Geese not susceptible to virulent subgroup J avian leukosis virus isolated from chickens.

To determine whether geese are susceptible to infection by avian leukosis virus (ALV), 702 serum samples from domestic and foreign goose breeds were screened for p27 antigen as well as being inoculated into DF-1 cell cultures to isolate ALV. Although 5.7% of samples were positive for p27 antigen, reactivity appeared to be non-specific because no ALV was detected in the corresponding DF-1 cultures. To further determine whether geese are susceptible to ALV-J isolated from chickens, ALV-J strain JS09GY7 was artificially inoculated into 10-day-old goose embryos, with one-day-old hatched goslings then screened for p27 antigen and the presence of ALV. In all cases, the results of both tests were negative. Liver tissues from the 1-day-old goslings were screened using a polymerase chain reaction-based assay, which failed to amplify ALV-J gene fragments from any of the samples. Further, no histopathological damage was observed in the liver tissues. ALV-J was further inoculated intraperitoneally into one-day-old goslings, with cloacal swabs samples and plasma samples then collected every 5 days for 30 days. All samples were again negative for the presence of p27 antigen and ALV, and liver tissues from the challenged geese showed no histopathological damage and were negative for the presence of ALV-J gene fragments. Furthermore, p27 antigen detection, PCR-based screening, and indirect immunofluorescence assays were all negative following the infection of goose embryo fibroblasts with ALV-J. Together, these results confirm that virulent chicken-derived ALV-J strains cannot infect geese, and that p27 antigen detection in goose serum is susceptible to non-specific interference.

Development of a duplex SYBR Green I-based quantitative real-time PCR assay for the rapid differentiation of goose and Muscovy duck parvoviruses.

BACKGROUND: Waterfowl parvoviruses, including goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), can cause seriously diseases in geese and ducks. Developing a fast and precise diagnosis assay for these two parvoviruses is particularly important. RESULTS: A duplex SYBR Green I-based quantitative real-time PCR assay was developed for the simultaneous detection and differentiation of GPV and MDPV. The assay yielded melting curves with specific single peak (Tm = 87.3 ± 0.26 °C or Tm = 85.4 ± 0.23 °C) when GPV or MDPV was evaluated, respectively. When both parvoviruses were assessed in one reaction, melting curves with specific double peaks were yielded. CONCLUSION: This duplex quantitative RT-PCR can be used to rapid identify of GPV and MDPV in field cases and artificial trials, which make it a powerful tool for diagnosing, preventing and controlling waterfowl parvovirus infections.

Detection of ALV p27 in cloacal swabs and virus isolation medium by sELISA.

BACKGROUND: Avian leukosis (AL), which is caused by avian leukosis virus (ALV), has led to substantial economic losses in the poultry industry. The kit used to detect all ALV-positive chickens in breeder flocks is very important for efficiently controlling AL. However, a new emerging ALV subtype is currently a severe challenge in the poultry industry. RESULTS: In this paper, we compared different enzyme-linked immunosorbent assay (ELISA) kits for detecting p27 of ALV in the same batch of meconium samples. Different positive samples were further analyzed by PCR or virus isolation. The results showed that 36 positive samples among the 1812 chicken meconium samples could be detected by a sandwich ELISA (sELISA) kit, but only 17 positive samples could be identified by a commercial kit. To verify this result, cloacal swabs and viruses isolated from the positive chickens (2 days old) were used to detect the presence of p27. The results showed that the positive rate of p27 was 100% for the swabs and 40% for virus isolation. Surprisingly, PCR and sequence analysis revealed that the env gene of ALV in these positive samples belonged to the novel subgroup K (ALV-K). CONCLUSION: These data not only demonstrate the relatively high sensitivity of the sELISA kit but also highlight the challenge of controlling ALV-K.

Alternate routes of influenza A virus infection in Mallard (Anas platyrhynchos).

The natural reservoir for all influenza A viruses (IAVs) is wild birds, particularly dabbling ducks. During the autumn, viral prevalence can be very high in dabbling ducks (> 30%) in the Northern Hemisphere, and individuals may be repeatedly infected. Transmission and infection is through the fecal-oral route, whereby birds shed viruses in feces and conspecifics are infected though feeding in virus-contaminated water. In this study we wanted to assess two alternative infection routes: cloacal drinking and preening. Using experimental infections, we assessed patterns of infection using a combination of virus shedding, as assessed by real-time PCR from cloacal swabs, and patterns of viral replication using virus-immunohistochemistry of gastrointestinal tissues. The cloacal drinking experiment consisted of two trials using cloacal inoculation at two different time points to account for age differences, as well as a trial whereby ducks were allowed to take up virus-laden water through the cloaca. All ducks became infected, and rather than the bursa of Fabricius being the main site of replication, the colon had the highest intensity of replication, as inferred through immunohistochemistry. In experiments assessing preening, feathers were contaminated with virus-laden water and all ducks became infected, regardless of whether they were kept individually or together. Further, naive contacts were infected by the individuals whose feathers were virus-contaminated. Overall, we reinforce that IAV transmission in dabbling ducks is multifactorial-if exposed to virus-contaminated water ducks may be infected through dabbling, preening of infected feathers, and cloacal drinking.

Genetic diversity of avian avulavirus 1 (Newcastle disease virus genotypes VIg and VIIb) circulating in wild birds in Kazakhstan.

In order to improve current understanding of the molecular epidemiology of avian avulavirus 1 (AAvV-1, formerly avian paramyxovirus 1) in wild birds in Kazakhstan, 860 cloacal swab samples were evaluated. Samples were collected from 37 families of wild birds in nine different regions in the years 2011 and 2014. Overall, 54 positive samples (4.2%) were detected from 17 different families of wild birds, and 16 AAvV-1 isolates were characterized. Three of the isolates contained the fusion protein cleavage site motif RRQKR, and 13 contained KRQKR, which is typical for pathogenic strains of AAvV-1. The AAvV-1 isolates were found to belong to the genotypes VIg and VIIb.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

Specific detection of Muscovy duck parvovirus infection by TaqMan-based real-time PCR assay.

BACKGROUND: Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in Muscovy ducks, with the pathogenesis of the virus still unknown in many respects. Specific MDPV detection is often rife with false positive results because of high identity at the genomic nucleotide level and antigenic similarity with goose parvovirus (GPV). The objective of this study was to develop a sensitive, highly specific, and repeatable TaqMan-based real-time PCR (qPCR) assay for facilitating the molecular detection of MDPV. RESULTS: The specific primers and probe were designed based on the conserved regions within MDPVs, but there was a variation in GPVs of the nonstructural (NS) genes after genetic comparison. After the optimization of qPCR conditions, the detection limit of this qPCR assay was 29.7 copies/µl. The assay was highly specific for the detection of MDPV, and no cross-reactivity was observed with other non-targeted duck-derived pathogens. Intra- and inter-assay variability was less than 2.21%, means a high degree of repeatability. The diagnostic applicability of the qPCR assay was proven that MDPV-positive can be found in cloacal swabs samples, Muscovy duck embryos and newly hatched Muscovy ducklings. CONCLUSIONS: Our data provided incidents that MDPV could be possible vertically transmitted from breeder Muscovy ducks to Muscovy ducklings. The developed qPCR assay in the study could be a reliable and specific tool for epidemiological surveillance and pathogenesis studies of MDPV.

Influenza A virus on oceanic islands: host and viral diversity in seabirds in the Western Indian Ocean.

Ducks and seabirds are natural hosts for influenza A viruses (IAV). On oceanic islands, the ecology of IAV could be affected by the relative diversity, abundance and density of seabirds and ducks. Seabirds are the most abundant and widespread avifauna in the Western Indian Ocean and, in this region, oceanic islands represent major breeding sites for a large diversity of potential IAV host species. Based on serological assays, we assessed the host range of IAV and the virus subtype diversity in terns of the islands of the Western Indian Ocean. We further investigated the spatial variation in virus transmission patterns between islands and identified the origin of circulating viruses using a molecular approach. Our findings indicate that terns represent a major host for IAV on oceanic islands, not only for seabird-related virus subtypes such as H16, but also for those commonly isolated in wild and domestic ducks (H3, H6, H9, H12 subtypes). We also identified strong species-associated variation in virus exposure that may be associated to differences in the ecology and behaviour of terns. We discuss the role of tern migrations in the spread of viruses to and between oceanic islands, in particular for the H2 and H9 IAV subtypes.

How Does Sampling Methodology Influence Molecular Detection and Isolation Success in Influenza A Virus Field Studies?

Wild waterfowl are important reservoir hosts for influenza A virus (IAV) and a potential source of spillover infections in other hosts, including poultry and swine. The emergence of highly pathogenic avian influenza (HPAI) viruses, such as H5N1 and H5N8, and subsequent spread along migratory flyways prompted the initiation of several programs in Europe, North America, and Africa to monitor circulation of HPAI and low-pathogenicity precursor viruses (low-pathogenicity avian influenza [LPAI] viruses). Given the costs of maintaining such programs, it is essential to establish best practice for field methodologies to provide robust data for epidemiological interpretation. Here, we use long-term surveillance data from a single site to evaluate the influence of a number of parameters on virus detection and isolation of LPAI viruses. A total of 26,586 samples (oropharyngeal, fecal, and cloacal) collected from wild mallards were screened by real-time PCR, and positive samples were subjected to isolation in embryonated chicken eggs. The LPAI virus detection rate was influenced by the sample type: cloacal/fecal samples showed a consistently higher detection rate and lower cycle threshold (Ct) value than oropharyngeal samples. Molecular detection was more sensitive than isolation, and virus isolation success was proportional to the number of RNA copies in the sample. Interestingly, for a given Ct value, the isolation success was lower in samples from adult birds than in those from juveniles. Comparing the results of specific real-time reverse transcriptase (RRT)-PCRs and of isolation, it was clear that coinfections were common in the investigated birds. The effects of sample type and detection methods warrant some caution in interpretation of the surveillance data.

Partial heterologous protection by low pathogenic H9N2 virus against natural H9N2-PB1 gene reassortant highly pathogenic H5N1 virus in chickens.

Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 10(6) EID50 of H9N2 virus were challenged with 10(6) EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co-circulating.

Reverse genetics based rgH5N2 vaccine provides protection against high dose challenge of H5N1 avian influenza virus in chicken.

An inactivated vaccine was developed using the rgH5N2 virus (6 + 2 reassortant) generated by plasmid based reverse genetics system (RGS) with WSN/33/H1N1 as backbone virus. Following mutation of the basic amino acid cleavage site RRRKKR*GLF to IETR*GLF, the H5-HA (haemagglutinin) gene of the selected donor H5N1 virus (A/chicken/West Bengal/80995/2008) of antigenic clade 2.2 was used along with the N2-NA gene from H9N2 field isolate (A/chicken/Uttar Pradesh/2543/2004) for generation of the rgH5N2 virus. A single dose (0.5 ml/bird) of the inactivated rgH5N2 vaccine protected 100% of the vaccinated chickens (n = 10) on 28(th) dpv (early challenge) and 90% of the vaccinated chickens (n = 10) on 200(th) dpv (late challenge) against high dose challenge with HPAI virus (10(9) EID50/bird). Challenge virus shedding via oropharynx and cloaca of the vaccinated chickens was detectable by realtime RT-PCR during 1-5 dpc and 1-9 days dpc in the early and the late challenge, respectively. The protective level of antibodies (mean HI titre > 128) was maintained without booster vaccination for 200 days. The present study provides the experimental evidence about the extent of protection provided by a reverse genetics based vaccine for clade 2.2 H5N1 viruses against challenge with high dose of field virus at two different time points (28 dpv and 200 dpv). The challenge study is uniquely different from the previous similar experiments on account of 1000 times higher dose of challenge and protection at 200 dpv. The protection and virus shedding data of the study may be useful for countries planning to use H5 vaccine in poultry especially against the clade 2.2 H5N1 viruses.

Chicken parvovirus viral loads in cloacal swabs from malabsorption syndrome-affected and healthy broilers.

Chicken parvovirus (ChPV) has been associated with malabsorption syndrome (MAS) in broilers. However, the participation of this virus in such syndrome is unclear, since it may be detected in diseased and healthy chickens. In the course of these studies, it was argued whether ChPV genome loads might be correlated to the occurrence of MAS. To check such a hypothesis, a SYBR green-based quantitative polymerase chain reaction was developed to detect and quantify ChPV genomes. Cloacal swabs from 68 broilers with MAS and 59 from healthy animals were collected from different poultry farms. Genomes of ChPV were detected in all samples, regardless of their health status. However, viral genome loads in MAS-affected broilers were significantly higher (1 × 105 genome copies per 100 ng DNA) than in healthy animals (1.3 × 103 GC/100 ng DNA). These findings indicate that there is an association between high ChPV genome loads and the occurrence of MAS in broilers.

Role of poultry in the spread of novel H7N9 influenza virus in China.

UNLABELLED: The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 10(6) 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity. IMPORTANCE: The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This study provides experimental data to show that quail and chickens are susceptible to infection, shed large amounts of virus, and are likely important in the spread of the virus to humans. Other poultry species can be infected and shed virus but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested to be a possible source of the virus because of isolation of the virus from several pigeons in poultry markets in China, but experimental studies show that they are generally resistant to infection and are unlikely to play a role in the spread of the virus.

Host-specific exposure and fatal neurologic disease in wild raptors from highly pathogenic avian influenza virus H5N1 during the 2006 outbreak in Germany.

Raptors may contract highly pathogenic avian influenza virus H5N1 by hunting or scavenging infected prey. However, natural H5N1 infection in raptors is rarely reported. Therefore, we tested raptors found dead during an H5N1 outbreak in wild waterbirds in Mecklenburg-Western Pomerania, Germany, in 2006 for H5N1-associated disease. We tested 624 raptors of nine species-common buzzard (385), Eurasian sparrowhawk (111), common kestrel (38), undetermined species of buzzard (36), white-tailed sea eagle (19), undetermined species of raptor (12), northern goshawk (10), peregrine falcon (6), red kite (3), rough-legged buzzard (3), and western marsh-harrier (1)-for H5N1 infection in tracheal or combined tracheal/cloacal swabs of all birds, and on major tissues of all white-tailed sea eagles. H5N1 infection was detected in two species: common buzzard (12 positive, 3.1%) and peregrine falcon (2 positive, 33.3%). In all necropsied birds (both peregrine falcons and the six freshest common buzzards), H5N1 was found most consistently and at the highest concentration in the brain, and the main H5N1-associated lesion was marked non-suppurative encephalitis. Other H5N1-associated lesions occurred in air sac, lung, oviduct, heart, pancreas, coelomic ganglion, and adrenal gland. Our results show that the main cause of death in H5N1-positive raptors was encephalitis. Our results imply that H5N1 outbreaks in wild waterbirds are more likely to lead to exposure to and mortality from H5N1 in raptors that hunt or scavenge medium-sized birds, such as common buzzards and peregrine falcons, than in raptors that hunt small birds and do not scavenge, such as Eurasian sparrowhawks and common kestrels.

Parrot bornavirus-2 and -4 RNA detected in wild bird samples in Japan are phylogenetically adjacent to those found in pet birds in Japan.

Bornaviruses (family Bornaviridae) are non-segmented negative-strand RNA viruses. Avian bornaviruses (ABVs), which are causative agents of proventricular dilatation disease, are a genetically diverse group with at least 15 genotypes, including parrot bornaviruses (PaBVs) and aquatic bird bornavirus 1(ABBV-1). Borna disease virus 1(BoDV-1), which infects mammals and causes neurological diseases, has also been reported to infect avian species, although the numbers of the cases have been markedly fewer than those of ABVs. In this study, we conducted genetic surveillance to detect ABVs (PaBV-1 to -5 and ABBV-1) and BoDV-1 in wild birds in Japan. A total of 2078 fecal or cloacal swab samples were collected from wild birds in 2006, 2007, 2008, and 2011, in two regions of Japan. The results demonstrated the presence of PaBV-2 and -4 RNA, while no positive results for other PaBVs, ABBV-1, and BoDV-1 were obtained. PaBV-2 and -4 RNA were detected in 18 samples (0.9 %) of the genera Anas, Grus, Larus, Calidris, Haliaeetus, and Emberiza, in which either PaBV-2 RNA or PaBV-4 RNA, or both PaBV-2 and -4 RNA were detected in 15 (0.7 %), 5 (0.2 %), and 2 (0.1 %) samples, respectively. The nucleotide sequences of PaBV-2 and -4 detected in these samples from wild birds are phylogenetically close to those found in samples from pet birds in Japan, with identities ranging from 99.8 to 100 % and from 98.2 to 99.4 %, respectively. To the best of our knowledge, this is the first report on the detection of PaBV-2 and -4 RNA detected in samples from wild birds.

Genetic characterization of natural reassortant H4 subtype avian influenza viruses isolated from domestic ducks in Zhejiang province in China from 2013 to 2014.

The H4 subtype of the influenza virus was first isolated in 1999 from pigs with pneumonia in Canada. H4 avian influenza viruses (AIVs) are able to cross the species barrier to infect humans. In order to better understand the genetic relationships between H4 AIV strains circulating in Eastern China and other AIV strains from Asia, a survey of domestic ducks in live poultry markets was undertaken in Zhejiang province from 2013 to 2014. In this study, 23 H4N2 (n = 14) and H4N6 (n = 9) strains were isolated from domestic ducks, and all eight gene segments of these strains were sequenced and compared to reference AIV strains available in GenBank. The isolated strains clustered primarily within the Eurasian lineage. No mutations associated with adaption to mammalian hosts or drug resistance was observed. The H4 reassortant strains were found to be of low pathogenicity in mice and able to replicate in the lung of the mice without prior adaptation. Continued surveillance is required, given the important role of domestic ducks in reassortment events leading to new AIVs.

Wood ducks (Aix sponsa) as potential reservoirs for avian influenza and avian paramyxoviruses.

Influenza A viruses (IAVs) and avian paramyxoviruses (APMVs) are important pathogens of poultry worldwide, and both commonly occur in wild waterfowl, especially ducks in the family Anatidae. Although wood ducks (Aix sponsa) are members of the Anatidae, their behaviour differs from most other species in this family, which could affect the transmission of IAVs and APMVs. We collected cloacal and oropharyngeal swab and blood samples from more than 700 wood ducks across nine states in the eastern United States of America. No IAVs were isolated, and based on blocking enzyme-linked immunoassay ELISA results, antibodies to IAVs were only detected in 0.2% of samples. In contrast, 23 (3%) APMVs were isolated (22 Newcastle disease virus and 1 APMV-6), and antibodies to multiple serotypes of APMVs were detected in more than 60% of the samples. After-hatch-year birds were more likely to be antibody positive for APMV-4 and APMV-6 compared to hatch-year birds. Female birds were more likely to be antibody positive for APMV-4 than were male birds. Our results indicate that wood ducks are probably not an important host for IAV but are frequently infected with APMVs.

Serological Surveillance of Wild Waterfowl in Northern Australia for Avian Influenza Virus Shows Variations in Prevalence and a Cyclical Periodicity of Infection.

The virological surveillance of 3582 wild waterfowl in northern Australia from 2004 to 2009 for avian influenza virus (AIV) found an apparent prevalence (AP) of 1% (31 of 2989 cloacal swabs; 95% CI: 0.71%-1.47%) using a Taqman Type A real-time reverse transcription polymerase chain reaction test and no viral isolations from 593 swabs tested by the embryonating chicken egg culture method. From serological testing using a nucleoprotein competitive enzyme-linked immunosorbent assay for AIV antibody, 1131 of 3645 sera had ≥ 40% inhibition, indicating an apparent seroprevalence of 31% (95% CI: 29.5%-32.6%). This value suggests that the low AP from virological testing does not reflect the dynamics of AIV infection in these populations. Spatiotemporal and species variations in seroprevalence were found at wetland sampling sites, with consistently higher values at Kununurra in Western Australia (AP  =  39%, 95% CI: 36.9%-41.4%) compared to other locations. At Kununurra, seroprevalence values had a two-year cyclical periodicity and suggest this location is a hotspot of AIV activity. From hemagglutination inhibition (HI) testing using multiple subtype antigens, the highest AP of HI reactions were to H6 and H5 subtypes. The phenomenon of cyclic periodicity in NP seroprevalence at Kununurra is hypothesized as being related to the prevalent H6 subtype that may have either become predominant or cycled back into a mostly AIV naïve flock. The inclusion of serological testing provided insight into the dynamics of AIV infection in wild birds such as species risk profiles and spatiotemporal patterns, important epidemiological information for a risk-based approach to surveillance.

Experimental infection of highly pathogenic avian influenza virus H5N1 in black-headed gulls (Chroicocephalus ridibundus).

Historically, highly pathogenic avian influenza viruses (HPAIV) rarely resulted in infection or clinical disease in wild birds. However, since 2002, disease and mortality from natural HPAIV H5N1 infection have been observed in wild birds including gulls. We performed an experimental HPAIV H5N1 infection of black-headed gulls (Chroicocephalus ridibundus) to determine their susceptibility to infection and disease from this virus, pattern of viral shedding, clinical signs, pathological changes and viral tissue distribution. We inoculated sixteen black-headed gulls with 1 × 10(4) median tissue culture infectious dose HPAIV H5N1 (A/turkey/Turkey/1/2005) intratracheally and intraesophageally. Birds were monitored daily until 12 days post inoculation (dpi). Oropharyngeal and cloacal swabs were collected daily to detect viral shedding. Necropsies from birds were performed at 2, 4, 5, 6, 7, and 12 dpi. Sampling from selected tissues was done for histopathology, immunohistochemical detection of viral antigen, PCR, and viral isolation. Our study shows that all inoculated birds were productively infected, developed systemic disease, and had a high morbidity and mortality rate. Virus was detected mainly in the respiratory tract on the first days after inoculation, and then concentrated more in pancreas and central nervous system from 4 dpi onwards. Birds shed infectious virus until 7 dpi from the pharynx and 6 dpi from the cloaca. We conclude that black-headed gulls are highly susceptible to disease with a high mortality rate and are thus more likely to act as sentinel species for the presence of the virus than as long-distance carriers of the virus to new geographical areas.

Characterization of genotype IX Newcastle disease virus strains isolated from wild birds in the northern Qinling Mountains, China.

This study was conducted to evaluate the virulence and evolution of genotype IX Newcastle disease virus (NDV) isolates obtained from wild birds in the northern Qinling Mountains of China. Five isolates were obtained from 374 larynx and cloacae swabs, which were collected from multiple asymptomatic wild bird species from August 2008 to July 2011, and were subsequently characterized by pathotype and genotype. Deduced amino acid sequences revealed that all five NDV isolates exhibited velogenic fusion protein cleavage sites motif (112)R-R-Q-R-R-F(117), shared as high as 99.8-99.9 % homology with each other, and varied in pathotype by intracerebral pathogenicity indices (ICPI) of 0.425-1.638. Phylogenetic analysis showed that all five isolates were clustered to genotype IX NDV. This is the first study to confirm multiple asymptomatic wild bird species as natural carriers of virulent genotype IX NDV. A novel NDV isolate from the Spotted-necked Dove (family Columbidae) exhibited discordance between its lentogenic ICPI and its virulent proteolytic cleavage site motif (112)R-R-Q-R-R-F(117). Although the five isolates underwent several amino acid mutations in the fusion protein, evidence of continuous evolutionary divergence did exist in the genotype IX NDV, which was always regarded as a conservative genotype.