Influenza Virus In Chickens Research Articles

Potency of an inactivated influenza vaccine prepared from a non-pathogenic H5N1 virus against a challenge with antigenically drifted highly pathogenic avian influenza viruses in chickens

Antigenic variants of H5N1 highly pathogenic avian influenza virus (HPAIV) have selected and are prevailing in poultry populations in Asia. In the present study, the potency of inactivated influenza vaccine prepared from a non-pathogenic H5N1 avian influenza virus, A/duck/Hokkaido/Vac-3/2007 (H5N1), was assessed by challenging with H5N1 HPAIV variants, A/muscovy duck/Vietnam/OIE-559/2011 (H5N1), A/whooper swan/Hokkaido/4/2011 (H5N1), and A/peregrine falcon/Hong Kong/810/2009 (H5N1) belonging to clades 1, 2.3.2.1, and 2.3.4, respectively. All chickens immunized with the Vac-3 vaccine survived without showing any clinical signs after intranasal challenge either with A/whooper swan/Hokkaido/4/2011 (H5N1) or A/muscovy duck/Vietnam/OIE-559/2011 (H5N1). After challenge with A/peregrine falcon/Hong Kong/810/2009 (H5N1), 10 out of 12 vaccinated chickens survived and the other 2 died on 4 or 7 post-challenge days. The Vac-3 vaccine of 2.4-fold antigen concentration conferred complete protective immunity in chickens against challenge with A/peregrine falcon/Hong Kong/810/2009 (H5N1).

Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds

Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we identified a novel genotype of H9N2 viruses in widespread circulation in poultry in Pakistan that contained polymerases (PB2, PB1 and PA) and non-structural (NS) gene segments identical to highly pathogenic H7N3 viruses. Here, we investigated the potential of these viruses to cause disease and assessed the transmission capability of the virus within and between poultry and wild terrestrial avian species. Groups of broilers, layers, jungle fowl, quail, sparrows or crows were infected with a representative strain (A/chicken/UDL-01/08) of this H9N2 virus and then mixed with naïve birds of the same breed or species, or different species to examine transmission. With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with quail showing the most pronounced clinical signs. Virus shedding was detected in all infected birds, with quail showing the greatest levels of virus secretion, but only very low levels of virus were found in directly infected crow samples. Efficient virus intra-species transmission was observed within each group with the exception of crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens and sparrows and vice versa and efficient transmission was seen in either direction. These results highlight the ease of spread of this group of H9N2 viruses between domesticated poultry and sparrows and show that sparrows need to be considered as a high risk species for transmitting H9N2 viruses between premises.

Amino Acid 316 of Hemagglutinin and the Neuraminidase Stalk Length Influence Virulence of H9N2 Influenza Virus in Chickens and Mice

H9N2 influenza viruses with an A316S substitution in hemagglutinin (HA) and a shorter neuraminidase (NA) stalk have become predominant in China. The A316S was shown to increase HA cleavage efficiency when combined with short stalk NA, and the short stalk NA improved NA enzyme activity and release of virus from erythrocytes. Single mutations or combinations of these mutations strengthened the virulence of H9N2 virus in chickens and mice.

Characterization of low-pathogenicity H5 and H7 Korean avian influenza viruses in chickens

To date, all isolated highly pathogenic avian influenza (HPAI) viruses that cause systemic infection with a high mortality rate in poultry species have been known to belong to either the H5 or H7 subtypes. The HPAI viruses may originate because of the insertion of multiple basic amino acids at the cleavage site of the hemagglutinin protein after the low-pathogenic H5 and H7 viruses have been introduced into poultry. In the present study, we investigated the phylogenetic characteristics of the H5 (n = 4) and H7 (n = 3) low-pathogenic avian influenza (LPAI) viruses isolated from wild birds in Korea by using nucleotide sequences of all 8 gene segments of the viral genome. Further, we evaluated the infectivity, transmissibility, and pathogenic potential of these viruses in chickens. Phylogenetic analysis showed that all viruses used in the study clustered in the Eurasian lineage and were similar to the viruses isolated in Asian countries that share the East Asian–Australasian migratory bird flyway. Our H5N2 isolates could not be replicated and transmitted in chickens, but the H7N8 isolates could efficiently be replicated and transmitted to contact-exposure chickens. In addition, because our H7N8 isolates caused watery diarrhea in chickens, these viruses cannot only serve as progenitors of novel HPAI strains but also potentially cause clinical disease in poultry. Although there have been no reports of LPAI mutation to HPAI in these regions, the wild bird surveillance effort should focus on monitoring the introduction and transmission of the HPAI H5N1 and LPAI H5 and H7 viruses.

Pinon shell polysaccharide enhances immunity against H9N2 avian influenza virus in chickens

This study investigated the potential of pinon shell polysaccharide (PSP) to enhance immunity against H9N2 subtype avian influenza virus (H9N2 AIV) in chickens. The effect of PSP treatment and H9N2 virus infection after PSP pretreatment were investigated by [3-(4, 5-dimethylthiazol-2-yl)-2, 3-diphenyl tetrazolium bromide] assay and analysis of major histocompatibility complex and cytokine mRNA expression. The in vivo effects on lymphocytes and serum antibody titers were also investigated. The IL-6 expression and antibody titers were enhanced in the first week after PSP treatment. These data indicate that prophylactic PSP administration reduces H9N2 AIV replication and promotes early humoral immune responses in young birds.

Effect of intranasal administration of Lactobacillus fermentum CJL-112 on horizontal transmission of influenza virus in chickens

The aim of this study was to determine whether intranasal administration of Lactobacillus sp. could prevent horizontal transmission of H9N2 avian influenza virus (AIV) in specific-pathogen-free chickens. Three-week-old chickens received 500 μL of 1.5 × 10(9) cfu of Lactobacillus fermentum CJL-112 strain (CJL) intranasally for 7 d before and 14 d after a challenge. Challenged chickens, each inoculated with H9N2 AIV, were kept in either direct or indirect contact with naive chickens, and morbidity and viral shedding were monitored. We demonstrated that the intranasal administration of CJL significantly decreased the number of chickens with viral shedding from the gastrointestinal tract in the indirect contact chickens (P < 0.001) and also significantly reduced viral shedding from the respiratory tract in the challenged (P < 0.05) and the direct contact chickens (P < 0.001) than those in the control group. Hence, the use of this lactobacilli strain may constitute a novel and effectively plausible alternative to prevent and control H9N2 AIV infection in chickens.

Prophylactic treatment with Toll-like receptor ligands enhances host immunity to avian influenza virus in chickens

Avian influenza viruses (AIV) pose a threat towards the health of both poultry and humans. To interrupt the transmission of the virus, novel prophylactic strategies must be considered which may reduce the shedding of AIV. One potential is the prophylactic use of Toll-like receptor (TLR) ligands. Many cells of the immune system express TLRs, and cellular responses to TLR stimulation include activation and the production of cytokines. TLR ligands have been employed as prophylactic treatments to enhance host resistance to pathogens both in mammals and chickens. Therefore, the present study was conducted to determine whether TLR ligands may be used prophylactically in chickens to enhance host immunity to AIV. Chickens received intramuscular injections of either low or high doses of the TLR ligands poly I:C, lipopolysaccharide (LPS) and CpG ODN. Twenty-four hours post-treatment, chickens were infected with the low pathogenic avian influenza virus H4N6, and both oropharyngeal and cloacal virus shedding were assessed on days 4 and 7 post-infection. To identify potential correlates of immunity, spleen and lungs were collected on days 2, 4 and 7 post-infection for RNA extraction. The results suggested that all of the TLR ligand treatments induced a significant reduction in virus shedding, with the TLR3 ligand poly I:C conferring the greatest AIV immunity compared to control birds, followed by CpG ODN and LPS. Furthermore, transcriptional analysis of gene expression in the spleen and lungs suggest IFN-α and IL-8 as correlates of immunity conferred by poly I:C, and IFN-γ for CpG ODN and LPS. In conclusion, TLR ligands, have the ability to enhance host immunity against AIV, and future studies should consider exploring the combinatory effects of poly I:C and CpG ODN prophylaxis in conjunction with AIV vaccination.

Role of Position 627 of PB2 and the Multibasic Cleavage Site of the Hemagglutinin in the Virulence of H5N1 Avian Influenza Virus in Chickens and Ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

Development of a recombinant ELISA using yeast (Pichia pastoris)-expressed polypeptides for detection of antibodies against avian influenza A subtype H5

Two truncated sequences (designated P1 and rHA1) of influenza A virus subtype H5 haemagglutinin (HA) were cloned and expressed in yeast Pichia pastoris (P. pastoris). These polypeptides were used in an indirect recombinant ELISA (rELISA) for detection of H5 antibodies in poultry. Serum samples obtained from broiler chickens vaccinated with commercial inactivated vaccine (H5N2) and control negative sera from non-vaccinated chickens against influenza were tested using rP1-ELISA, rHA1-ELISA, whole H5N1-ELISA, Western blot, agar gel immunodiffusion (AGID) and haemagglutination inhibition (HI) tests. The rHA1-ELISA proved to be highly sensitive and specific. To study the validity of rHA1-ELISA, a total of 179 serum samples obtained from commercial broiler chickens vaccinated previously with commercial H5N2 inactivated vaccines, were tested by rHA1-ELISA, commercial ELISA (cELISA) and HI. The relative sensitivity and specificity between rHA1-ELISA, and HI tests were 100% and 70%, respectively, and between cELISA and HI were 100% and 57%, respectively. The agreement ratio between rHA1-ELISA and HI was 84.9% and between cELISA and HI tests was 76.5%. Serum samples obtained from ducks vaccinated with commercial inactivated H5N2 were tested by rHA1-ELISA and the results showed significant reactivity with duck sera. In conclusion, the results demonstrate the potential applicability of the rELISA for the determination of antibodies to H5 influenza virus in chickens and ducks.

Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 Å resolution and two structures of HP HA at 2.95 and 3.10 Å resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

Evaluation of the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 influenza viruses in chickens

Despite the long-term vaccination programs implemented in China, H9N2 avian influenza viruses (AIVs) continue to persist in chicken populations, even in vaccinated flocks. We previously demonstrated that H9N2 AIV isolated from chickens in China also underwent antigenic drift and evolved into distinct antigenic groups (C, D and E). To understand whether antigenic drift of viruses away from the vaccine strain partially contributed to the circulation of H9N2 AIV in China, we evaluated the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 AIV. Challenge experiments using vaccinated chickens indicated that the vaccine prevented shedding of antigenic group C viruses, but not those of the more recent groups D and E. Vaccinated chickens, even those with vaccine-induced HI titers of 1:1024, shed virus after being infected with A/chicken/Shandong/ZB/2007, a representative virus of antigenic group D. Genetic analysis showed that the representative viruses of antigenic groups D and E possessed greater numbers of amino acid substitutions in the hemagglutinin protein compared to the vaccine strain and the antigenic group C virus, and many of which were located in antigenic sites. Our results indicated that the persistence of H9N2 AIV in China might be due to incomplete vaccine protection, and that the avian influenza vaccine should be regularly evaluated and updated to maintain optimal protection. Furthermore, the avian influenza vaccination policy also needs to be re-assessed, and increased veterinary biosecurity on farms, rather than vaccine application alone, should be implemented to prevent and control avian influenza.

Emergence of Avian Influenza Viruses with Enhanced Transcription Activity by a Single Amino Acid Substitution in the Nucleoprotein during Replication in Chicken Brains

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP (I)109(T)). By analyzing viruses constructed by the reverse-genetic method, we established that the NP (I)109(T) substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP (I)109(T) substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP (I)109(T) substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP (I)109(T) mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains.

Bursopentine as a novel immunoadjuvant enhances both humoral and cell-mediated immune responses to inactivated H9N2 Avian Influenza virus in chickens.

There is an urgent need for identification of a new adjuvant capable of selectively promoting an efficient immune response for use with vaccines and especially subunit vaccines. Our pervious study showed that Bursopentine (BP5) is a novel immunomodulatory peptide and has the ability to significantly stimulate an antigen-specific immune response in mice. In this study, the potential adjuvant activities of BP5 were examined in chickens by coinjection of BP5 and an inactivated avian influenza virus (AIV) (A/Duck/Jiangsu/NJ08/05 [AIV H9N2 subtype]). The results suggested that BP5 markedly elevated serum hemagglutination inhibition (HI) titers and antigen-specific antihemagglutinin (anti-HA) antibody (IgG) levels, induced both Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ])- and Th2 (IL-4)-type cytokines, promoted the proliferation of peripheral blood lymphocytes, and increased populations of CD3(+) T cells and their subsets CD4(+) (CD3(+) CD4(+)) T cells and CD8(+) (CD3(+) CD8(+)) T cells. Furthermore, a virus challenge experiment revealed that BP5 contributes to protection against homologous avian influenza virus challenge by reducing viral replication in chicken lungs. This study indicates that the combination of inactivated AIVs and BP5 gives a strong immune response at both the humoral and cellular levels and implies that BP5 is a novel immunoadjuvant suitable for vaccine design.

NP Body Domain and PB2 Contribute to Increased Virulence of H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens

The molecular basis of pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in chickens remains largely unknown. H5N1 A/chicken/Yamaguchi/7/2004 virus (CkYM7) replicates rapidly in macrophages and vascular endothelial cells in chickens, causing sudden death without fever or gross lesions, while H5N1 A/duck/Yokohama/aq10/2003 virus (DkYK10) induces high fever, severe gross lesions, and a prolonged time to death, despite the 98% amino acid identity between the two viruses. To explore the molecular basis of this difference in pathogenicity, a series of eight single-gene reassortant viruses from these HPAI viruses were compared for pathogenicity in chickens. Two reassortants possessing the NP or PB2 gene from DkYK10 in the CkYM7 background reduced pathogenicity compared to other reassortants or CkYM7. Inversely, reassortants possessing the NP or PB2 gene of CkYM7 in the DkYK10 background (rgDkYK-PB2(Ck), rgDkYK-NP(Ck)) replicated quickly and reached higher titers than DkYK10, accompanied by more rapid and frequent apoptosis of macrophages. The rgDkYK-NP(Ck) and rgDkYK-PB2(Ck) reassortants also replicated more rapidly in chicken embryo fibroblasts (CEFs) than did rgDkYK10, but replication of these viruses was similar to that of CkYM7 and DkYK10 in duck embryo fibroblasts. A comparison of pathogenicities of seven rgDkYK10 mutants with a single amino acid substitution in NP(Dk) demonstrated that valine at position 105 in the NP(Ck) was responsible for the increased pathogenicity in chickens. NP(Ck), NP(105V), and PB2(Ck) enhanced the polymerase activity of DkYK10 in CEFs. These results indicate that both NP and PB2 contribute to the high pathogenicity of the H5N1 HPAI viruses in chickens, and valine at position 105 of NP may be one of the determinants for adaptation of avian influenza viruses from ducks to chickens.

Chicken cyclophilin A is an inhibitory factor to influenza virus replication

BackgroundThe importance of enhancing influenza resistance in domestic flocks is quite clear both scientifically and economically. Chicken is very susceptible to influenza virus. It has been reported that human cellular cyclophilin A (CypA) impaired influenza virus infection in 293T cells. Whether chicken CypA (chCypA) inhibits influenza virus replication is not known. The molecular mechanism of resistance in chicken to influenza virus remains to be studied.ResultsThe chCypA gene was isolated and characterized in the present study. It contained an ORF of 498 bp encoding a polypeptide of 165 amino acids with an estimated molecular mass of 17.8 kDa sharing high identity with mammalian CypA genes. The chCypA demonstrated an anti-influenza activity as expected. ChCypA protein was shown to be able to specifically interact with influenza virus M1 protein. Cell susceptibility to influenza virus was reduced by over-expression of chCypA in CEF cells. The production of recombinant influenza virus A/WSN/33 reduced to one third in chCypA expressing cells comparing to chCypA absent cells. ChCypA was widely distributed in a variety of chicken tissues. It localized in cytoplasm of chicken embryo fibroblast (CEF) cells. Avian influenza virus infection induced its translocation from cytoplasm into nucleus. ChCypA expression was not significantly up-regulated by avian influenza virus infection. The present study indicated that chCypA was an inhibitory protein to influenza virus replication, suggesting a role as an intrinsic immunity factor against influenza virus infection.ConclusionThe present data demonstrates that chCypA possesses anti-influenza virus activity which allows the consideration of genetic improvement for resistance to influenza virus in chickens.

The pathogenesis of low pathogenicity H7 avian influenza viruses in chickens, ducks and turkeys

BackgroundAvian influenza (AI) viruses infect numerous avian species, and low pathogenicity (LP) AI viruses of the H7 subtype are typically reported to produce mild or subclinical infections in both wild aquatic birds and domestic poultry. However relatively little work has been done to compare LPAI viruses from different avian species for their ability to cause disease in domestic poultry under the same conditions. In this study twelve H7 LPAI virus isolates from North America were each evaluated for their comparative pathogenesis in chickens, ducks, and turkeys.ResultsAll 12 isolates were able to infect all three species at a dose of 106 50% egg infectious doses based on seroconversion, although not all animals seroconverted with each isolate-species combination. The severity of disease varied among isolate and species combinations, but there was a consistent trend for clinical disease to be most severe in turkeys where all 12 isolates induced disease, and mortality was observed in turkeys exposed to 9 of the 12 viruses. Turkeys also shed virus by the oral and cloacal routes at significantly higher titers than either ducks or chickens at numerous time points. Only 3 isolates induced observable clinical disease in ducks and only 6 isolates induced disease in chickens, which was generally very mild and did not result in mortality. Full genome sequence was completed for all 12 isolates and some isolates did have features consistent with adaptation to poultry (e.g. NA stalk deletions), however none of these features correlated with disease severity.ConclusionsThe data suggests that turkeys may be more susceptible to clinical disease from the H7 LPAI viruses included in this study than either chickens or ducks. However the severity of disease and degree of virus shed was not clearly correlated with any isolate or group of isolates, but relied on specific species and isolate combinations.

The pathogenesis of H3N8 canine influenza virus in chickens, turkeys and ducks

Canine influenza virus (CIV) of the H3N8 subtype has emerged in dog populations throughout the United States where it has become endemic in kennels and animal shelters in some regions. It has not previously been determined whether the canine-adapted H3N8 influenza virus can be transmitted to chickens, turkeys or ducks which are economically important animals that are susceptible to type A influenza virus from numerous species. Four-week-old specific pathogen-free (SPF) chickens, 3-week-old SPF turkeys and 2-week-old commercial Pekin ducks were inoculated with 10(5·2) 50% tissue culture infectious doses of CIV per bird by the intra-choanal route. The birds were observed daily, and at 2 and 4 days post-inoculation (DPI), two inoculated birds and one sham-inoculated control bird were euthanized and necropsied to evaluate gross lesions and to collect tissues for microscopic examination. Cloacal and oral swabs were collected at 2, 4 and 7 DPI to evaluate virus shed by real-time RT-PCR (rRT-PCR). Two weeks post-infection, sera were collected from all remaining birds for type A influenza antibody detection by hemagglutination inhibition assay. Clinical signs and gross lesions were not observed in any of the birds of any species nor did any seroconvert. Oral and Cloacal swab material was negative for virus by rRT-PCR. Chickens, turkeys and Pekin ducks are not susceptible to infection with CIV by simulated respiratory exposure route with the dose administered.

Phenotypes influencing the transmissibility of highly pathogenic avian influenza viruses in chickens

To identify critical phenotypes that affect avian influenza virus transmission in chickens, we compared the transmissibility of three H5N1 highly pathogenic viruses of different pathogenicity in chickens by monitoring the exact time of death using wireless thermo-sensors. This study showed that, despite quick deaths, the most virulent H5N1 A/chicken/Yamaguchi/7/2004 transmitted quickly in chickens via contact and airborne routes. Intermediate virulent H5N1 A/chicken/Miyazaki/K11/2007 spread moderately and less virulent H5N1 A/duck/Yokohama/aq10/2003, causing severe clinical signs and a long period to death, spread slowly among the animals. The transmissibility was correlated with virus titres of oropharyngeal and cloacal swabs, and the time for swab virus titres to reach 50 % chicken infective dose affected the transmission speed. These results demonstrate that peak virus titres excreted and the time required for virus titres to reach a minimal chicken infectious dose may be the critical phenotypes influencing the transmissibility of highly pathogenicity avian influenza viruses in chickens.

Diversified reassortant H9N2 avian influenza viruses in chicken flocks in northern and eastern China

According to our previous study of the M genes of H9N2 avian influenza viruses (AIV) in infected chickens, A/Quail/Hong Kong/G1/97 (G1 97)-like M genes newly emerged in northern and eastern China in addition to the existing A/chicken/Hong Kong/Y280/97 (Y280)-like lineage M genes. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of seventeen H9N2 isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the Y280-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.

Adaptation of a Mallard H5N2 Low Pathogenicity Influenza Virus in Chickens with Prior History of Infection with Infectious Bursal Disease Virus

The influenza A/Mallard/Pennsylvania/10218/1984 (H5N2) virus is unable to replicate in 3-wk-old immunocompetent specific-pathogen-free chickens when a dose of 5 x 10(6) 50% egg infectious dose/ml is used. In contrast, this mallard virus shows limited replication in 3-wk-old chickens that had been previously infected at 2 days of age with, and recovered from, the immunosuppressive agent infectious bursal disease virus (IBDV; herein referred to as IBDV chickens). This limited replication in IBDV chickens allowed for the serial passage of the mallard influenza virus in chickens. After 22 passages (P22) in IBDV chickens, the resulting chicken-adapted influenza virus replicated in both immunocompetent and IBDV chickens more efficiently than the mallard influenza virus. Analysis of the outcomes of infection and the lesions caused by the two viruses at the microscopic level in a time-point study showed that the P22 virus is more virulent than the parental mallard virus in both immunocompetent and IBDV chickens. Our studies provide evidence that a previous history of IBDV infection in chickens may render them more susceptible to avian influenza virus (AIV) infections, allowing for the potential introduction of AIVs in an otherwise resistant population.