Transcription phase, protein characteristics of DEV UL45 and prokaryotic expression, antibody preparation of the UL45 des-transmembrane domain

<p>Abstract</p> <p>Background</p> <p>Some UL45 gene function of Herpesvirus was reported. While there was no any report of the duck enteritis virus (DEV) UL45 protein as yet.</p> <p>Results</p> <p>The UL45 gene and des-transmembrane domain of UL45 (named UL45Δ gene, 295-675bp of UL45) of DEV were amplified by PCR and subcloned into the prokaryotic expression vector pET-32a(+). The constructed recombinant plasmids were transformed into the host strain BL21(DE3) PLysS and induced by IPTG. SDS-PAGE analysis showed the UL45 gene couldn't express while UL45Δ gene was highly expressed. His Purify Kit or salting-out could purify the protein effectively. Using the purified protein to immunize New-Zealand rabbits and produce polyclonal antibody. The agar diffusion reaction showed the titer of antibody was 1:32. Western blot analysis indicated the purified rabbit anti-UL45Δ IgG had a high level of specificity and the UL45 gene was a part of DEV genome. The transcription phase study of UL45 gene showed that expression of UL45 mRNA was at a low level from 0 to 18 h post-infection (pi), then accumulated quickly at 24 h pi and peaked at 42 h pi. It can be detected till 72 h pi. Besides, western blot analysis of purified virion and different viral ingredients showed that the UL45 protein resided in the purified virion and the viral envelope.</p> <p>Conclusions</p> <p>The rabbit anti-UL45Δ IgG was produced successfully and it can serve as a good tool for penetrating studies of the function of DEV UL45 protein. The transcription phase and protein characteristics analysis indicated that DEV UL45 gene was a late gene and UL45 protein may be a viral envelope protein.</p

Characterization of the duck enteritis virus UL55 protein

<p>Abstract</p> <p>Background</p> <p>Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function.</p> <p>Results</p> <p>The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells.</p> <p>Conclusions</p> <p>In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene.</p

Cloning, expression and characterization of gE protein of Duck plague virus

<p>Abstract</p> <p>Background</p> <p>The gE protein of duck plague virus is the important membrane glycoprotein, its protein characterization has not been reported. In this study, we expressed and presented the characterization of the DPV gE product.</p> <p>Results</p> <p>According to the sequence of the gE gene, a pair of primers were designed, and the DNA product with 1490bp in size was amplified by using the polymerase chain reaction (PCR). The PCR product was cloned into pMD18-T vector, and subcloned into pET32a(+), generating the recombinant plasmid pET32a/DPV-gE. SDS-PAGE analysis showed that the fusion pET32a/DPV-gE protein was highly expressed after induction by 0.2 mM IPTG at 30°C for 4.5 h in Rosseta host cells. Over expressed 6×His-gE fusion protein was purified by nickel affinity chromatography, and used to immunize the rabbits for the preparation of polyclonal antibody. The result of the intracellular localization revealed that the gE protein was appeared to be in the cytoplasm region. The real time PCR, RT-PCR analysis and Western blotting revealed that the gE gene was produced most abundantly during the late phase of replication in DPV-infected cells.</p> <p>Conclusions</p> <p>In this work, the DPV gE protein was successfully expressed in a prokaryotic expression system, and we presented the basic properties of the DPV gE product for the first time. These properties of the gE protein provided a prerequisite for further functional analysis of this gene.</p

Host-Virus Interactions of Infectious Laryngotracheitis Virus Infection in Cultured Cells

Infectious laryngotracheitis virus (ILTV; Gallid herpesvirus 1) causes upper respiratory diseases in mainly chickens and exhibits 90-100% of high morbidity and up to 70% of mortality, resulting in huge economic losses in the poultry industry worldwide. To study host-ILTV interactions, the changes in genome-wide gene expressions in response to wild-type and vaccine ILTV infections in primary chicken embryo lung cells were investigated using microarray analysis. Results provide crucial insights into host cell pathogenic and immunogenic responses against wild-type and vaccine ILTV infections. Using microarray method and Ingenuity Pathway Analysis (IPA) bioinformatics tool, 273 and 306 differentially expressed genes were identified responding to wild-type and vaccine ILTV infections, respectively. Further integrated analysis to compare differentially expressed genes revealed that eight host genes including coagulation factor II (thrombin) receptor-like 1 (F2RL1), bone morphogenetic protein 2 (BMP2), inhibitor of NF-kB (IkB) kinase subunit beta (IKBKB) interacting protein (IKBIP), thymidylate synthetase (TYMS), chromosome 8 open reading frame 79 (C8orf79), coagulation factor X (F10), prostaglandin-endoperoxide synthase 2 (PTGS2) and neuropeptide Y (NPY) were regulated differently between wild-type and vaccine ILTV infections in an opposite direction, suggesting that these host factors may play important roles in host immune responses against ILTV infection. In addition, the transcriptome changes of ILTV encoding genes were studied during infection time courses using quantitative PCR. In this study, infected-cells polypeptide (ICP) 4 showed the highest expression level and UL21 and UL42 showed unique expression patterns, unlike most of the other ILTV gene which exhibited continuous elevation of expression during lytic infection. Kinetic analysis of ILTV gene expression in host cells may provide new knowledge to understand ILTV pathogenesis

Development of antibody therapeutic approaches for poultry diseases using avian influenza as a disease model

One of the main threats to poultry is avian influenza virus (AIV), causing significant economic losses worldwide and threatening human populations due to its zoonotic potential. To reduce disease impact, vaccination of poultry is carried out; however, most of the vaccines are insufficient to induce sterile immunity, leading to enzootic disease prevalence worldwide. In addition, due to virus evolution, new virus variants continuously arise, further compromising vaccine effectiveness. The aim of this study was to assess if monoclonal antibodies could be used as prophylactic treatment to reduce avian influenza disease impact as an alternative to vaccination in emergency situations, and to investigate approaches which could be employed for delivery of antibodies as antiviral therapeutics for poultry. A panel of monoclonal antibodies, specific to the AIV H9N2 subtype (A/chicken/Pakistan/UDL-01/2008) (UDL-1/08) major antigenic surface glycoprotein hemagglutinin (HA), generated from mouse hybridomas, were gene sequenced for their variable domains that were subsequently used for recombinant antibody production in cultured cell supernatants. Functional activities (HA binding affinity and AIV neutralizing activity) of the recombinant antibodies were evaluated against homologous and heterologous viruses. Three antibodies retained functional activity matching that of the natural antibody isotype after conversion to single chain variable fragment (scFv) format, suggesting the antibody fragment crystallizable (Fc) region did not mediate function for these antibodies, but that function was dependent on direct antigen recognition. Next, scFv antibodies were chosen for passive immunization purposes in vivo due to their small molecule size and potentially reduced immunogenicity. scFvs were administered to birds intranasally 24 h before challenge with H9N2 AIV representative UDL-1/08 and treatment was continued for seven days post-infection. Results indicated reduced morbidity and virus shedding in treated birds. Moreover, compared to non-treated birds, treated chickens also produced lower levels of IL-6, a known pro-inflammatory cytokine induced in response to virus infection. This data suggests treated birds experienced overall reduced impact of disease. Nevertheless, like in vivo vaccine induced antibodies, the antibody treatment also provoked the virus to generate HA antibody escape mutants likely to overcome the neutralizing activity of therapeutic antibodies. Finally, this study investigated if herpesvirus of turkeys (HVT), which is used as a viral vaccine vector in poultry, could act as a vector for therapeutic antibody delivery to poultry. A recombinant virus encoding a transgene of a broadly AIV-neutralizing antibody was generated using a CRISPR/Cas9 approach. It was found that antibody gene insertion into HVT altered recombinant virus growth kinetics, resulting in reduced replication when compared to a wildtype virus control. Antibody levels secreted in rHVT infected cell culture supernatant were retained after 20 virus passages. Next, to investigate antibody expression and tolerability in vivo, rHVT was administered to day-old birds; however, no detectable systemic antibody circulation was identified throughout 42-days post rHVT delivery. Instead, an anti-antibody response was generated, suggesting only a low level of expression occurred that was sufficient to act as an antigen. Taken together, this work has built a proof-of-concept suggesting that passive immunization for poultry can reduce weight loss in infected birds and overall disease burden, but selection of antibodies targeting different antigens and epitopes is crucial to avoid virus escape mutant formation. For the first time it was also demonstrated that HVT can act as an efficient vector for antigen but not antibody delivery. This information can be relevant not only to AIV but also other pathogens affecting poultry