Influenza Virus In Chickens Research Articles

Biologic Characterization of Chicken-Derived H6N2 Low Pathogenic Avian Influenza Viruses in Chickens and Ducks

Low pathogenic avian influenza H6N2 viruses were biologically characterized by infecting chickens and ducks in order to compare adaptation of these viruses in these species. We examined the clinical signs, virus shedding, and immune response to infection in 4-wk-old white leghorn chickens and in 2-wk-old Pekin ducks. Five H6N2 viruses isolated between 2000 and 2004 from chickens in California, and one H6N2 virus isolated from chickens in New York in 1998, were given intrachoanally at a dose of 1 x 10(6) 50% embryo infectious dose per bird. Oral-pharyngeal and cloacal swabs were taken at 2, 4, and 7 days postinoculation (PI) and tested by real-time reverse-transcriptase polymerase chain reaction for presence of virus. Serum was collected at 7, 14, and 21 days PI and examined for avian influenza virus antibodies by commercial enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) testing. Virus shedding for all of the viruses was detected in the oral-pharyngeal swabs from chickens at 2 and 4 days PI, but only three of the five viruses were detected at 7 days PI. Only two viruses were detected in the cloacal swabs from the chickens. Virus shedding for four of the five viruses was detected in the oral-pharyngeal cavity of the ducks, and fecal shedding was detected for three of the viruses (including the virus not shed by the oral-pharyngeal route) in ducks at 4 and 7 days PI. All other fecal swabs from the ducks were negative. Fewer ducks shed virus compared to chickens. Both the chickens and the ducks developed antibodies, as evidenced by HI and ELISA titers. The data indicate that the H6N2 viruses can infect both chickens and ducks, but based on the number of birds shedding virus and on histopathology, the viruses appear to be more adapted to chickens. Virus shedding, which could go unnoticed in the absence of clinical signs in commercial chickens, can lead to transmission of the virus among poultry. However, the viruses isolated in 2004 did not appear to replicate or cause more disease than earlier virus isolates.

Biologic Characterization of Chicken-Derived H6N2 Low Pathogenic Avian Influenza Viruses in Chickens and Ducks

Biologic Characterization of Chicken-Derived H6N2 Low Pathogenic Avian Influenza Viruses in Chickens and Ducks

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

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

A Genetically Engineered Waterfowl Influenza Virus with a Deletion in the Stalk of the Neuraminidase Has Increased Virulence for Chickens

A deletion of about 20 amino acids in the stalk of the neuraminidase (NA) is frequently detected upon transmission of influenza A viruses from waterfowl to domestic poultry. Using reverse genetics, a recombinant virus derived from a wild duck influenza virus isolate, A/Mallard/Marquenterre/Z237/83 (MZ), and an NA stalk deletion variant (MZ-delNA) were produced. Compared to the wild type, the MZ-delNA virus showed a moderate growth advantage on avian cultured cells. In 4-week-old chickens inoculated intratracheally with the MZ-delNA virus, viral replication in the lungs, liver, and kidneys was enhanced and interstitial pneumonia lesions were more severe than with the wild-type virus. The MZ-delNA-inoculated chickens showed significantly increased levels of mRNAs encoding interleukin-6 (IL-6), transforming growth factor-beta4 (TGF-beta4), and CCL5 in the lungs and a higher frequency of apoptotic cells in the liver than did their MZ-inoculated counterparts. Molecular mechanisms possibly underlying the growth advantage of the MZ-delNA virus were explored. The measured enzymatic activities toward a small substrate were similar for the wild-type and deleted NA, but the MZ-delNA virus eluted from chicken erythrocytes at reduced rates. Pseudoviral particles expressing the MZ hemagglutinin in combination with the MZ-NA or MZ-delNA protein were produced from avian cultured cells with similar efficiencies, suggesting that the deletion in the NA stalk does not enhance the release of progeny virions and probably affects an earlier step of the viral cycle. Overall, our data indicate that a shortened NA stalk is a strong determinant of adaptation and virulence of waterfowl influenza viruses in chickens.

Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza viruses in chickens

Highly pathogenic (HP) H5N1 avian influenza has become endemic in several countries in Asia and Africa, and vaccination is being widely used as a control tool. However, there is a need for efficacious vaccines preferably utilizing a DIVA (differentiate infected from vaccinated animals) marker strategy to allow for improved surveillance of influenza in vaccinated poultry. Using a reverse genetics approach, we generated Asian rgH5N9 vaccine strain deriving the hemagglutinin gene from A/chicken/Indonesia/7/2003 (H5N1) with modification of the cleavage site to be low pathogenic (LP) and N9 neuraminidase gene from the North American LP A/turkey/Wisconsin/1968 (H5N9) virus. The recombinant rgH5N9, A/turkey/Wisconsin/1968 (H5N9) A/chicken/Hidalgo/232/1994 (H5N2), and wild type HP A/chicken/Indonesia/7/2003 (H5N1) viruses were used to prepare inactivated oil-emulsified whole virus vaccines. Two weeks after vaccination, chickens were challenged with either Asian HP H5N1 viruses, A/chicken/Indonesia/7/2003 (W.H.O. clade 2.1) or A/chicken/Supranburi Thailand/2/2004 (W.H.O. clade 1.0). The H5 HA1 of the North American vaccine strains exhibited 12% amino acid differences including amino acid changes in the major antigenic sites as compared to the Asian HP H5N1 challenge viruses, serologically exhibited substantial antigenic difference, but still provided 100% protection from mortality. However, challenge virus shedding was significantly higher in chickens immunized with antigenically distinct American lineage vaccines as compared to the antigenically matched Asian rgH5N9 and the wild type Asian H5N1 vaccine. The antibody response to the heterologous subtype neuraminidase proteins were discriminated in vaccinated and infected chickens using a rapid fluorescent 2′-(4-methylumbelliferyl)-α- d- N-acetylneuraminic acid sodium salt as substrate for neuraminidase inhibition assay. This study demonstrates the value of using a vaccine containing antigenically matched H5 hemagglutinin for control of HP H5N1 avian influenza in poultry and the potential utility of a heterologous neuraminidase as a DIVA marker.

A single substitution in amino acid 184 of the NP protein alters the replication and pathogenicity of H5N1 avian influenza viruses in chickens

Changes in the NP gene of H5N1 highly pathogenic avian influenza (HPAI) viruses have previously been shown to affect viral replication, alter host gene expression levels and affect mean death times in infected chickens. Five amino acids at positions 22, 184, 400, 406, and 423 were different between the two recombinant viruses studied. In this study, we individually mutated the five amino acids that differed and determined that the difference in virus pathogenicity after NP gene exchange was a result of an alanine to lysine change at position 184 of the NP protein. Infection with viruses containing a lysine at NP 184 induced earlier mortality in chickens, increased virus titers and nitric oxide levels in tissues, and resulted in up-regulated host immune genes, such as alpha-interferon (IFN-alpha), gamma-interferon (IFN-gamma), orthomyxovirus resistance gene 1 (Mx1), and inducible nitric oxide synthase (iNOS). This study underlines the importance of the NP in avian influenza virus replication and pathogenicity.

Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses

The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.

Evolution analysis of the matrix (M) protein genes of 17 H9N2 chicken influenza viruses isolated in northern China during 1998–2008

Matrix (M) protein genes of 17 H9N2 avian influenza viruses (AIVs) isolated from chickens in northern China during the last 10 years were completely sequenced and phylogenetically analyzed. Homology of nucleotide sequences in the M gene of 17 isolates was 92.7-99.9%. Phylogenetic analysis showed that 11 of the tested M genes belong to the A/chicken/HongKong/Y280/97 (Y280)-like lineage, while the other six belong to the A/Quail/HongKong/G1/97 (G1)-like lineage. This is also the first time that a G1-like M gene of a H9N2 virus was detected in chicken flocks in northern China. These newly appearing changes in M genes may be due to reassortment events of AIVs, or they may have come from the H9N2 strains of southern China which surged in northern China after translocation. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S31N in two isolates, which is the molecular characterization of amantadine resistance in AIVs. Results of this study suggest that long-term monitoring should be continued to track the transmission and evolution of H9N2 AIVs in chickens in China.

Genetic characterization and protective immunity of cold-adapted attenuated avian H9N2 influenza vaccine

H9N2 influenza viruses are endemic in many Asian countries including China and Korea, and cause a considerable economic loss to chicken industry by reduction in egg production and about 30% mortality. Here we developed live cold-adapted attenuated H9N2 influenza vaccine by adaptation of viruses in hen’s eggs at 25 °C. Genetic analysis shows that the cold-adapted H9N2 (A/Chicken/Korea/S1/03) viruses contain a total of 44 amino acid substitutions, of which 7 amino acids are identical to the loci identified in the cold-adapted H2N2 (A/Ann Arbor/6/60) vaccine strain compared to genes in wild-type H9N2 (A/Chicken/Korea/S1/03) influenza viruses. When cold-adapted H9N2 (A/Chicken/Korea/S1/03) influenza viruses were inoculated in layers viruses were detectable in the tracheas, not in the lungs, no reduction of egg production and mortality was observed in contrast to the infection of wild-type H9N2 influenza viruses, and CD8+ T lymphocytes expressing IFN-γ were induced. When layers vaccinated with cold-adapted attenuated H9N2 (A/Chicken/Korea/S1/03) influenza viruses were challenged with wild-type H9N2 (A/Chicken/Korea/S21/04) influenza viruses, they were protected from the loss of egg production and mortality. Our results suggest that cold-adapted attenuated H9N2 vaccine can be used for controlling the infection of H9N2 influenza viruses in chickens.

NP, PB1, and PB2 Viral Genes Contribute to Altered Replication of H5N1 Avian Influenza Viruses in Chickens

The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.

A vaccine prepared from a non-pathogenic H7N7 virus isolated from natural reservoir conferred protective immunity against the challenge with lethal dose of highly pathogenic avian influenza virus in chickens

During 2001-2004, 41 H7 influenza viruses (2 H7N1 and 39 H7N7 strains) were isolated from fecal samples of migratory ducks that flew from Siberia in the autumn of each year to Japan and Mongolia. A phylogenetic analysis of the hemagglutinin (HA) genes of the nine representative isolates revealed that they belonged to the Eurasian lineage and the deduced amino acid sequence at the cleavage site of the HAs represented apathogenic profiles. One of the H7 isolates A/duck/Mongolia/736/02 (H7N7) was chosen from these H7 isolates for the preparation of the test vaccine. To improve the growth potential of A/duck/Mongolia/736/02 (H7N7) in chicken embryos, A/duck/Hokkaido/Vac-2/04 (H7N7) was generated by genetic reassortment between A/duck/Mongolia/736/02 (H7N7) as the donor of the PB2, PB1, PA, HA, NA, and NS genes and A/duck/Hokkaido/49/98 (H9N2) as that of NP and M genes. The test vaccine was prepared as follows; A/duck/Hokkaido/Vac-2/04 (H7N7) was propagated in chicken embryos and the virus in the allantoic fluid was inactivated and adjuvanted to form an oil-in-water emulsion. The test vaccine conferred immunity to chickens, completely protecting the manifestation of clinical signs against the challenge with lethal dose of H7 highly pathogenic avian influenza virus. These results indicate that influenza viruses isolated from natural reservoirs are useful for vaccine strains.

Protective efficacy of several vaccines against highly pathogenic H5N1 avian influenza virus under experimental conditions

Although several vaccines have been developed to protect against highly pathogenic avian influenza of subtype H5N1 'Asia' their efficiency has primarily been assessed individually. Thus, a direct comparison of their performance is still lacking. The following study was conducted to compare the protective efficacy of three commercially available inactivated vaccines based on influenza virus strains of subtypes H5N2 (vaccine A), H5N9 (vaccine B), and H5N3 (vaccine C), as well as two hemagglutinin expressing experimental vector vaccines (modified vaccinia virus Ankara-H5 and Newcastle disease virus-H5) against a lethal dose of highly pathogenic H5N1 avian influenza virus in chickens. To assess their potential as emergency vaccines, a single immunisation was performed for all vaccines, despite the recommendation of a double-vaccination schedule for commercial vaccines B and C. Overall, all vaccines induced clinical protection against challenge infection 3 weeks after immunisation. No mortality was observed in chickens immunised with vaccine A and viral shedding could not be detected. Immunisation with NDV-H5, vaccine C and MVA-H5 conferred also protection against lethal challenge. However, viral RNA was detected by real-time RT-PCR in swabs of 10%, 20% and 50% of animals, and 0%, 10% and 30% of animals, respectively, shed infectious virus. Immunisation with vaccine B was less protective since 50% of the vaccinated animals shed infectious virus after challenge and 20% of the chickens succumbed to disease. These results indicate that the NDV-H5 vectored vaccine is similarly effective as the best inactivated vaccine. Considering the advantage of live NDV which can be administered via spray or drinking water as well as the potential use of this H5 expressing vector vaccine for an easy DIVA (differentiating infected from vaccinated animals) strategy, NDV-H5 could represent an alternative for extensive vaccination against avian influenza in chickens.

A Naturally Occurring Deletion in Its NS Gene Contributes to the Attenuation of an H5N1 Swine Influenza Virus in Chickens

In 2001 and 2003, we isolated two H5N1 viruses, A/swine/Fujian/1/01 (SW/FJ/01) and A/swine/Fujian/1/03 (SW/FJ/03), from pigs in Fujian Province, southern China. Genetically, these two viruses are similar, although the NS gene of the SW/FJ/03 virus has a 15-nucleotide deletion at coding positions 612 to 626. The SW/FJ/01 virus is highly lethal for chickens, whereas the SW/FJ/03 virus is nonpathogenic for chickens when administrated intravenously or intranasally. To understand the molecular basis for the difference in virulence, we used reverse genetics to create a series of single-gene recombinants of both viruses. We found that a recombinant virus containing the mutated NS gene from the SW/FJ/03 virus in the SW/FJ/01 virus background was completely attenuated in chickens. We also found that viruses expressing the mutant NS1 protein of SW/FJ/03 did not antagonize the induction of interferon (IFN) protein. Conversely, only the recombinant virus containing the wild-type SW/FJ/01 NS gene in the SW/FJ/03 background was lethal in chickens and antagonized IFN protein levels. Further, we proved that the NS1 genes of the two viruses differ in their stabilities in the host cells and in their abilities to interact with the chicken cleavage and polyadenylation specificity factor. These results indicate that the deletion of amino acids 191 to 195 of the NS1 protein is critical for the attenuation of the SW/FJ/03 virus in chickens and that this deletion affects the ability of the virus to antagonize IFN induction in host cells.

Detection of antibodies against avian influenza virus in chickens in ninevah province iraq

Detection of antibodies against avian influenza virus in chickens in ninevah province iraq

The NS1 Gene Contributes to the Virulence of H5N1 Avian Influenza Viruses

In the present study, we explored the genetic basis underlying the virulence and host range of two H5N1 influenza viruses in chickens. A/goose/Guangdong/1/96 (GS/GD/1/96) is a highly pathogenic virus for chickens, whereas A/goose/Guangdong/2/96 (GS/GD/2/96) is unable to replicate in chickens. These two H5N1 viruses differ in sequence by only five amino acids mapping to the PA, NP, M1, and NS1 genes. We used reverse genetics to create four single-gene recombinants that contained one of the sequence-differing genes from nonpathogenic GS/GD/2/96 and the remaining seven gene segments from highly pathogenic GS/GD/1/96. We determined that the NS1 gene of GS/GD/2/96 inhibited the replication of GS/GD/1/96 in chickens, while the substitution of the PA, NP, or M gene did not change the highly pathogenic properties of GS/GD/1/96. Conversely, of the recombinant viruses generated in the GS/GD/2/96 background, only the virus containing the NS1 gene of GS/GD/1/96 was able to replicate and cause disease and death in chickens. The single-amino-acid difference in the sequence of these two NS1 genes resides at position 149. We demonstrate that a recombinant virus expressing the GS/GD/1/96 NS1 protein with Ala149 is able to antagonize the induction of interferon protein levels in chicken embryo fibroblasts (CEFs), but a recombinant virus carrying a Val149 substitution is not capable of the same effect. These results indicate that the NS1 gene is critical for the pathogenicity of avian influenza virus in chickens and that the amino acid residue Ala149 correlates with the ability of these viruses to antagonize interferon induction in CEFs.

Genetic Analysis of the Nonstructural (NS) Genes of H9N2 Chicken Influenza Viruses Isolated in China During 1998–2002

H9N2 subtype avian influenza viruses are widespread in domestic poultry. Genetic analysis indicated that three lineages of H9N2 viruses have been established in Eurasia and only one lineage is present on chicken farms in mainland China. Here, NS1 genes of eight H9N2 chicken influenza viruses, isolated in mainland China during 1998-2002, were completely sequenced and phylogenetically analyzed. By comparison, the homology of the NS1 of the A/chicken/Neimenggu/ZH/02 (Ck/NM/ZH/02) strain had a high identity (93.8%) with that of A/chicken/Korea/323/96 (Ck/Kor/323/96), which is an A/duck/Hong Kong/Y439/97 (Dk/HK/Y439/97)-like virus. NS1 peptides of seven strains possessed 217 amino acids, while that of the strain Ck/NM/ZH/02 coded 230 amino acids. Except for the amino acid at position 225, the additional amino acid sequence (13 AAs) of NS1 of Ck/NM/Zh/02 at the carboxy-terminus is identical with that of Ck/Kor/323/96. Phylogenetic analysis showed that seven of the tested strains belong to the A/duck/Hong Kong/Y280/97 (DK/HK/Y280/97)-like lineage, while the NS1 gene of Ck/NM/Zh/02 belongs to the Dk/HK/Y439/97-like lineage and has a close relationship with that of the Ck/Kor/323/96-like viruses. Therefore, although most of the H9N2 influenza viruses circulating on chicken farms in mainland China belong to the DK/HK/Y280/97-like lineage, the present results indicate that the other two of the three H9N2 lineage viruses also circulate in the chicken population in mainland China.

Molecular determinants within the surface proteins involved in the pathogenicity of H5N1 influenza viruses in chickens.

Although it is established that the cleavage site and glycosylation patterns in the hemagglutinin (HA) play important roles in determining the pathogenicity of H5 avian influenza viruses, some viruses exist that are not highly pathogenic despite possessing the known characteristics of high pathogenicity (i.e., their HA contains multiple basic amino acids at the cleavage site and has glycosylation patterns similar to that of the highly pathogenic H5 viruses). Currently little is known about the H5N1 viruses that fall into this intermediate category of pathogenicity. We have identified strains of H5N1 avian influenza viruses that have markers typical of high pathogenicity but distinctly differ in their ability to cause disease and death in chickens. By analyzing viruses constructed by reverse-genetic methods and containing recombinant HAs, we established that amino acids 97, 108, 126, 138, 212, and 217 of HA, in addition to those within the cleavage site, affect pathogenicity. Further investigation revealed that an additional glycosylation site within the neuraminidase (NA) protein globular head contributed to the high virulence of the H5N1 virus. Our findings are in agreement with previous observations that suggest that the activities of the HA and NA proteins are functionally linked.

Isolation and identification of swine influenza recombinant A/Swine/Shandong/1/2003(H9N2) virus

Ten influenza virus isolates were obtained from infected pigs from different places in Shandong province showing clinical symptoms from October 2002 to January 2003. All 10 isolates were identified in China's National Influenza Research Center as influenza A virus of H9N2 subtype. The complete genome of one isolate, designated A/Swine/Shandong/1/2003(H9N2), was sequenced and compared with sequences available in GenBank. The results of analyses indicated that the sequence of A/Swine/Shandong/1/2003(H9N2) was similar to those of several chicken influenza viruses and duck influenza viruses recently prevalent in South China. According to phylogenetic analysis of the complete gene sequences, A/Swine/Shandong/1/2003(H9N2) possibly originated from the reassortment of chicken influenza viruses and duck influenza viruses. It was found that the amino acid sequence at the HA cleavage site in Sw/SD/1/2003 is R-S-L-R-G, differing clearly from that of other H9N2 subtype isolates of swine influenza and avian influenza, which is R-S-S-R-G.

Molecular determinants of the pathogenicity of H5N1 influenza viruses in chickens

We have identified strains of H5N1 viruses that have typical markers of high pathogenicity, but distinctly differ in their ability to cause disease and death in chickens. To investigate the function of viral surface proteins in the pathogenicity of avian H5N1 viruses, we analyzed viruses constructed by reverse-genetic methods. Our results established that amino acid differences in the viral surface proteins play a significant role in pathogenicity.

Histopathological Study of Intratracheally Inoculated A/Chicken/Iran/259/1998 (H9N2) Influenza Virus in Chicken

Histopathological Study of Intratracheally Inoculated A/Chicken/Iran/259/1998 (H9N2) Influenza Virus in Chicken