection, and the pandemic virus H1N1pdm09 is a weak stimulator of RIG1 gene. Avian influenza H5N2 and H9N2 viruses are released by rapid induction of the inflammatory response in macrophages. At the late stages of infection, we observed a minor increase in IL-10 secretion in macrophages and, probably, the polarization of a part of the population in type M2. The studied influenza A viruses are weak inductors of IFN in THP-PMA macrophages. In the culture medium of THP-PMA macrophages infected with H9N2 and H5N2 viruses, MTT test revealed high levels of toxic factors causing the death of Caco-2 cells. In contrast to avian viruses, pandemic virus H1N1pdm09 did not induce production of toxic factors.

Evaluation of defense strategy against Influenza A in cell line models

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 virus is getting increasingly infective for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild aquatic birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS. Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialilation to prevent infection. Tissue specific expression of the sialidases could protect domestic birds from infection.

Genetic and biological characterization of three poultry-origin H5N6 avian influenza viruses with all internal genes from genotype S H9N2 viruses.

During surveillance for avian influenza viruses, three H5N6 viruses were isolated in chickens obtained from live bird markets in eastern China, between January 2015 and April 2016. Sequence analysis revealed a high genomic homology between these poultry isolates and recent human H5N6 variants whose internal genes were derived from genotype S H9N2 avian influenza viruses. Glycan binding assays revealed that all avian H5N6 viruses were capable of binding to both human-type SAα-2,6Gal receptors and avian-type SAα-2,3Gal receptors. Their biological characteristics were further studied in BALB/c mice, specific-pathogen-free chickens, and mallard ducks. All three isolates had low pathogenicity in mice but were highly pathogenic to chickens, as evidenced by 100% mortality 36-120 hours post infection at a low dose of 103.0EID50 and through effective contact transmission. Moreover, all three poultry H5N6 isolates caused asymptomatic infections in ducks, which may serve as a reservoir host for their maintenance and dissemination; these migrating waterfowl could cause a potential global pandemic. Our study suggests that continuous epidemiological surveillance in poultry should be implemented for the early prevention of future influenza outbreaks.

Pathogenicity and transmissibility of three avian influenza A (H5N6) viruses isolated from wild birds

Since 2013, highly pathogenic H5N6 avian influenza viruses (AIVs) have emerged in poultry and caused sporadic human infections in Asia. The recent discovery of three new avian H5N6 viruses – A/oriental magpie-robin/Guangdong/SW8/2014 (H5N6), A/common moorhen/Guangdong/GZ174/2014 (H5N6) and A/Pallas's sandgrouse/Guangdong/ZH283/2015 (H5N6) – isolated from apparently healthy wild birds in Southern China in 2014–2015 raises great concern for the spread of these highly pathogenic AIVs (HPAIVs) and their potential threat to human and animal health. In our study, we conducted animal experiments and tested their pathogenicity in ducks, chickens and mice. Ducks can carry and shed the H5N6 HPAIVs, but show no ill effects. On the other hand, these H5N6 HPAIVs can efficiently infect, transmit and cause death in chickens. Due to the overlap of habitats, domestic ducks play an important role in circulating AIVs between poultry and wild birds. Our results raise the possibility that wild birds disseminate these H5N6 HPAIVs to poultry along their flyways and thus pose a great threat to the poultry industry. These viruses are also highly pathogenic to mice, suggesting they pose a potential threat to mammals and, thus, public health. One virus isolated in 2015 replicates much more efficiently and is more lethal in these animals than the two other viruses isolated in 2014. It seems that the H5N6 viruses tend to be more lethal as time passes. Therefore, it is necessary to vigilantly monitor H5N6 HPAIVs in wild birds and poultry.

Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice.

The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro, but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology.

Evidence of infection with avian, human, and swine influenza viruses in pigs in Cairo, Egypt.

The majority of the Egyptian swine population was culled in the aftermath of the 2009 H1N1 pandemic, but small-scale growing remains. We sampled pigs from piggeries and an abattoir in Cairo. We found virological evidence of infection with avian H9N2 and H5N1 viruses as well as human pandemic H1N1 influenza virus. Serological evidence suggested previous exposure to avian H5N1 and H9N2, human pandemic H1N1, and swine avian-like and human-like viruses. This raises concern about potential reassortment of influenza viruses in pigs and highlights the need for better control and prevention of influenza virus infection in pigs.

Increased virulence of a PB2/HA mutant of an avian H9N2 influenza strain after three passages in porcine differentiated airway epithelial cells

We analyzed the adaptation of influenza viruses to growth in differentiated airway epithelial cells of a new host by passaging an avian H9N2 virus three times in porcine precision-cut lung slices (PCLS). Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins, and two in the HA protein. In this study, we characterized the PB2 mutation G685R by generating recombinant H9N2 viruses containing the PB2 single mutation alone or in combination with one of the HA mutations (A190V or T212I). When analyzed in porcine cells − a tracheal cell line (NPTr) or PCLS − the PB2-685 mutant did not provide a growth advantage and had no effect on the ciliary activity which is a virulence marker of swine influenza viruses. Pathogenicity for mice was also not increased by the single PB2 mutation. However, both double mutants (HA-190+PB2-685 and HA-212+PB2-685) showed significantly increased virulence in mice. Therefore, the mutations in the HA and PB2 proteins may confer early adaptation of an avian H9N2 virus to a mammalian host. In conclusion, we expect that a broader ensemble of mutations will be required to render an H9N2 virus virulent for pigs.

Influenza Virus Hemagglutinin Stalk-Specific Antibodies in Human Serum are a Surrogate Marker for In Vivo Protection in a Serum Transfer Mouse Challenge Model.

ABSTRACTThe immunogenicity of current influenza virus vaccines is assessed by measuring an increase of influenza virus-specific antibodies in a hemagglutination inhibition assay. This method exclusively measures antibodies against the hemagglutinin head domain. While this domain is immunodominant, it has been shown that hemagglutination inhibition titers do not always accurately predict protection from disease. In addition, several novel influenza virus vaccines that are currently under development do not target the hemagglutinin head domain, but rather more conserved sites, including the hemagglutinin stalk. Importantly, antibodies against the hemagglutinin stalk do not show activity in hemagglutination inhibition assays and will require different methods for quantification. In this study, we tested human serum samples from a seasonal influenza virus vaccination trial and an avian H5N1 virus vaccination trial for antibody activities in multiple types of assays, including binding assays and also functional assays. We then performed serum transfer experiments in mice which then received an H1N1 virus challenge to assess the in vivo protective effects of the antibodies. We found that hemagglutinin-specific antibody levels measured in an enzyme-linked immunosorbent assay (ELISA) correlated well with protection from weight loss in mice. In addition, we found that weight loss was also inversely correlated with the level of serum antibody-dependent cellular cytotoxicity (ADCC) as measured in a reporter assay. These findings indicate that protection is in part conferred by Fc-dependent mechanisms. In conclusion, ELISAs can be used to measure hemagglutinin-specific antibody levels that could serve as a surrogate marker of protection for universal influenza virus vaccines.

Heterosubtypic cross-reactivity of HA1 antibodies to influenza A, with emphasis on nonhuman subtypes (H5N1, H7N7, H9N2).

Epidemics of influenza A vary greatly in size and age distribution of cases, and this variation is attributed to varying levels of pre-existing immunity. Recent studies have shown that antibody-mediated immune responses are more cross-reactive than previously believed, and shape patterns of humoral immunity to influenza A viruses over long periods. Here we quantify antibody responses to the hemagglutinin subunit 1 (HA1) across a range of subtypes using protein microarray analysis of cross-sectional serological surveys carried out in the Netherlands before and after the A/2009 (H1N1) pandemic. We find significant associations of responses, both within and between subtypes. Interestingly, substantial overall reactivity is observed to HA1 of avian H7N7 and H9N2 viruses. Seroprevalence of H7N7 correlates with antibody titers to A/1968 (H3N2), and is highest in persons born between 1954 and 1969. Seroprevalence of H9N2 is high across all ages, and correlates strongly with A/1957 (H2N2). This correlation is most pronounced in A/2009 (H1N1) infected persons born after 1968 who have never encountered A/1957 (H2N2)-like viruses. We conclude that heterosubtypic antibody cross-reactivity, both between human subtypes and between human and nonhuman subtypes, is common in the human population.