Interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failures in sows and respiratory diseases in growing pigs, resulting in huge economic loss for the pig production worldwide. The nonstructural protein 9 (nsp9) and nonstructural protein 2 (nsp2) of PRRSV are known to play important roles in viral replication. Cellular interleukin-2 enhancer binding factor 2 (ILF2) participates in many cellular pathways and involves in life cycle of some viruses. In the present study, we analyzed the interaction of cellular ILF2 with the nsp9 and nsp2 of PRRSV in vitro and explored the effect of ILF2 on viral replication. METHODS: The interaction of ILF2 with the nsp9 or nsp2 of PRRSV was analyzed in 293FT cells and MARC-145 cells by co-immunoprecipitation (Co-IP) and the co-localization of ILF2 with the nsp9 or nsp2 of PRRSV in MARC-145 cell and pulmonary alveolar macrophages (PAMs) was examined by confocal immunofluorescence assay. The effect of ILF2 knockdown and over-expression on PRRSV replication was explored in MARC-145 cells by small interfering RNA (siRNA) and lentivirus transduction, respectively. RESULTS: The interaction of ILF2 with nsp9 or nsp2 was first demonstrated in 293FT cells co-transfected with ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while three truncated nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs. Finally, our analysis indicated that knockdown of ILF2 favored the replication of PRRSV, while over-expression of ILF2 impaired the viral replication in MARC-145 cells. CONCLUSION: Our findings are the first to confirm that the porcine ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV. Our present study provides more evidence for understanding the roles of the interactions between cellular proteins and viral proteins in the replication of PRRSV.

Genetic characterization of two novel megriviruses in geese.

Two goose megriviruses (W18 and HN56) were detected and sequenced. Both viruses possessed megrivirus-like genomic features, including unusually long genomes (9840 and 10 101 nt). W18 and HN56 were very similar to duck megrivirus (DMV) in the P2 and P3 regions, but much less similar in the P1 and 2A1 regions. HN56 may be a potential recombinant virus, with a distinct P1 region possibly originating from an unknown picornavirus. W18 may represent a novel type of DMV, showing a P1 sequence identity of 67 %. Similar levels (64-68 %) of P1 sequence identity were also displayed by melegrivirus A with W18 and DMV, demonstrating an equal genetic separation of the capsid region among W18, DMV and melegrivirus A. For the 2A1 region, the divergence among W18, HN56 and DMV was remarkable, involving point mutations and a long insertion/deletion. The present work contributes to the understanding of unique features and phylogeny of megriviruses.

Isolation, identification, and whole genome sequencing of reticuloendotheliosis virus from a vaccine against Marek's disease.

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.

The DEAD-box RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9 in vitro.

The nonstructural protein 9 (Nsp9) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to play important roles in viral replication. The present study first screened that the DEAD-box RNA helicase 5 (DDX5) was a cellular protein interacting with the Nsp9 of PRRSV by a yeast two-hybrid method in a pulmonary alveolar macrophages (PAMs) cDNA library. Next, DDX5 was shown to interact with viral Nsp9 in the co-transfected HEK293 cells with the DDX5- and Nsp9-expressing plasmids, and the interaction between endogenous DDX5 and Nsp9 was also confirmed in MARC-145 cells infected with the Nsp9-expressing lentiviruses. Then, the interacting domains between DDX5 and Nsp9 were determined to be the DEXDc and HELICc domains in DDX5 and the RdRp domain in Nsp9, respectively. Moreover, in the HEK293 cells, MARC-145 cells and PAM cell lines co-transfected with the DDX5- and Nsp9-expressing plasmids, Nsp9 was shown to co-localize with DDX5 in the cytoplasm with a perinuclear pattern, and meanwhile in PRRSV-infected MARC-145 cells and PAMs, endogenous DDX5 was also found to co-localize with Nsp9. Finally, silencing the DDX5 gene in MARC-145 cells significantly impacted the replication of PRRSV, and while the over-expression of DDX5 could slightly enhance viral replication. These findings indicate that DDX5 positively regulates the replication of PRRSV via its interaction with viral Nsp9 in vitro.

Biological properties of a duck enteritis virus attenuated via serial passaging in chick embryo fibroblasts.

To gain a better understanding of the genetic changes required for attenuation of duck enteritis virus (DEV), the Chinese standard challenge strain of DEV (DEV CSC) was serially passaged 80 times in chick embryo fibroblasts. We plaque-purified the virus after the 25th passage (DEV p25) and the 80th passage (DEV p80) and investigated its in vitro and in vivo properties. Average plaque sizes for DEV p25 and p80 were significantly smaller than those for their parental DEV CSC. The results from an in vivo experiment revealed that DEV p25 and p80 were avirulent in ducks and protected them from virulent DEV challenge. The complete genome sequence of DEV p80 was determined and compared with that of the parent virus. An 1801-bp deletion was identified in the genome of DEV p80, which affected the genes encoding gI and gE. Moreover, there were 11 base substitutions, which led to seven amino acid changes in open reading frames LORF9, UL51, UL9, UL7, UL4, ICP4 and US3. Further DNA sequence analysis showed that the 1801-bp deletion was also present in DEV p25. Our findings suggest that DEV gE and/or gI are nonessential for virus growth and might, as with other herpesviruses, play an important role in cell-to-cell spread and virulence. Our experiments provide more genetic information about DEV attenuation and further advance our understanding of the molecular basis of DEV pathogenesis.

The risk factors for avian influenza on poultry farms: a meta-analysis.

BACKGROUND: Avian influenza is a severe threat both to humans and poultry, but so far, no systematic review on the identification and evaluation of the risk factors of avian influenza infection has been published. The objective of this meta-analysis is to provide evidence for decision-making and further research on AI prevention through identifying the risk factors associated with AI infection on poultry farms. METHODS: The results from 15 selected studies on risk factors for AI infections on poultry farms were analyzed quantitatively by meta-analysis. RESULTS: Open water source (OR=2.89), infections on nearby farms (OR=4.54), other livestock (OR=1.90) and disinfection of farm (OR=0.54) have significant association with AI infection on poultry farms. The subgroup analysis results indicate that there exist different risk factors for AI infections in different types of farms. CONCLUSIONS: The main risk factors for AI infection in poultry farms are environmental conditions (open water source, infections on nearby farms), keeping other livestock on the same farm and no disinfection of the farm.

Interactome profile of the host cellular proteins and the nonstructural protein 2 of porcine reproductive and respiratory syndrome virus.

The nonstructural protein 2 (NSP2) is considered to be one of crucial viral proteins in the replication and pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV). In the present study, the host cellular proteins that interact with the NSP2 of PRRSV were immunoprecipitated with anti-Myc antibody from the MARC-145 cells infected by a recombinant PRRSV with 3xMyc tag insertion in its NSP2-coding region, and then 285 cellular proteins interacting with NSP2 were identified by LC-MS/MS. The Gene Ontology and enriched KEGG Pathway bioinformatics analyses indicated that the identified proteins could be assigned to different subcellular locations and functional classes. Functional analysis of the interactome profile highlighted cellular pathways associated with infectious disease, translation, immune system, nervous system and signal transduction. Two interested cellular proteins-BCL2-associated athanogene 6 (BAG6) and apoptosis-inducing factor 1 (AIF1) which may involve in transporting of NSP2 to Endoplasmic reticulum (ER) or PRRSV-driven apoptosis were validated by Western blot. The interactome data between PRRSV NSP2 and cellular proteins contribute to the understanding of the roles of NSP2 in the replication and pathogenesis of PRRSV, and also provide novel cellular target proteins for elucidating the associated molecular mechanisms of the interaction of host cellular proteins with viral proteins in regulating the viral replication.

Comparative genomic sequence analysis between a standard challenge strain and a vaccine strain of duck enteritis virus in China.

Here, we present the complete genomic sequence of the Chinese standard challenge strain (CSC) of duck enteritis virus (DEV), which was isolated in China in 1962. The DEV CSC genome is 162,131 bp long and contains 78 predicted open reading frames (ORFs). Comparison of the genomic sequences of DEV CSC and DEV live vaccine strain K at passage 63 (DEV K p63) revealed that the DEV CSC genome is 4,040 bp longer than the DEV K p63 genome, mainly because of 3,513-bp and 528-bp insertions at the 5' and 3' ends of the unique long segment, respectively. At the nucleotide level, 63 of the 76 ORFs in the DEV CSC genome were 100 % identical to the ORFs in the DEV K p63 genome. Two ORFs (UL56 and US10) had frameshift mutations in the C-terminal regions, while LORF5 was unique to the DEV K p63 genome. It is difficult to assign attenuated virulence to changes in specific genes. However, the complete DEV CSC genome will further advance our understanding of the genes involved in virulence and evolution. The DEV CSC genome sequence has been deposited in GenBank under accession number JQ673560.

Transcription factor regulation and cytokine expression following in vitro infection of primary chicken cell culture with low pathogenic avian influenza virus.

BACKGROUND: Avian influenza virus (AIV) induced proinflammatory cytokine expression is believed to contribute to the disease pathogenesis following infection of poultry. However, there is limited information on the avian immune response to infection with low pathogenic avian influenza virus (LPAIV). METHODS: To gain a better understanding of the early viral-host interactions of LPAIV in chickens, primary chicken embryo hepatocytes (CEH) were infected with four different LPAIVs of U.S. origin. Kinetics of virus replication, transcription factor (c-Jun, p50 and IRF-3) activation and immune response gene (IL-6, IL-1beta, IFN-alpha and Mx) expression were studied at four different time points (6, 12, 24 and 48 hours) post infection and compared to non-infected controls. RESULTS: CEH can support growth of the tested LPAIVs when with trypsin supplementation. All four immune response genes tested were upregulated following infection as were transcription factors c-Jun, p50 and IRF-3. Amplification of these genes was dependant on virus replication (e.g. inclusion of trypsin), such that immune response genes and transcription factors were upregulated as viral titers increased. CONCLUSION: The results of these studies demonstrate the requirement of virus replication for innate immune regulation and broaden our understanding of transcription factor responses related to LPAIV infection in chickens.

Complete Genome Sequence of Reticuloendotheliosis Virus Strain MD-2, Isolated from a Contaminated Turkey Herpesvirus Vaccine.

Here, we present the complete genomic sequence of a reticuloendotheliosis virus (REV) isolated from a contaminated turkey herpesvirus (HVT) vaccine. This report will be helpful for epidemiological studies on REV infection in avian flocks.

Complete genome sequence of an attenuated duck enteritis virus obtained by in vitro serial passage.

Here, we present the complete genome sequence of an attenuated duck enteritis virus (DEV) obtained by serial chicken embryo passage. Compared with a virulent DEV, there is a serial deletion in unique long open reading frame 11 (LORF11) and unique long region 2 (UL2). This study will aid in further exploration of the molecular pathogenesis of DEV.

Subgroup J avian leukosis virus infection inhibits autophagy in DF-1 cells.

BACKGROUND: Subgroup J avian leukosis virus (ALV-J) infection can induce tumor-related diseases in chickens. Previous studies by our laboratory demonstrated that ALV-J infection of DF-1 cells resulted in altered activity and phosphorylation of AKT. However, little is known about the subsequent activation of host DF-1 cells. RESULTS: In the current study, autophagy inhibition was observed for ALV-J infected DF-1 cells. Our data showed that the autophagosome protein, microtubule-associated protein 1 light chain 3-II (LC3-II), was reduced considerably in DF-1 cells infected with active ALV-J, while no change was observed for cells infected with inactivated ALV-J. Autophagy inhibition was also confirmed by fluorescence microscopy and transmission electron microscopy. Interestingly, when autophagy was promoted by rapamycin, the titers of ALV-J replication were decreased, and the replication level of ALV-J was significantly enhanced when atg5 (autophagy-related gene 5) was knocked out. CONCLUSIONS: These results suggested that ALV-J infection could down-regulate autophagy in DF-1 cells during viral replication. This study is the first to report on the relationship between ALV-J infection and autophagy in DF-1 cells.

Rapid detection of newly isolated Tembusu-related Flavivirus by reverse-transcription loop-mediated isothermal amplification assay.

BACKGROUND: From April 2010 to January 2011, a severe new viral disease had devastated most duck-farming regions in China. This disease affected not only laying ducks but also meat ducks, causing huge economic losses for the poultry industry. The objective of this study is to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of the new virus related to Tembusu-related Flavivirus. RESULTS: The RT-LAMP assay is very simple and rapid, and the amplification can be completed within 50 min under isothermal conditions at 63°C by a set of 6 primers targeting the E gene based on the sequences analysis of the newly isolated viruses and other closely related Flavivirus.The monitoring of gene amplification can also be visualized by using SYBR green I fluorescent dye. In addition, the RT-LAMP assay for newly isolated Tembusu-related Flavivirus showed higher sensitivity with an RNA detection-limit of 2 copies/µL compared with 190 copies/µL of the conventional RT-PCR method. The specificity was identified without cross reaction to other common avian pathogens. By screening a panel of clinical samples this method was more feasible in clinical settings and there was higher positive coincidence rate than conventional RT-PCR and virus isolation. CONCLUSION: The RT-LAMP assay for newly isolated Tembusu-related Flavivirus is a valuable tool for the rapid and real-time detection not only in well-equipped laboratories but also in general conditions.

Isolation and characterization of emerging subgroup J avian leukosis virus associated with hemangioma in egg-type chickens.

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.

The NS segment of an H5N1 highly pathogenic avian influenza virus (HPAIV) is sufficient to alter replication efficiency, cell tropism, and host range of an H7N1 HPAIV.

A reassortant avian influenza virus (designated FPV NS GD), carrying the NS-segment of the highly pathogenic avian influenza virus (HPAIV) strain A/Goose/Guangdong/1/96 (GD; H5N1) in the genetic background of the HPAIV strain A/FPV/Rostock/34 (FPV; H7N1), was rescued by reverse genetics. Remarkably, in contrast to the recombinant wild-type FPV (rFPV), the reassortant virus was able to replicate more efficiently in different human cell lines and primary mouse epithelia cells without prior adaptation. Moreover, FPV NS GD caused disease and death in experimentally infected mice and was detected in mouse lungs; in contrast, rFPV was not able to replicate in mice effectively. These results indicated an altered host range and increased virulence. Furthermore FPV NS GD showed pronounced pathogenicity in chicken embryos. In an attempt to define the molecular basis for the apparent differences, we determined that NS1 proteins of the H5N1 and H7N1 strains bound the antiviral kinase PKR and the F2F3 domain of cleavage and polyadenylation specificity factor 30 (CPSF30) with comparable efficiencies in vitro. However, FPV NS GD infection resulted in (i) increased expression of NS1, (ii) faster and stronger PKR inhibition, and (iii) stronger beta interferon promoter inhibition than rFPV. Taken together, the results shed further light on the importance of the NS segment of an H5N1 strain for viral replication, molecular pathogenicity, and host range of HPAIVs and the possible consequences of a reassortment between naturally occurring H7 and H5 type HPAIVs.

Recombinant fowlpox virus vector-based vaccine completely protects chickens from H5N1 avian influenza virus.

With the widespread presence of influenza virus H5N1 in poultry and wildlife species, particularly migrating birds, vaccination has become an important control strategy for avian influenza (AI). In this study, the immune efficacy and hemagglutination inhibition (HI) antibody responses induced by a recombinant fowlpox virus (FPV) vector-based rFPV-HA-NA vaccine was evaluated in SPF and commercial chickens. Four-week old SPF chickens vaccinated with one dose of vaccine containing 2 x 10(3) plaque forming units (PFU) of virus were completely protected from H5N1 AI virus 1 week after vaccination, and protective immunity lasted for at least 40 weeks. Two-week old commercial layer chickens were vaccinated with the rFPV-HA-NA vaccine and boosted with the same dose of vaccine following an interval of 18 weeks. The HI antibody titers higher than 4log2 lasted for 52 weeks after the booster immunization. We also examined the efficacy of the rFPV-HA-NA vaccine in SPF chickens administrated by different routes. The results showed that effective application of rFPV-HA-NA vaccine in poultry may be restricted to wing-web puncture, intramuscular or subcutaneous injection. These results demonstrate that the rFPV-HA-NA vaccine is effective in the prevention of infection of H5N1 AI virus.

H5N1 influenza marker vaccine for serological differentiation between vaccinated and infected chickens.

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.

Specific small interfering RNAs-mediated inhibition of replication of porcine encephalomyocarditis virus in BHK-21 cells.

Encephalomyocarditis virus (EMCV) is recognized as a pathogen inducing acute myocarditis and sudden death in preweaned piglets and severe reproductive failure in sows. In this study, eight specific small interfering RNA (siRNA) duplexes targeting different genomic regions of EMCV BJC3 were designed and their ability to inhibit virus replication in BHK-21 cells was investigated. The results showed that BHK-21 cells transfected with siRNA duplexes to 2C gene (JH-4,666, BJC-1,739), 2B gene (BJC-807), 3C gene (BJC-2,363) and 3D gene (BJC-3269) were specifically resistant to EMCV infection when exposed to 500 times the 50% cell culture infective dose (CCID(50)) of EMCV. The levels of the 3D gene in the transfected cells were obviously decreased. IFA and Western blotting analysis confirmed that the expression of VP1 protein in cell culture transfected with the siRNAs was apparently reduced. Of the five siRNAs, JH-4,666, BJC-2,363 and BJC-3,269 were the most effective. Combination of the siRNA duplexes enhanced the inhibition of EMCV replication. Our data indicated that specific siRNAs are able to inhibit the replication of porcine encephalomyocarditis virus in BHK-21 cells, suggesting that RNAi might provide a new approach to prevent EMCV infection.

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.

Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector.

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.