Determination of antiviral action of long non-coding RNA loc107051710 during infectious bursal disease virus infection due to enhancement of interferon production.

The functions and profiles of lncRNAs during infectious bursal disease virus (IBDV) infection have not been determined, yet. The objectives of this study were to determine the antiviral action of loc107051710 lncRNA during IBDV infection by investigating the relationship between loc107051710 and IRF8, Type I IFN, STATs, and ISGs. DF-1 cells were either left untreated as non-infected controls (n = 1) or infected with IBDV (n = 3). RNA sequencing was applied for analysis of mRNAs and lncRNAs expression. Differentially expressed genes were verified by RT-qPCR. Then identification, of 230 significantly different expressed genes (182 mRNAs and 48 lncRNA) by pairwise comparison of the infected and control groups, was carried out. The functions of differentially expressed lncRNAs were investigated by selection of lncRNAs and mRNAs significantly enriched in the aforementioned biological processes and signaling pathways for construction of lncRNA-mRNA co-expression networks. The techniques of gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways were applied. It was suggested that these differentially expressed genes were involved in the interaction between the host and IBDV. Loc107051710 was found to have potential antiviral effects. RT-qPCR and western blot were applied and revealed that loc107051710 was required for induction of IRF8, type I IFN, STAT, and ISG expression, and its knockdown promoted IBDV replication. By fluorescence in situ hybridization, it was found that loc107051710 was translocated from the nucleus to the cytoplasm after infection with IBDV. Overall, loc107051710 promoted the production of IFN-α and IFN-ß by regulating IRF8, thereby promoting the antiviral activity of ISGs.

Estrogen receptor alpha regulates the Wnt/ß-catenin signaling pathway in colon cancer by targeting the NOD-like receptors.

It has been reported that estrogen receptors (ERs) participate in carcinogenesis by directly regulating NOD-like receptors (NLRs). However, the expression profiles of ERs and NLRs in tumor and the ER-NLR regulated signaling pathway are not clear. In this study, we summarized gene expression profiles of ERs and NLRs across normal and tumor tissue by comprehensive data mining. Then we explored the ER-NLR regulated signaling pathway by RNA sequencing (RNA-seq). The results showed that the NLRs and ERs were differentially expressed in different neoplasm tissues. Such expression discrepancies might influence inflammatory regulation and tumorigenesis. Importantly, we identified that ER-NLR regulate Wnt/ß-catenin pathway in colon cancer. Taking colon adenocarcinoma (COAD) as example, we found that Wnt2b/LRP8/Dvl1/Axin2/GSK3a/APC/ß-catenin genes were differentially expressed in ER-/- mouse colon tissue and colon cancer cells. The selective ERα antagonist could significantly decrease Wnt2b/LRP8/Dvl1 expression, increase destruction complex (Axin2/GSK3a/APC) expression, and promote degradation of ß-catenin in colon carcinoma cell by inhibited NLRP3 expression. In short, the research demonstrates that NLRs are potential biomarkers for cancer, and ERs can regulate the Wnt/ß-catenin signaling pathway in cancer by targeting the NLRs. Our results provide a possible signaling pathway in which ER-NLR is correlated with Wnt/ß-catenin.

Estrogen receptors participate in carcinogenesis signaling pathways by directly regulating NOD-like receptors.

Increasing evidence indicates that the NOD-like receptors (NLRs) family may act as critical back-up defenses and provide synergistic responses when confronted with persistent danger. However, the precise regulatory mechanism of NLRs and the contribution of NLRs to cancer are still unknown. In our previous study, we found that estrogen receptors (ERs) have a close connection with NLRs in the inflammatory response. Here, ERs are first identified as NLRs transcription regulation factors, both regulate NLRs expression and promote inflammasome co-localization. Furthermore, we identified that NLRP3 was differentially expressed in colon normal and cancer cells, selective ERα antagonist could significantly decrease pro-inflammatory cytokines expression, suppress proliferation and promote apoptosis by inhibited NLRP3 expression and inflammasome activity. In short, the research demonstrates that ERs participate in the NLR-associated signaling pathway in cancer by directly regulating NLRs. Our results provide novel insight into ERs as therapeutic targets in NLR-related inflammation and cancer.

Oral Immunization against PEDV with Recombinant Lactobacillus casei Expressing Dendritic Cell-Targeting Peptide Fusing COE Protein of PEDV in Piglets.

Porcine epidemic diarrhea (PED) is a highly contagious disease in newborn piglets. In our previous study, a genetically engineered Lactobacillus casei oral vaccine (pPG-COE-DCpep/L393) expressing a dendritic cell (DC)-targeting peptide fused with porcine epidemic diarrhea virus (PEDV) COE antigen was developed. This vaccine induced significant levels of anti-PEDV specific IgG and IgA antibody responses in mice, indicating a potential strategy against PEDV infection. In this study, pPG-COE-DCpep/L393 was used for oral vaccination of newborn piglets against PEDV. We then assessed the immune responses and protection efficacy of pPG-COE-DCpep/L393. An indirect enzyme-linked immunosorbent assay (ELISA) showed that the recombinant Lactobacillus vaccine elicits a specific systemic and mucosal immune response. The T-helper cells mediated by pPG-COE-DCpep/L393 and PEDV infection display a Th1 phenotype. The histopathological results showed that pPG-COE-DCpep/L393 promotes lymphocyte proliferation and effectively protects piglets against PEDV infection. The transforming growth factor-ß level indicated that the recombinant Lactobacillus vaccine plays a role in anti-inflammatory responses in mesenteric lymph nodes during PEDV infection. These results show that pPG-COE-DCpep/L393 is a potential vaccine against PEDV infection.

The neglected avian hepatotropic virus induces acute and chronic hepatitis in ducks: an alternative model for hepatology.

Duck Hepatitis A Virus (DHAV) belongs to the Avihepatovirus, which is also classified into Picornaviridae with Hepatovirus, Hepatitis A Virus (HAV). In humans, the pathogenesis of HAV is not well understood because of limited work with animal models. Here, we investigated the progress of duck viral hepatitis caused by DHAV and their potential for dissecting the pathogenesis of HAV. During the course of infection, the duck model had undergone hepatocellular lesions (vacuolation, acidophilic degeneration and steatosis), lymphocytes recruitment (neutrophil granulocytes, heterophilic granulocytes and T cells or plasm cells) and repair (activation of hepatic stellate cells, fibrosis and regeneration). Coincident with liver injury, the serum biomarkers, aspartate aminotransferase and alanine transaminase were significantly increased. Moreover, comparatively lower CD4+ and CD8+ T-cells were recruited to the liver, which might lead to a persistent infection (40 wk). Because DHAV and HAV have similar genomic structure, biological phenotypes and can easily replicate in liver. And half of fibrosis-related genes had high homology between humans and ducks. Considering these similarity in pathological and virological phenotypes, we proposed that the ducks might be an alternatively small animal model that would provide insight into the pathogenesis of viral hepatitis, fibrosis and liver regeneration.

Viral-host interaction in kidney reveals strategies to escape host immunity and persistently shed virus to the urine.

Hepatitis A virus is one of five types of hepatotropic viruses that cause human liver disease. A similar liver disease is also identified in ducks caused by Duck Hepatitis A virus (DHAV). Notably, many types of hepatotropic viruses can be detected in urine. However, how those viruses enter into the urine is largely unexplored. To elucidate the potential mechanism, we used the avian hepatotropic virus to investigate replication strategies and immune responses in kidney until 280 days after infection. Immunohistochemistry and qPCR were used to detect viral distribution and copies in the kidney. Double staining of CD4+ or CD8+ T cells and virus and qPCR were used to investigate T cell immune responses and expression levels of cytokines. Histopathology was detected by standard HE staining. In this study, viruses were persistently located at scattered renal tubules. No CD4+ or CD8+ T cells were recruited to the kidney, which was only accompanied by transient cytokine storms. In conclusion, the extremely scattered infection was the viral strategy to escape host immunity and may persistently shed virus into urine. The deletion of Th or Tc cell responses and transient cytokine storms indeed provide an advantageous renal environment for their persistent survival.

Development and evaluation of indirect ELISAs for the detection of IgG, IgM and IgA1 against duck hepatitis A virus 1.

Duck hepatitis A virus 1 (DHAV-1) is the principal pathogen that causes duck viral hepatitis (DHV), a highly fatal infectious disease in ducklings. Given the importance of the humoral immune response in the clearance of DHAV-1, indirect enzyme-linked immunosorbent assays (I-ELISAs) to detect immune indices, including IgG, IgM and IgA1, were developed and evaluated in this study. The optimal concentrations of coating-antigen were 1.79µg/ml, 2.23µg/ml and 2.23µg/ml for IgG, IgM and IgA1, respectively. Meanwhile, the optimal dilutions of sera were 1:80, 1:40 and 1:40, respectively; and of the conjugates were 1:300, 1:1800 and 1:800, respectively. Based on these conditions, three linear regression equations, y=1.363+1.954x (r2=0.983), y=1.141+2.228x (r2=0.970) and y=1.103+1.559x (r2=0.995) were derived for IgG, IgM and IgA1, respectively. Analytical sensitivities of the new methods were 1:2560, 1:1280 and 1:640 for IgG, IgM and IgA1, respectively. The concordances between the I-ELISAs and serum-neutralization were 95.2% for IgG and IgA1, and 75% for IgM. Although there was a weak cross-reaction with DHAV-3 positive serum for the IgG and IgA1 tests, it didn't affect the ability to detect DHAV-1 specific antibodies. Thus, these new I-ELISAs were shown to be potentially convenient methods to survey the status of humoral immune response to DHAV-1.

Genome-Wide Analysis of the Synonymous Codon Usage Patterns in Riemerella anatipestifer.

Riemerella anatipestifer (RA) belongs to the Flavobacteriaceae family and can cause a septicemia disease in poultry. The synonymous codon usage patterns of bacteria reflect a series of evolutionary changes that enable bacteria to improve tolerance of the various environments. We detailed the codon usage patterns of RA isolates from the available 12 sequenced genomes by multiple codon and statistical analysis. Nucleotide compositions and relative synonymous codon usage (RSCU) analysis revealed that A or U ending codons are predominant in RA. Neutrality analysis found no significant correlation between GC12 and GC3 (p > 0.05). Correspondence analysis and ENc-plot results showed that natural selection dominated over mutation in the codon usage bias. The tree of cluster analysis based on RSCU was concordant with dendrogram based on genomic BLAST by neighbor-joining method. By comparative analysis, about 50 highly expressed genes that were orthologs across all 12 strains were found in the top 5% of high CAI value. Based on these CAI values, we infer that RA contains a number of predicted highly expressed coding sequences, involved in transcriptional regulation and metabolism, reflecting their requirement for dealing with diverse environmental conditions. These results provide some useful information on the mechanisms that contribute to codon usage bias and evolution of RA.

A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3.

Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains.

The 2A2 protein of Duck hepatitis A virus type 1 induces apoptosis in primary cell culture.

Duck hepatitis A virus type 1, (DHAV-1) 2A2pro, is one of the most highly conserved viral proteins within the DHAV serotypes. However, its effect on host cells is unclear. We predicted that DHAV-1 2A2pro was a GTPase-like protein based on the results of multiple sequence alignment and homologous modeling analysis. Upon transfection of a recombinant plasmid expressing DHAV-1 2A2, cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-sized DNA ladder, and positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining; hence, cell death has the characteristics of apoptosis. By staining cells with fluorescein Annexin V-FITC and PI, it is possible to distinguish and quantitatively analyze nonapoptotic cells, early apoptotic cells, late apoptotic/necrotic cells, and dead cells through flow cytometry and fluorescence microscopy. The percentage of apoptotic cells gradually increased and reached a maximum after 48 h of transfection. In conclusion, apoptosis induced by this GTPase-like protein may contribute to DHAV-1 pathogenesis.

A phase trial of the oral Lactobacillus casei vaccine polarizes Th2 cell immunity against transmissible gastroenteritis coronavirus infection.

Transmissible gastroenteritis coronavirus (TGEV) is a member of the genus Coronavirus, family Coronaviridae, order Nidovirales. TGEV is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. An oral Lactobacillus casei (L. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. In this L. casei vaccine, repetitive peptides expressed by L. casei (specifically the MDP and tuftsin fusion protein (MT)) were repeated 20 times and the D antigenic site of the TGEV spike (S) protein was repeated 6 times. Immunization with recombinant Lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. The first immunization is more important than the last immunization in the series. The recombinant Lactobacillus elicited specific systemic and mucosal immune responses. Recombinant L. casei had a strong potentiating effect on the cellular immunity induced by the oral L. casei vaccine. However, during TGEV infection, the systemic and local immune responses switched from Th1 to Th2-based immune responses. The systemic humoral immune response was stronger than the cellular immune response after TGEV infection. We found that the recombinant Lactobacillus stimulated IL-17 expression in both the systemic and mucosal immune responses against TGEV infection. Furthermore, the Lactobacillus vaccine stimulated an anti-TGEV infection Th17 pathway. The histopathological examination showed tremendous potential for recombinant Lactobacillus to enable rapid and effective treatment for TGEV with an intestinal tropism in piglets. The TGEV immune protection was primarily dependent on mucosal immunity.

Influenza A virus acquires enhanced pathogenicity and transmissibility after serial passages in swine.

Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs. Importance: We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.

Hemagglutinin mutation D222N of the 2009 pandemic H1N1 influenza virus alters receptor specificity without affecting virulence in mice.

The D222N hemagglutinin (HA) mutation within the receptor-binding site was detected with higher frequencies in severe cases of 2009 pandemic H1N1 (pdmH1N1) infections. The impact of this mutation was investigated in vitro and in vivo using recombinant viruses. The recombinant D222N virus grew to significantly lower viral titers than the WT in A549 but not in MDCK cells. A dose-dependent glycan array analysis with the D222N virus showed a modest increase in the binding avidity to human-like (α-2,6 sialylated glycan) receptors and avian-like (α-2,3 sialylated glycan) receptors than the WT virus. The D222N HA mutation resulted in slight weight loss, lower lung titers, inflammatory cytokines and alveolar inflammation in mice than the WT virus. This may or may not be associated with severe clinical outcomes reported in humans.

Effects of diethylphthalate and di-(2-ethyl)hexylphthalate on the physiology and ultrastructure of cucumber seedlings.

Phthalic acid esters (PAEs) are one kind of persistent organic pollutants. This study was conducted to investigate the effects of diethylphthalate (DEP) and di(2-ethyl)hexylphthalate (DEHP) with different concentrations (0, 30, 50, 100, and 200 mg L(-1)) on early seedling growth of Cucumis sativus L. Physiological, biochemical, and ultrastructure of seedling leaves were examined for 7-day exposure. The three antioxidant enzymes' activities was stimulated at low-DEP treatments and decreased under higher levels (>200 mg L(-1)) compared to the controls. Furthermore, MDA and H2O2 gradually enhanced with the elevation of DEP and DEHP concentration. Significant impact on the chloroplast and mitochondrion was visible, possibly as a consequence of free radical generation. DEP induced bigger and more starch grains in chloroplasts than DEHP. This study concluded that the effects of DEP and DEHP on cucumber seedlings represented the adverse impacts of DEP and DEHP on the ecosystem and agricultural production. The environmental harm caused by DEP was severer than DEHP.

A shift to Th2 immune response caused by constitutive expression of IPSE/alpha-1 in transfected pig fibroblasts in mice.

The IPSE/alpha-1 gene (IL-4-inducing principle of Schistosoma mansoni eggs) is a major secreted glycoprotein of S. mansoni eggs that has a potent IL-4-inducing effect. To test the hypothesis that the immune evasion mechanism can be used to overcome the xenograft immune response, the IPSE/alpha-1 gene was transferred into pig fibroblasts, and the transgenic cells were transplanted into KM mice by subcutaneously injecting 10(5)cells per mouse. Cytokine levels were measured to examine the immune response polarization by real-time PCR and ELISA. Mice injected with pig fibroblasts containing a pIRES2-EGFP expression vector were used as a control group. In this group, both cellular and humoral immune responses were activated to reject the grafts alongside increases in all measured cytokine levels. In contrast, the experimental group injected with cells constitutively expressing the IPSE/alpha-1 gene demonstrated a significant decrease in Th1 response cytokines and a significant increase in Th2 response cytokines compared with the control group. These results imply that constitutive IPSE/alpha-1 expression can shift the Th1/Th2 balance of xenograft rejections toward the Th2 response while suppressing the Th1 response. In conclusion, IPSE/alpha-1 could influence the polarization of immune responses during xenograft rejection and suppress the Th1 response. Therefore, this parasitic immune evasion mechanism could be helpful in overcoming xenograft rejection.

Acute respiratory distress syndrome induced by a swine 2009 H1N1 variant in mice.

BACKGROUND: Acute respiratory distress syndrome (ARDS) induced by pandemic 2009 H1N1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 H1N1 infection. However, no animal model has been developed for ARDS caused by infection with 2009 H1N1 virus. Here, we present a mouse model of ARDS induced by 2009 H1N1 virus. METHODOLOGY PRINCIPAL FINDINGS: Mice were inoculated with A/swine/Shandong/731/2009 (SD/09), which was a 2009 H1N1 influenza variant with a G222D mutation in the hemagglutinin. Clinical symptoms were recorded every day. Lung injury was assessed by lung water content and histopathological observation. Arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. Mice infected with SD/09 virus showed typical ARDS symptoms characterized by 60% lethality on days 8-10 post-inoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines. CONCLUSIONS/SIGNIFICANCE: These results suggested that we successfully established an ARDS mouse model induced by a virulent 2009 H1N1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ARDS caused by 2009 H1N1 virus.

Characterization of an artificial swine-origin influenza virus with the same gene combination as H1N1/2009 virus: a genesis clue of pandemic strain.

Pandemic H1N1/2009 influenza virus, derived from a reassortment of avian, human, and swine influenza viruses, possesses a unique gene segment combination that had not been detected previously in animal and human populations. Whether such a gene combination could result in the pathogenicity and transmission as H1N1/2009 virus remains unclear. In the present study, we used reverse genetics to construct a reassortant virus (rH1N1) with the same gene combination as H1N1/2009 virus (NA and M genes from a Eurasian avian-like H1N1 swine virus and another six genes from a North American triple-reassortant H1N2 swine virus). Characterization of rH1N1 in mice showed that this virus had higher replicability and pathogenicity than those of the seasonal human H1N1 and Eurasian avian-like swine H1N1 viruses, but was similar to the H1N1/2009 and triple-reassortant H1N2 viruses. Experiments performed on guinea pigs showed that rH1N1 was not transmissible, whereas pandemic H1N1/2009 displayed efficient transmissibility. To further determine which gene segment played a key role in transmissibility, we constructed a series of reassortants derived from rH1N1 and H1N1/2009 viruses. Direct contact transmission studies demonstrated that the HA and NS genes contributed to the transmission of H1N1/2009 virus. Second, the HA gene of H1N1/2009 virus, when combined with the H1N1/2009 NA gene, conferred efficient contact transmission among guinea pigs. The present results reveal that not only gene segment reassortment but also amino acid mutation were needed for the generation of the pandemic influenza virus.

Cryptosporidium andersoni is the predominant species in post-weaned and adult dairy cattle in China.

Dairy industry plays an important role in the agricultural economy of China. To estimate the prevalence and public health significance of cryptosporidiosis in post-weaned and adult dairy cattle in China, during four consecutive years (from 2006 to 2009), a total of 1315 fecal samples from 22 dairy cattle farms in ten prefectures in Henan Province were examined for the presence of Cryptosporidium oocysts. The overall prevalence of Cryptosporidium was 7.9%, with the highest infection rate (11.3%) in 3 to 11-month-old calves and the lowest infection rate (1.0%) in >2-year-old cows (p<0.01). Cryptosporidium-positive samples (n=104) were analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene, and 25 representative samples were further analyzed by DNA sequencing of the PCR products. Cryptosporidium bovis and Cryptosporidium andersoni were identified. C. andersoni (84/104) was the predominant species and was found in all age groups, whereas C. bovis (20/104) was only detected in 3 to 11-month-old calves. Thus, C. andersoni appears to be the dominant species in weaned dairy calves and heifers in China, in contrast with its common occurrence in adult cattle in other parts of the world.

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

BACKGROUND: Some UL45 gene function of Herpesvirus was reported. While there was no any report of the duck enteritis virus (DEV) UL45 protein as yet. RESULTS: 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. CONCLUSIONS: 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.

Establishment of a multiplex RT-PCR assay to detect different lineages of swine H1 and H3 influenza A viruses.

Classical swine H1N1, emerging European avian-like H1N1 and human-like H3N2 lineages are co-circulating in the swine population in China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an effective method for use in influenza surveillance. In this study, a multiplex RT-PCR method was developed for simultaneous identification of hemagglutinin (HA) genes derived from the three lineages of swine influenza viruses. Three primer sets were designed and aimed specifically at HA genes of these viral lineages. The specificity of the assay showed that the established methods could efficiently differentiate the HA genes of classical swine H1N1, European avian-like H1N1, and human-like H3N2 viruses while other viruses such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, and porcine circovirus type 2, could not be detected. The assay showed a sensitivity of 1 x 10(2.5) 50% egg infectious dose for each virus lineage. The comparison of the results with those obtained from the analysis of 300 swine tracheal swab samples by means of virus isolation showed a high level of agreement. This multiplex RT-PCR method provides a rapid and specific swine influenza diagnostic tool that also has the potential for investigating the epidemiology of different lineages of swine influenza virus prevalent currently in China.