Expression and characterization of duck enteritis virus gI gene

<p>Abstract</p> <p>Background</p> <p>At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein.</p> <p>Results</p> <p>The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into <it>E.coli </it>BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-β-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively.</p> <p>Conclusions</p> <p>The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by <it>E.coli </it>BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.</p

Different linkages in the long and short regions of the genomes of duck enteritis virus Clone-03 and VAC Strains

<p>Abstract</p> <p>Background</p> <p>Duck enteritis virus (DEV) is an unassigned member in the family <it>Herpesviridae</it>. To demonstrate further the evolutionary position of DEV in the family <it>Herpesviridae</it>, we have described a 42,897-bp fragment. We demonstrated novel genomic organization at one end of the long (L) region and in the entire short (S) region in the Clone-03 strain of DEV.</p> <p>Results</p> <p>A 42,897-bp fragment located downstream of the <it>LOFR11 </it>gene was amplified from the Clone-03 strain of DEV by using 'targeted gene walking PCR'. Twenty-two open reading frames (ORFs) were predicted and determined in the following order: 5'<it>-LORF11-RLORF1</it>-<it>ORF1</it>-<it>ICP4</it>-<it>S1-S2-US1-US10-SORF3-US2-MDV091.5-like-US3-US4-US5-US6-US7-US8-ORFx-US1-S2-S1-ICP4 </it>-3'. This was different from that of the published VAC strain, both in the linkage of the L region and S region, and in the length of the US10 and US7 proteins. The <it>MDV091.5-like </it>gene, <it>ORFx </it>gene, <it>S1 </it>gene and <it>S2 </it>gene were first observed in the DEV genome. The lengths of DEV US10 and US7 were determined to be 311 and 371 amino acids, respectively, in the Clone-03 strain of DEV, and these were different from those of other strains. The comparison of genomic organization in the fragment studied herein with those of other herpesviruses showed that DEV possesses some unique characteristics, such as the duplicated US1 at each end of the US region, and the US5, which showed no homology with those of other herpesviruses. In addition, the results of phylogenetic analysis of ORFs in the represented fragment indicated that DEV is closest to its counterparts VZV (<it>Varicellovirus</it>) and other avian herpesviruses.</p> <p>Conclusion</p> <p>The molecular characteristics of the 42,897-bp fragment of Clone-03 have been found to be different from those of the VAC strain. The phylogenetic analysis of genes in this region showed that DEV should be a separate member of the subfamily <it>Alphaherpesvirinae</it>.</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

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

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

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

Antiviral chemotherapeutic agents against equine herpesvirus type 1: the mechanism of antiviral effects of porphyrin derivatives

Equine Herpesvirus-1 (EHV-1) is an important ubiquitous enzootic equine pathogen, causing significant economic losses to the horse industry. Despite extensive vaccination protocols, EHV-1 continues to be a major cause of epidemic abortion, perinatal mortality, respiratory disease and neurologic disease. EHV-1 infections are usually dealt with by using management practices that limit spread of the disease and secondary complications, providing symptomatic relief to infected horses, but no specific treatment is available. New therapeutic or virucidal agents could have great utility in slowing both the progression and spread of the disease in an epidemic situation. A number of porphyrins and their derivatives have been tested to have activity against HIV, vaccinia, and coronavirus. Porphyrin based compounds were suggested to inhibit virus infection by reducing the fusogenic potential of the virus (Vzorov et al., 2002). However, the mechanism of action of porphyrin-based compounds is not well understood. While current antiherpetic agents target viral DNA replication, interference with the upstream replicative events such as fusion would not adversely affect the host cell metabolism, and makes them important targets for chemotherapeutic intervention of virus dissemination. We screened a number of porphyrin and platinum compounds for EHV-1 antiviral activity by testing their ability to interfere with EHV-1 infection of rabbit kidney and equine cell cultures during the entry and post entry events of the viral life cycle in order to determine if compounds act at the level of binding, penetration, replication, or egress. We identified Cu (III) tetrasulfonated phenylporphyrin and Fe (II) tetrasulfonated phenylporphyrin as lead candidate antiviral compounds on the basis of their in vitro efficacy, cytotoxicity and therapeutic index. These compounds exhibited high antiviral potency during virus-to-cell fusion events, as well as no apparent cytotoxicity in cell culture assays at EHV-1 inhibitory concentrations. Specifically, selected porphyrin compounds inhibited free virus, gB-mediated virus entry, reduced the extent of virus spread, and cell-to-cell fusion in the virus-free cell fusion system. The EHV-1 antiviral properties and other pharmacological characteristics make porphyrins auspicious candidates for the treatment of EHV-1 infections and may promote understanding of membrane fusion events of EHV-1 life cycle

Interaction of host and viral microRNAs with infectious laryngotracheitis virus transcripts

Infectious Laryngotracheitis Virus (ILTV) is an Alphaherpesvirus of the domesticated chicken and other economically important fowl such as pheasants, peafowl and turkeys. It causes an upper respiratory disease that is clinically characterised by dyspnoea, rales and expulsion of a thick, sometimes hemorrhagic, tracheal exudate. Incidences of mortality range from 10 – 70 % whilst morbidity ranges from 50 – 100 %. The disease causes significant financial losses to the poultry industry through bird death, stunted growth and a marked decrease in egg production. Due to its economic importance, attenuated live-vaccines have been developed by serial passage of virus either in eggs or tissue culture. These have the ability to protect birds against ILTV however they do not stop latent infection which can result in reactivation of the virus termed ‘vaccinal laryngotracheitis’. The molecular biology underlying virus-host interactions for ILTV is poorly understood and there are large gaps in knowledge regarding the pathogenesis of ILTV infection. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally regulate gene expression through targeting of specific mRNAs. Several herpesviruses have been shown to encode miRNAs that have the ability to regulate both viral and cellular gene expression which can impact virus-host interactions. Previous work in the literature has shown that ILTV encodes for 10 miRNAs with sparse data on what they may be regulating. It was hypothesised that the virus-encoded miRNAs may have an effect upon the pathogenesis of the virus by targeting both cellular and viral mRNAs. To investigate this hypothesis initially, the biochemical technique CLASH (Cross-Linking and Sequencing of Hybrids) was attempted however a lack of suitable reagents such as physiologically relevant cell lines of chicken origin made this technically challenging and this approach was halted. Instead, a bioinformatic approach was developed and split into two avenues of research. Firstly, it was hypothesised that miRNAs encoded by ILTV would target virally derived transcripts. As the virus genome is poorly annotated, transcripts for all 79 open reading frames (ORFs) were created manually using an arbitrary system of 1000 bp upstream of the ATG start site and 50 bp downstream of the designated PolyA tail. These were then fed into the online algorithm RNA Hybrid alongside sequences for all 10 virus-encoded miRNAs. Results from the bioinformatic predictions were then sorted and filtered using pre-defined conditions. This left a total of 227 predicted interactions. These were then filtered again leaving 28 novel targets that were screened in a reporter based system. Three of the predicted interactions showed a decrease in luciferase-reporter activity compared to the siRNA control (UL24, UL29 and UL46/48), however only the latter two showed statistically significant decreases in activity of 15 % and 20 % respectively. Mutation of the seed sequences in both UL29 and UL46/48 targets abrogated the effects of the miRNA mimic. Further work on UL29 and its interaction with ILTV-miR-I2 looked at validating this interaction by western blotting however these results were inconclusive. Investigations into the interaction between UL46/48 and ILTV-miR-I6-5p first confirmed by RT-PCR that UL46 was targeted by ILTV-miR-I6-5p. Validation of the interaction between UL46 and ILTV-miR-I6-5p by western blotting was inconclusive. Investigations into the interplay between UL46, UL48 and the ICP4 promoter were also characterised with UL46 able to negatively modulate the effects of UL48 on ICP4 promoter activity in a reporter-based system. Secondly, the same viral transcripts were then used in conjunction with high confidence chicken miRNAs as per MiRBase (Release 21, Jun 2014). The sorting and filtering of results mirrored that of the viral transcript study giving a final list of 103 predicted targets. From the list, three targets were picked that were all targeted by the cellular miRNA ggamiR- 133a-3p and tested using the same reporter system. Two targets, one in UL20 and one in the coding region of ICP4 showed no statistical difference between the miRNA mimic and siRNA control. In contrast, one target, located in the 5’UTR of ICP4 and confirmed by RTPCR to be within the expressed mRNA transcript was found to cause a 55 % reduction in luciferase activity. This effect was then abrogated upon mutation of the miRNA seed sequence. Further investigations found that this miRNA can cause an apparent reduction in virus titer and a statistically significant decrease in plaque size morphology when virus is harvested from cells transfected with the miRNA mimic and used to infect naïve cells. Moreover, a combination RT-qPCR and sequencing was used to confirm the sequence of gga-miR-133a-3p in several tissues of the chicken including the Dorsal Root Ganglia (DRG) and Harderian gland (HG). These are of importance to ILTV biology as the DRG is a site of latent infection and the HG is a secondary lymphoid organ (SLO) in the bird which monitors the upper respiratory tract, the site of lytic replication/clinical symptoms. Finally, CRISPR-Cas9 genome editing was used to delete a cluster of five miRNAs from the viral genome. Guide RNAs (sgRNAs) were designed to target the miRNA cluster and shown to efficiently direct cleavage of target DNA in an in vitro system. Following transfection/infection of cells, virus was harvested and subsequent sequencing showed that this approach was successful in creating a recombinant ILTV. This was detectable after passage of the virus through naïve cells although a pure population of recombinant virus was not obtained due to a lack of time

Studies on the US3 and UL13 Protein Kinase Genes of Herpes Simplex Virus Type 1

The availability of the complete sequence of HSV-1 has permitted interpretation of HSV-1 gene function. As a result of database searches, US3 and UL13, the two HSV-1 genes studied during the course of this research, were found to possess amino acid sequence motifs characteristic of the protein kinase family. The US3 gene has indeed been shown to encode a protein kinase present in the cytoplasm of infected cells, however, protein kinase activity has not yet been assigned to the UL13 gene product, although the protein does correlate with a novel protein kinase activity in the nuclei of HSV-1 infected cells. Protein kinases constitute a very important class of enzymes which are responsible for the regulation of many cellular processes, thus, it is considered likely that these two genes play a significant role in the life cycle of HSV-1. This was investigated by constructing HSV-1 viruses which contain the US3 or UL13 genes disrupted by the insertion of the Escherichia coli lacZ gene. This insertional mutagenesis technique has been used successfully to investigate the functions of several other HSV-1 genes. During the course of this research three lacZ insertion mutants were constructed, a US3-lacZ insertion mutant, a UL13-lacZ insertion mutant and a UL13-US3 double insertion mutant. The effects of these mutations on the growth properties of HSV-1 and on the phosphoprotein profiles of infected cell extracts and virion preparations were then investigated. The US3-lacZ insertion mutant used in the experiments described in this thesis was found, at a late stage of the work, to be contaminated with a low level of wt virus, despite 5/6 rounds of plaque purification. Nevertheless, the titres of the US3-lacZ virus were significantly reduced compared to wt, indicating that, although the gene is not essential for virus growth, its disruption does impair virus growth. This reduction in growth does not appear to be due to a decrease in the amount of DNA synthesised by the US3-lacZ virus but to a decrease in the infectivity of the virus particles (as indicated by the significantly higher particle:pfu ratio for the US3-lacZ virus). Examination of the phosphoprotein profiles of US3-lacZ infected cells, following in vitro phosphorylation, showed a reduction in the degree of phosphorylation of several proteins with estimated MWs of 200K, 80K and 30K. These proteins, which represent potential substrates of the US3 protein kinase, have not yet been identified, although the 30K protein may correspond to the UL34 gene product, which has recently been reported to be a substrate of the US3 protein kinase. The growth of the UL13-lacZ insertion mutant, both in vitro and in vivo, does not differ significantly from that of wt, indicating that UL13 is not essential for virus growth, despite its conservation throughout the three herpesvirus families. Comparison of silver-stained gels of wt and UL13-lacZ virion preparations identified the 57K UL13 gene product as a relatively abundant component of the tegument, probably corresponding to VP18.8 - a phosphoprotein previously mapped to this region of the genome. The phosphoprotein profiles of both ULl3-iacZ-infected cells (nuclear extracts) and UL13-lacZ virions showed a reduction in the phosphorylation of several proteins, of MW 160K, 106K, 60K, 45K and 38K, indicating that the UL13 gene product does possess protein kinase activity. One of these potential substrates of UL13, the 38K phosphoprotein, has been mapped, using intertypic recombinants, to gene UL49, the product of which is known to be the virion tegument protein VP22. UL13-lacZ infected cell nuclear extracts were also found to contain two hyper- phosphorylated proteins with estimated MWs of 45K and 70K. The origins of these proteins are not known. Preliminary experiments with the UL13-US3 double insertion mutant have indicated that the growth of the virus is impaired to a much greater extent than expected from the growth properties of the two single lacZ mutants. One possible explanation for this is that the two kinases have common substrates and can therefore substitute for each others absence in the two single mutants, however this is considered unlikely as the phosphoprotein profiles of the two single mutants have not revealed any common substrates. Alternatively, the possibility exists that the two kinases may carry out similar functions in the virus life cycle. Thus, the research presented in this thesis provides evidence that the UL13 gene product, a relatively abundant virion protein, possesses protein kinase activity, and identifies the UL49 gene product as a major substrate for this activity. The research has also indicated several potential substrates for the US3 protein kinase, although these have not yet been identified. Finally, preliminary experiments with the UL13-US3 double insertion mutant have revealed a considerable reduction in virus growth compared to the two single mutants