Flexibility In Vitro of Amino Acid 226 in the Receptor-Binding Site of an H9 Subtype Influenza A Virus and Its Effect In Vivo on Virus Replication, Tropism, and Transmission.

Influenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 2015-2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from avian-like to human-like terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred an in vivo replication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variants in vivo and in vitroIMPORTANCE A single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.

Isolation of Ontario aquatic bird bornavirus 1 and characterization of its replication in immortalized avian cell lines.

BACKGROUND: Aquatic bird bornavirus 1 (ABBV-1) has been associated with neurological diseases in wild waterfowls. In Canada, presence of ABBV-1 was demonstrated by RT-qPCR and immunohistochemistry in tissues of waterfowls with history of neurological disease and inflammation of the central and peripheral nervous tissue, although causation has not been proven by pathogenesis experiments, yet. To date, in vitro characterization of ABBV-1 is limited to isolation in primary duck embryo fibroblasts. The objectives of this study were to describe isolation of ABBV-1 in primary duck embryonic fibroblasts (DEF), and characterize replication in DEF and three immortalized avian fibroblast cell lines (duck CCL-141, quail QT-35, chicken DF-1) in order to evaluate cellular permissivity and identify suitable cell lines for routine virus propagation. METHODS: The virus was sequenced, and phylogenetic analysis performed on a segment of the N gene coding region. Virus spread in cell cultures, viral RNA and protein production, and titres were evaluated at different passages using immunofluorescence, RT-qPCR, western blotting, and tissue culture dose 50% (TCID50) assay, respectively. RESULTS: The isolated ABBV-1 showed 97 and 99% identity to European ABBV-1 isolate AF-168 and North American ABBV-1 isolates 062-CQ and CG-N1489, and could infect and replicate in DEF, CCL-141, QT-35 and DF-1 cultures. Viral RNA was detected in all four cultures with highest levels observed in DEF and CCL-141, moderate in QT-35, and lowest in DF-1. N protein was detected in western blots from infected DEF, CCL-141 and QT-35 at moderate to high levels, but minimally in infected DF-1. Infectious titre was highest in DEF (between approximately 105 to 106 FFU / 106 cells). Regarding immortalized cell lines, CCL-141 showed the highest titre between approximately 104 to 105 FFU / 106 cells. DF-1 produced minimal infectious titre. CONCLUSIONS: This study confirms the presence of ABBV-1 among waterfowl in Canada and reported additional in vitro characterization of this virus in different avian cell lines. ABBV-1 replicated to highest titre in DEF, followed by CCL-141 and QT-35, and poorly in DF-1. Our results showed that CCL-141 can be used instead of DEF for routine ABBV-1 production, if a lower titre is an acceptable trade-off for the simplicity of using immortalized cell line over primary culture.

Whole genome characterisation of quail deltacoronavirus detected in Poland.

Quail deltacoronavirus (QdCoV) described for the first time in the United Arab Emirates in 2018 belongs to the same deltacoronavirus species as viruses discovered in swine and tree sparrows. The full-length genome of QdCoV detected in quails with enteritis in Poland has similar organization as Middle Eastern viruses although there is no NSP7c gene. The overall degree of nucleotide sequence identity was 92.4-92.6% between Polish PL/G032/2015 and Middle Eastern UAE-HKU30 QdCoV isolates. The sequences of the individual genes show similar nucleotide identities in the range of 91.4-94.7% with the exception of the S gene with lower identity of 85.6-85.7%. The most variable part of the S gene is its fragment encoding the N-terminal domain of the S protein which is responsible for receptor binding. The amino acid homology in this region between PL/G032/2015 and UAE-HKU30 QdCoVs was 74.5-74.7%. In contrast, the C-terminal domain of the S protein which is responsible for membrane fusion had an amino acid homology of 96.9%. In the phylogenetic tree, PL/G032/2015 branched separately but clustered with the UAE-HKU30 QdCoV isolates. These data suggest that PL/G032/2015 could be a new genetic/serologic variant of QdCoV.

Experimental model of oral transmissible AA amyloidosis in quails.

In poultry and zoo birds, mass outbreaks of amyloid A (AA) amyloidosis are often reported, and horizontal transmission is considered as one of the causes. However, oral transmission of avian AA amyloidosis in nature has been unclear. In order to clarify the horizontal transmission of avian AA amyloidosis, basic research using an appropriate oral transmission model is necessary. In this study, we developed an oral transmission model of AA amyloidosis using quails, and assessed the oral transmission efficiency of AA amyloidosis in quails and mice. Young quails, adult quails, and young mice received inflammatory stimulation with lipopolysaccharide; simultaneously, homogeneous amyloid fibrils were orally or intravenously administered. By histological examination, induction of amyloidosis by oral or intravenous administration of amyloid was confirmed in all species. Furthermore, both quail and murine AA amyloidosis were orally transmitted in a dose-dependent manner. These results support the possibility of horizontal transmission of avian AA amyloidosis in nature. This model will be able to contribute to the elucidation of spontaneous horizontal transmission of avian AA amyloidosis in the future. RESEARCH HIGHLIGHTS Quail AA amyloidosis was orally transmitted in a dose-dependent manner. Oral transmission was less efficient than intravenous transmission. In-cage horizontal transmission did not occur during 4-week cohabitation. Amyloid deposition in tissues of quail was grossly visible.

Pathology of clade 2.3.2.1 avian influenza virus (H5N1) infection in quails and ducks in Bangladesh.

We performed pathological and molecular virological investigation of three outbreaks of highly pathogenic avian influenza (HPAI) in a quail farm and two duck farms of Mymensingh and Netrokona districts of Bangladesh in 2011. HPAI viruses of subtype H5N1 were detected from all three outbreaks and phylogenetic analysis of HA gene sequence placed the viruses into clade 2.3.2.1. The outbreak in the quail farm was characterized by acute death with 100% mortality within two days. Marked haemorrhages and congestion with necrotic and inflammatory lesions in the respiratory tract, liver, pancreas and kidneys were the major gross and histopathological lesions. In the case of ducks, nervous signs were the remarkable clinical manifestations and the mortality was around 10%. No significant gross lesions were observed at necropsy. Non-purulent encephalitis with gliosis and neuronal degeneration was observed on histopathological examination. By immunohistochemistry, viral antigen could be detected in different organs of both quails and ducks. This study records varying clinical and pathological manifestations of HPAI in ducks and quails following natural infection with the same strain of the virus. RESEARCH HIGHLIGHTS HPAIV of clade 2.3.2.1 was detected from clinical outbreaks in quails and ducks Sudden death with severe haemorrhages in various organs was found in quails Pronounced nervous signs with non-purulent encephalitis were observed in ducks Viral antigen could be localized in different organs by immunohistochemistry.

Characterization of H7N9 influenza A viruses isolated from humans.

Avian influenza A viruses rarely infect humans; however, when human infection and subsequent human-to-human transmission occurs, worldwide outbreaks (pandemics) can result. The recent sporadic infections of humans in China with a previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern owing to the appreciable case fatality rate associated with these infections (more than 25%), potential instances of human-to-human transmission, and the lack of pre-existing immunity among humans to viruses of this subtype. Here we characterize two early human A(H7N9) isolates, A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9); hereafter referred to as Anhui/1 and Shanghai/1, respectively. In mice, Anhui/1 and Shanghai/1 were more pathogenic than a control avian H7N9 virus (A/duck/Gunma/466/2011 (H7N9); Dk/GM466) and a representative pandemic 2009 H1N1 virus (A/California/4/2009 (H1N1pdm09); CA04). Anhui/1, Shanghai/1 and Dk/GM466 replicated well in the nasal turbinates of ferrets. In nonhuman primates, Anhui/1 and Dk/GM466 replicated efficiently in the upper and lower respiratory tracts, whereas the replicative ability of conventional human influenza viruses is typically restricted to the upper respiratory tract of infected primates. By contrast, Anhui/1 did not replicate well in miniature pigs after intranasal inoculation. Critically, Anhui/1 transmitted through respiratory droplets in one of three pairs of ferrets. Glycan arrays showed that Anhui/1, Shanghai/1 and A/Hangzhou/1/2013 (H7N9) (a third human A(H7N9) virus tested in this assay) bind to human virus-type receptors, a property that may be critical for virus transmissibility in ferrets. Anhui/1 was found to be less sensitive in mice to neuraminidase inhibitors than a pandemic H1N1 2009 virus, although both viruses were equally susceptible to an experimental antiviral polymerase inhibitor. The robust replicative ability in mice, ferrets and nonhuman primates and the limited transmissibility in ferrets of Anhui/1 suggest that A(H7N9) viruses have pandemic potential.

Biological characterization of wild-bird-origin avian avulavirus 1 and efficacy of currently applied vaccines against potential infection in commercial poultry.

Newcastle disease virus (NDV), the type member of the species Avian avulavirus 1 (formerly known as avian paramyxovirus serotype 1), causes a highly contagious and economically important disease in a myriad of avian species around the globe. While extensive vaccination programs have been implemented in ND-endemic countries, the disease is continuously spreading in commercial, backyard, and wild captive poultry. In order to investigate the evolution of the virus and assess the efficiency of the vaccine regimens that are currently being applied in commercial poultry, four wild-bird-origin NDV strains were characterized biologically, based on mean death time and intracerebral pathogenicity index, and genetically, based on the cleavage motif (112RRQKRF117) in the fusion (F) protein. Based on these features, all of the isolates were characterized as velogenic strains of NDV. Phylogenetic analysis based on the complete genome sequence revealed clustering of these isolates within class II, genotype VII. This class of NDV remains the predominant genotype in the Egyptian poultry industry, as well as in those of many Asian and African countries. To investigate the potential of these wild-bird-origin NDV isolates to cause infection in domesticated poultry and to assess the efficacy of currently available vaccines for protection of commercial poultry, an extensive animal challenge experiment was performed. Cumulative clinicopathological and immunological investigations of virus-challenged chickens indicate that these isolates can potentially be transmitted between chicken and cause systemic infections, and the currently applied vaccines are unable to prevent clinical disease and virus shedding. Taken together, the data represent a comprehensive evaluation of the ability of Egyptian wild-bird-origin NDV strains to cause infection in commercial poultry and highlights the need for a continuous and large-scale surveillance as well as revised vaccine approaches. These integrated and multifaceted strategies would be crucial in any efforts to control and eradicate the disease globally.

Molecular characterization of avipoxviruses circulating in Mozambique, 2016-2018.

Samples from 45 chickens, two turkeys, one peacock and one quail with symptoms of fowlpox were collected in Mozambique between November 2016 and January 2018. Phylogenetic analysis revealed that the samples contained avipoxviruses belonging to both clade A1 and clade A2. In addition, all of the Clade A1 viruses were positive by PCR for the integration of reticuloendotheliosis virus, while the clade A2 avipoxvirus samples were negative. This study confirms the circulation of clade A1 avipoxviruses in Mozambique in addition to identifying clade A2 for the first time in the country.

Prevalence of avian influenza (H9N2) in commercial quail, partridge, and turkey farms in Iran, 2014-2015.

Avian influenza virus (AIV) H9N2 subtype is endemic in Iran and causes substantial economic loss to the growing poultry industry within the country. In this study, a cross-sectional analysis was carried out to determine the sero-prevalence of H9N2 in several commercial farms between the years 2014 and 2015. The comparison of the mean of serum titers and the ratio of sero-positive birds between all units were analyzed using one-way ANOVA test. In 2014, a total of 77 farms (58 turkey farms, 14 quail farms, and 5 partridge farms) and 894 birds (682 turkeys, 154 quails, and 58 partridges) were sampled while in 2015, a total of 69 farms (54 turkey farms, 8 quail farms, and 7 partridge farms) and 856 birds (675 turkeys, 105 quails, and 76 partridges) were sampled. Of that, 52 of 77 sampled farms (67.5%) and 437 of 894 samples (48.9%) were positive for H9N2 in 2014 while. Forty-one of 69 farms (59.4%) and 307 of 856 sera (35.9%) were positive in 2015. Furthermore, the mean titer of partridge farms was significantly lower than that of turkey farms (p < 0.01) and the mean percentage of sero-positive turkey farms was significantly higher than partridge farms (p < 0.01) in 2014. In 2015, no significant difference was observed between the mean sera titer amongst farms and percentage of sero-positive birds (p > 0.05). Our results indicated that H9N2 is circulating in these farms. Since many more such farms are being established for operations, in addition to the threat of emergence and continuous reemergence of the disease in these farms, enhanced veterinary biosecurity measures on farms are required for mitigation.

Susceptibility of chickens, quail, and pigeons to an H7N9 human influenza virus and subsequent egg-passaged strains.

H7N9 human influenza virus A/Anhui/1/2013 (Anhui2013) showed low pathogenicity in chickens, quail, and pigeons, with quail being the most susceptible among the species tested. IVPIE1-1, which was recovered from a dead chicken after intravenous inoculation of Anhui 2013, had broader tissue tropism in chickens than did the original inoculum, as well as amino acid substitutions in the polymerase acidic gene and neuraminidase gene segments, but its pathogenicity was not enhanced. Viruses obtained after passage of Anhui 2013 in 10- and 14-day-old embryonated eggs showed rapid accumulation of amino acid substitutions at the receptor-binding site of the hemagglutinin protein. Two strains obtained through egg passage, 10E4/14E17 and 10E4/10E13, replicated better in intranasally infected chickens than did the original Anhui 2013 strain, yet the new isolates showed low pathogenicity in chickens despite their amino acid substitutions. The increased virus replication in chickens of 10E4/14E17 and 10E4/10E13 was not correlated with temperature-sensitive replication, given that virus replication was suppressed at increased temperatures. The existence of highly susceptible hosts, such as quail, which permit asymptomatic infection, facilitates increased mutation of the virus through amino acid substitution at the receptor-binding site, and this might be one of the mechanisms underlying the prolonged circulation of H7N9 influenza virus.

Pathogenicity of the Korean H5N8 highly pathogenic avian influenza virus in commercial domestic poultry species.

In 2014, the highly pathogenic avian influenza (HPAI) virus H5N8 triggered outbreaks in wild birds and poultry farms in South Korea. In the present study, we investigated the pathogenicity of the H5N8 HPAI virus, belonging to the clade 2.3.4.4, in different species of poultry. For this, we examined clinical signs and viral shedding levels following intranasal inoculation of the virus in 3-week-old commercial layer chickens and quails, 10-week-old Korean native chickens, and 8-week-old Muscovy ducks. Intranasal inoculation with 10(6.0) viruses at 50% egg-infective dose resulted in 100% mortality in the layer chickens (8/8) and quails (4/4), but 60% and 0% deaths in the Korean native chickens (3/5) and Muscovy ducks (0/4), respectively. In addition, transmission of the inoculated virus to contact-exposed birds was evident in all the species used in this study. Based on our results, we conclude that the H5N8 HPAI virus has lower pathogenicity and transmissibility in poultry species compared with previously reported H5N1 HPAI viruses.

Replication and transmission of mammalian-adapted H9 subtype influenza virus in pigs and quail.

Influenza A virus is a major pathogen of birds, swine and humans. Strains can jump between species in a process often requiring mutations and reassortment, resulting in outbreaks and, potentially, pandemics. H9N2 avian influenza is predominant in poultry across Asia and occasionally infects humans and swine. Pandemic H1N1 (H1N1pdm) is endemic in humans and swine and has a history of reassortment in pigs. Previous studies have shown the compatibility of H9N2 and H1N1pdm for reassortment in ferrets, a model for human infection and transmission. Here, the effects of ferret adaptation of H9 surface gene segments on the infectivity and transmission in at-risk natural hosts, specifically swine and quail, were analysed. Reassortant H9N1 and H9N2 viruses, carrying seven or six gene segments from H1N1pdm, showed infectivity and transmissibility in swine, unlike the wholly avian H9N2 virus with ferret-adapted surface genes. In quail, only the reassortant H9N2 with the six internal gene segments from the H1N1pdm strain was able to infect and transmit, although less efficiently than the wholly avian H9N2 virus with ferret-adapted surface genes. These results highlight that ferret-adapted mutations on the haemagglutinin of H9 subtype virus do not restrict the ability of the virus to infect swine and quail, and that the ability to transmit in these species depends on the context of the whole virus. As such, this study emphasizes the threat that H9N2 reassortant viruses pose to humans and agricultural species and the importance of the genetic constellation of the virus to its ability to replicate and transmit in natural hosts of influenza.

Genetic characterization of influenza A virus subtype H7N1 isolated from quail, Thailand.

In Thailand, surveillance for the highly pathogenic avian influenza virus H5N1 (HPAI-H5N1) has revealed high prevalence of the virus in quail in live-bird markets. This study monitored avian influenza viruses (AIVs) in quail farms in an area at high risk for HPAI-H5N1 over a 12-month period from 2009 to 2010. One-step real-time RT-PCR (rRT-PCR) results showed that 1.18 % of swab samples (24/2,040) were AIV positive. Among the rRT-PCR positive samples, three samples were identified as subtype H7N1. One Thai H7N1 virus designated "A/quail/Thailand/CU-J2882/2009 (H7N1)" was subjected to whole genome sequencing and genetic characterization. Phylogenetic analysis showed that the HA gene of the Thai H7N1 virus groups with those of the H7 Eurasian viruses. Interestingly, the NA gene of the virus was found to be closely related to those of the HPAI-H5N1 viruses from Vietnam and Thailand. This study constitutes the first report on AIV H7N1 in Thailand. Our results suggest the possibility of genetic reassortment between AIV-H7NX and HPAI-H5N1 in quail. The HA cleavage site of the Thai H7N1 virus contains no multiple amino acid insertions, suggesting low pathogenic characteristics for this virus.

Characterization of the amantadine-resistant H5N1 highly pathogenic avian influenza variants isolated from quails in Southern China.

Highly pathogenic H5N1 avian influenza viruses have spread in poultry and wild birds in Asia, Europe, and Africa since 2003. To evaluate the role of quails in the evolution of influenza A virus, we characterized three H5N1 viruses isolated from quails (QA viruses) in southern China. Phylogenetic analysis indicated that three QA viruses derived from the A/goose/Guangdong/1/96-like lineage and most closely related to HA clade 4 A/chicken/Hong Kong/31.4/02-like viruses. Molecular analysis suggested that QA viruses and clade 4 H5N1 viruses carried consistent residue signatures, such as the characteristic M2 Ser31Asn amantadine-resistance mutation, implying a common origin of these viruses. As revealed by viral pathogenicity tests, these QA viruses could replicate in intranasally infected mice, but were not lethal to them, showing low pathogenicity in mammals. However, they killed all intravenously inoculated chickens, showing high pathogenicity in poultry. Results from amantadine sensitivity tests of wild-type QA viruses and their reverse genetic viruses demonstrated that all QA viruses were resistant to amantadine, and the M2 Ser31Asn mutation was determined as the most likely cause of the increased amantadine-resistance of H5N1 QA viruses. Our study confirmed experimentally that the amino acid at residue 31 in the M2 protein plays a major role in determining the amantadine-resistance phenotype of H5N1 influenza viruses. Our findings provide further evidence that quails may play important roles in the evolution of influenza A viruses, which raises concerns over possible transmissions of H5N1 viruses among poultry, wild birds, and humans.

Genetic and antigenic characteristics of H4 subtype avian influenza viruses in Korea and their pathogenicity in quails, domestic ducks and mice.

In Korea, a nationwide surveillance programme was implemented in 2003 to identify highly pathogenic avian influenza viruses (AIVs). AIVs belonging to one of the most common haemagglutinin subtypes, H4, were isolated from two domestic ducks and 52 wild birds between 2004 and 2010. These H4 AIVs could be further classified into three neuraminidase subtypes: H4N6 (94.4%), H4N2 (3.7%) and H4N3 (1.9%). Phylogenetic analysis revealed that the H4 AIVs had a variety of genetic constellations, with at least nine different genotypes represented. The pathogenicity of these H4 viruses was assessed in quails, domestic ducks and mice. None of the H4 AIVs induced clinical signs in quails or domestic ducks. Viral shedding in quails was relatively high, and virus was recovered up to 5-7 days post-inoculation (p.i.) in oropharyngeal swabs, but the viruses replicated poorly in domestic ducks. Quails may act as an intermediate host in which AIVs are amplified and transmitted to other species. In mice, all of the AIVs were recovered efficiently at relatively high titres from the lungs up to 7 days p.i., demonstrating the potential for AIVs to infect mice directly without prior adaptation. None of the AIVs induced clinical signs nor was any lethal to infected mice. However, there was significant loss of body weight in mice infected with viruses of duck origin. It is suggested that the active surveillance of influenza viruses needs to be enhanced in domestic poultry as well as in wild birds, and that it should include assessment of pathogenicity in animal models.

Adaptation of a duck influenza A virus in quail.

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)--and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.

Quantification of heterosubtypic immunity between avian influenza subtypes H3N8 and H4N6 in multiple avian host species.

Low-pathogenicity avian influenza virus (LPAIV) can lead to epizootics that cause economic losses in poultry or the emergence of human-infectious strains. LPAIVs experience a complex immunity landscape as they are endemic in numerous host species, and many antigenically distinct strains co-circulate. Prevention and control of emergence of detrimental strains requires an understanding of infection/transmission characteristics of the various subtypes in different hosts, including interactions between subtypes. In order to develop analytical frameworks for examining control efficacy, quantification of heterosubtypic immunity interactions is fundamental. However, these data are scarce, especially for wild avian subtypes in natural hosts. Consequently, in this study, three host species (mallards, quail and pheasants) were infected with two LPAIV subtypes isolated from wild birds: H3N8 and H4N6. The recovered hosts were also reinfected with the alternate subtype to measure the effects of heterosubtypic immunity. Oropharyngeal and cloacal swabs were collected and viral RNA load was quantified by real-time RT-PCR. For secondary infections in recovered hosts, peak viral load was up to four orders of magnitude lower and shedding length was up to 4 days shorter. However, both the magnitude and presence of heterosubtypic immunity varied across specific host species/subtype combinations. Using a mathematical model of virus replication, the variation in virus replication dynamics due to host individuals was quantified. It was found that accounting for individual heterogeneity is important for drawing accurate conclusions about treatment effects. These results are relevant for developing epidemiological models to inform control practices and for analysing virus replication data.

Isolation of H9N2 avian influenza virus from bobwhite quail (Colinus virginianus) in Egypt.

This study describes the first isolation of H9N2 avian influenza virus (AIV) from commercial bobwhite quail (Colinus virginianus) in Egypt. Infected birds showed neither clinical signs nor mortality. Virus isolation and real-time reverse transcription polymerase chain reaction confirmed the presence of the H9N2 virus in cloacal swab samples collected at 35 days of age and the absence of other AIV subtypes, including H5 and H7. The hemagglutinin and neuraminidase genes of the isolated virus showed 99.1% and 98.2% nucleotide identity and 97.3% and 100% amino acid identity, respectively, to those of H9N2 viruses currently circulating in poultry in the Middle East. Phylogenetically, the Egyptian H9N2 virus was closely related to viruses of the G1-like lineage isolated from neighbouring countries, indicating possible epidemiological links.

Extensive mammalian ancestry of pandemic (H1N1) 2009 virus.

We demonstrate that the novel pandemic influenza (H1N1) viruses have human virus-like receptor specificity and can no longer replicate in aquatic waterfowl, their historic natural reservoir. The biological properties of these viruses are consistent with those of their phylogenetic progenitors, indicating longstanding adaptation to mammals.

Increased pathogenicity and shedding in chickens of a wild bird-origin low pathogenicity avian influenza virus of the H7N3 subtype following multiple in vivo passages in quail and turkey.

In order to investigate viral adaptation mechanisms to poultry, we performed serial in vivo passages of a wild bird low pathogenicity avian influenza isolate of the H7N3 subtype (A/mallard/Italy/33/01) in three different domestic species (chicken, turkey, and Japanese quail). The virus under study was administered via natural routes at the dose of 10(6) egg infective dose50/ 0.1 ml to chickens, turkeys, and quails in order to investigate the clinical susceptibility and the shedding levels after infection. Multiple in vivo passages of the virus were performed by serially infecting groups of five naive birds of each species, with samples collected from a previously infected group. Quails and turkeys were susceptible to infection for 10 serial passages, whereas chickens were susceptible to two cycles of infection only. Infection of chicken with the quail- and turkey-adapted viruses showed an increased pathogenicity and/or shedding, causing more severe clinical signs and/or higher levels of viral excretion compared to the original strain. The data obtained herein suggest that infection of selected avian species may facilitate the adaptation of avian influenza viruses originating from the wild bird reservoir to chicken. This is the first time turkey has been shown to act as a species in which a virus from the wild reservoir can increase its replication activity in other domestic species.