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According to the requirements of the Ministry of Agriculture of China, all vaccines must be screened for exogenous virus contamination before commercialization. A freeze-dried vaccine against Marek's disease was used to inoculate specific pathogen-free chickens, from which serum samples were collected after 42 days. The results were positive for reticuloendotheliosis virus antibody, which was indicative of reticuloendotheliosis virus contamination. After neutralization with serum positive for Marek's disease virus, chicken embryo fibroblasts were inoculated with the vaccine. Afterward, viral isolation and identification were performed. One reticuloendotheliosis virus strain (MD-2) was isolated and verified using an immunofluorescence assay. Polymerase chain reaction amplification of the provirus MD-2 genome was performed using seven overlapping fragments as primers. The amplified products were sequenced and spliced to obtain the whole MD-2 genome sequence. The full genome length of MD-2 was 8,284 bp, which had an identity greater than 99% with the prairie chicken isolate APC-566 from the US, the goose-derived isolate 3410/06 from Taiwan, and the chicken-derived reticuloendotheliosis virus isolate HLJR0901 from Heilongjiang Province, China. The MD-2 was phylogenetically close to these isolates. The identity with REV isolate HA9901 from Jiangsu Province of China was 96.7%. The MD-2 had the lowest identity with duck-derived Sin Nombre virus from the United States, with the value of only 93.5%. The main difference lay in the U3 region of the long terminal repeat. The present research indicated that some vaccines produced during specific periods in China might be contaminated by reticuloendotheliosis virus. The reticuloendotheliosis virus strain isolated from the vaccine was phylogenetically close to the prevalent strain, with only minor variations., (© 2015 Poultry Science Association Inc.)

Subgroup J avian leukosis virus (ALV-J), first isolated in 1989, predominantly causes myeloid leukosis (ML) in meat-type or egg-type chicken. Since 2006, the clinical cases of hemangioma rather than ML in commercial layer flocks associated with ALV-J have been reported, but it was still not clear whether the novel oncogenic ALV-J had emerged. We characterized SCAU-HN06 isolate of ALV-J from hemangioma in commercial Roman layers through animal experiment and full-length proviral genome sequence analysis. The SPF white leghorn egg-type chickens infected with SCAU-HN06 in ovo at day 11 of incubation showed an overall incidence of 56% hemangioma and 8% renal tumor throughout the 22-week trial, the mortality rate was 16%. Most genes of SCAU-HN06 isolate showed high nucleotide sequence identity to JS09GY6 which was isolated from Hy-Line Variety Brown layers suffering hemangioma. The 19-bp insertion in leader sequence and one key deletion in E element were the common features of SCAU-HN06 and JS09GY6. SCAU-HN06 and those ALV-Js associated with hemangioma, possibly recombinants of ALV-J and other avian retrovirus, may share the same ancestor., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Using plasmid-based reverse genetics, we generated a molecularly altered virus, H5N1/PR8-5B19, containing modified HA and NA genes from A/Goose/Guangdong/1/96 (GS/GD/1/96). In the H5N1/PR8-5B19 virus, the HA cleavage site was modified to resemble that of low-pathogenic avian strains and a portion of the NA stalk region was replaced by the immunodominant 5B19 epitope of the S2 glycoprotein of murine hepatitis virus (MHV). H5N1/PR8-5B19 is not lethal to embryonated eggs or chickens. Chickens immunized with the H5N1/PR8-5B19 inactivated vaccine produced high levels of HI antibody and a measurable antibody response against the MHV 5B19 epitope, and were fully protected against subsequent challenge with different highly pathogenic H5N1 avian influenza viruses. H5N1/PR8-5B19 is therefore an attractive marker vaccine candidate, eliciting a strong, protective antibody response and enabling serological discrimination between vaccinated and wild-type virus-infected chickens.

H5N1 influenza viruses have caused significant disease and deaths in various parts of the world in several species, including humans. Vaccination combined with culling can provide an attractive method for outbreak containment. Using synthesized oligos and overlapping extension PCR techniques, we constructed an H5 HA gene, optiHA, containing chicken biased codons based on the HA amino acid sequence of the highly pathogenic H5N1 virus A/goose/Guangdong/1/96 (GS/GD/96). The optiHA and wild-type HA genes were inserted into plasmids pCI or pCAGGS, and designated as pCIoptiHA, pCAGGoptiHA, pCIHA and pCAGGHA, respectively. To evaluate vaccine efficacy, groups of 3-week-old specific pathogen free (SPF) chickens were intramuscularly injected with the four plasmids. Sera were collected on a weekly basis post-vaccination (p.v.) for hemagglutination inhibition (HI) assays and neutralization (NT) antibody detection. All chickens receiving pCAGGoptiHA and pCAGGHA developed high levels of HI and NT antibodies at 3 weeks p.v., and were completely protected from lethal H5 virus challenge, while only partial protection was induced by inoculation with the other two plasmids. A second experiment was conducted to evaluate if a lower dose of the pCAGGoptiHA vaccine could be effective, results indicated that two doses of 10 microg of pCAGGoptiHA could induce complete protection in chickens against H5 lethal virus challenge. Based on our results, we conclude that construction optimization could dramatically increase the H5 HA gene DNA vaccine efficacy in chickens, and therefore, greatly decrease the dose necessary for inducing complete protection in chickens.

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.

Since the first detection of highly pathogenic H5N1 avian influenza virus from sick goose in Guangdong province in China in 1996, scientists in China started to develop vaccines for avian influenza pandemic preparedness. An H5N2 inactivated vaccine was produced from a low pathogenic virus, A/turkey/England/N-28/73, and was used for the buffer zone vaccination in the H5N1 outbreaks in 2004 in China. We also generated a low pathogenic H5N1 reassortant virus A/Harbin/Re-1/2003 (Re-1) that derives its HA and NA genes from GSGD/96 virus and six internal genes from the high-growth A/Puerto Rico/8/34 (PR8) virus by using plasmid-based reverse genetics. The inactivated vaccine derived from Re-1 strain could induce more than 10 months protective immune response in chickens after one dose inoculation, and most importantly, this vaccine is immunogenic for geese and ducks. An H5N1 fowlpox vectored live vaccine was also generated by inserting the HA and NA genes of GSGD/96 virus in the genome of a fowlpox vaccine strain. Laboratory tests indicated that after one dose of immunization of this vaccine, chickens could develop an over than 40 weeks protective immune response against H5N1 virus challenge.

An H7N2 avian influenza virus was isolated from chickens during routine surveillance in northern China in 2002. To understand the origin of this virus, we completely sequenced its genome. The PB1, PA, HA, and M genes of this virus were highly homologous with those of the wild bird virus A/Africa starling/Eng-Q/983/79 (H7N1). The NP and NS genes were closely related to those of two other wild bird viruses isolated 30 years ago. The closest relatives of the PB2 and NA genes of the virus were those of the A/swine/Germany/2/81 (H1NI) and A/Leningrad/134/57 (H2N2), respectively. Animal inoculation tests showed that the virus cannot replicate efficiently in chickens. However, after intranasal inoculation, the virus induced 20% weight loss and replicated well in the lungs of mice. The virus was also recovered from the hearts and brains of the mice. These results suggest that the influenza virus isolated in chickens in northern China in 2002 originated in wild birds and may pose a threat for both avian species and mammalian hosts.

Abstract: We generated a high-growth H5N1/PR8 virus by plasmid-based reverse genetics. The virulence associated multiple basic amino acids of the HA gene were removed, and the resulting virus is attenuated for chickens and chicken eggs. A formalin-inactivated oil-emulsion vaccine was prepared from this virus. When SPF chickens were inoculated with 0.3 ml of the vaccine, the hemagglutinin-inhibition (HI) antibody became detectable at 1 week post-vaccination (p.v.) and reached a peak of 10log2 at 6 weeks p.v. then slowly declined to 4log2 at 43 weeks p.v. Challenge studies performed at 2, 3 and 43 weeks p.v. indicated that all of the chickens were completely protected from disease signs and death. Ducks and geese were completely protected from highly pathogenic H5N1 virus challenge 3 weeks p.v. The duration of protective immunity in ducks and geese was investigated by detecting the HI antibody of the field vaccinated birds, and the results indicated that 3 doses of the vaccine inoculation in geese could induce a 34 weeks protection, while 2 doses induced more than 52 weeks protection in ducks. We first reported that an oil-emulsion inactivated vaccine derived from a high-growth H5N1 vaccine induced approximately 10 months of protective immunity in chickens and demonstrated that the oil-emulsion inactivated avian influenza vaccine is immunogenic for geese and ducks. These results provide useful information for the application of vaccines to the control of H5N1 avian influenza in poultry, including chickens and domestic waterfowl. [Copyright &y& Elsevier]