Development of an Indirect Dot-PPA-ELISA using glutamate dehydrogenase as a diagnostic antigen for the rapid and specific detection of Streptococcus suis and its application to clinical specimens.

Streptococcus suis is an important zoonotic pathogen causing infections in pigs and humans. Bacterial surface-related proteins are often explored as potential vaccine candidates and diagnostic antigens. In the present study, glutamate dehydrogenase, a highly conserved immunogenic extracellular protein, was used to establish a dot horseradish peroxidase enzyme-linked staphylococcal protein A immunosorbent assay (Dot-PPA-ELISA) for diagnosis of S. suis infection. The antigen-antibody reaction was optimised through checkerboard titration involving serial dilutions, followed by selective blocking tests and evaluations of cross-reaction, repeatability, and stability. Comparative analysis by using a conventional plate ELISA kit showed that the specificity and sensitivity of the Dot-PPA-ELISA were 97.5 and 96.6%, respectively. Furthermore, dynamic changes in the levels of antibody in rabbits immunised with a propolis inactivated vaccine were monitored by Dot-PPA-ELISA. A total seroprevalence of 73.1% in 305 pig serum samples indicated the method's applicability to detect S. suis infection. Cumulatively, the results suggested that Dot-PAA-ELISA is a convenient, rapid, sensitive, and specific diagnostic method suitable for studying large numbers of samples obtained from clinical and epidemiological studies, thereby helping reduce important economic losses.

Circulation and in vivo distribution of duck hepatitis A virus types 1 and 3 in infected ducklings.

The circulation of duck hepatitis A virus types 1 (DHAV-1) and 3 (DHAV-3) in Southeast Asia has resulted in a continuously changing epidemiological scenario. In this study, a duplex real-time PCR assay for simultaneous quantitative detection of DHAV-1 and DHAV-3 was established, and 200 liver samples from dead ducklings collected from 31 different flocks in Shandong province, China, were tested. Fifty-eight (29.0 %) samples from 13 flocks were positive for DHAV-1 single infection, 113 (56.5 %) samples from 13 other flocks were positive for DHAV-3 single infection, and 24 samples (12.0 %) from four flocks were positive for both viruses. DHAV-1 and DHAV-3 were detected with high viral loads in all of the organs tested (liver, spleen, pancreas, kidney, heart, thymus, bursa of Fabricius and brain). No significant difference in DHAV-1 and DHAV-3 viral loads was found between singly infected and coinfected samples, and there was no correlation between the viral loads of the two viruses and the age of dead ducklings. To the best of our knowledge, this is the first report about the in vivo distribution of DHAV-1 and DHAV-3 in clinically infected ducklings.

Complete genome sequence of a novel porcine parvovirus in China.

The porcine parvovirus JT strain (PPV-JT) was isolated from a piglet showing nonsuppurative myocarditis in Shandong, China, in 2010. The complete genomic sequence of PPV-JT, 4,941 bp long, was determined from clones made from replicative form (RF) DNA. The genomic analysis demonstrated that the PPV-JT might be involved in a recombination event, which will help us understand the molecular characteristics and evolutionary of PPV in China.

Alternative splicing of histone demethylase Kdm6bb mediates temperature-induced sex reversal in the Nile tilapia.

Sex determination in many fish species is remarkably plastic and temperature sensitive. Nile tilapia display a genetic sex-determination system (XX/XY). However, high-temperature treatment during critical thermosensitive periods can induce XX females into XXm pseudo-males, and this phenomenon is termed temperature-induced sex reversal (TISR). To investigate the molecular mechanism of TISR in Nile tilapia, we performed Iso-seq analysis and found a dramatic effect of high temperature on gene alternative splicing (AS). Kdm6bb histone demethylase showed a novel AS at intron 5 that generates Kdm6bb_tv1 transcripts without intron 5 and Kdm6bb_tv2 with intron 5. Kdm6bb_tv1 encodes a full-length protein while Kdm6bb_tv2 encodes a truncated protein. Expression analysis revealed that intron 5 splicing of Kdm6bb is male and gonad biased at larval stage, and only gonad biased at adult stage. High-temperature treatment induced intron 5 splicing in the gonads of XX and XY fish, resulting in increased Kdm6bb_tv1 expression. To directly test the role of Kdm6bb_tv1 in Nile tilapia TISR, we knocked out expression of Kdm6bb_tv1. However, Kdm6bb_tv1-/- homozygous mutants showed embryonic lethality. Overexpression of Kdm6bb_tv1, but not Kdm6bb_tv2, induced sex reversal of XX females into pseudo-males. Overexpression of Kdm6bb_tv1, as with high-temperature treatment, modified the promotor region of Gsdf and Dmrt1 by demethylating the trimethylated lysine 27 of histone 3 (H3K27me3), thereby increasing expression. Collectively, these studies demonstrate that AS of Kdm6bb intron 5 increases the expression of Kdm6bb_tv1, which acts as a direct link between high temperature and activation of Gsdf and Dmrt1 expression, leading to male sex determination.

Analysis of avian hepatitis E virus from chickens, China.

Avian hepatitis E virus (HEV) has been identified in chickens; however, only 4 complete or near-complete genomic sequences have been reported. We found that the near-complete genomic sequence of avian HEV in chickens from China shared the highest identity (98.3%) with avian HEV from Europe and belonged to avian HEV genotype 3.

Tandem repeat sequence of duck circovirus serves as downstream sequence element to regulate viral gene expression.

Duck circovirus (DuCV) has a small, single-stranded circular DNA genome of approximately 1.99 kb. Through a genome sequence analysis using the dottup program, we found that a quadruple tandem repeat sequence (QTR) in the intergenic region between the rep and cap genes of the DuCV genome, but not in other circoviruses. The QTR was also substantially different and evolutionarily conserved in the genotype 1 and 2 DuCV strains. Furthermore, a luciferase reporter assay demonstrated that QTR functioned as a downstream sequence element (DSE) of polyadenylation signals to enhance mRNA stability, which was dependent on four copies but not the QTR direction. Cap and Rep expression derived by subgenomic constructs also revealed a critical role of QTR in regulating viral gene expression. Finally, a reverse genetic study of a DuCV-based minicircle DNA technique found that a deletion of QTR induced a significant deficiency in viral genes transcription and replication. Our findings were the first to report that QTR only exists in the DuCV genome and serves as a novel molecular marker of DuCV genotyping, and has revealed its crucial biological function in regulating viral gene expression.

IGF2BP1 Significantly Enhances Translation Efficiency of Duck Hepatitis A Virus Type 1 without Affecting Viral Replication.

As a disease characterized by severe liver necrosis and hemorrhage, duck viral hepatitis (DVH) is mainly caused by duck hepatitis A virus (DHAV). The positive-strand RNA genome of DHAV type 1 (DHAV-1) contains an internal ribosome entry site (IRES) element within the 5' untranslated region (UTR), structured sequence elements within the 3' UTR, and a poly(A) tail at the 3' terminus. In this study, we first examined that insulin-like growth factor-2 mRNA-binding protein-1 (IGF2BP1) specifically interacted with the DHAV-1 3' UTR by RNA pull-down assay. The interaction between IGF2BP1 and DHAV-1 3' UTR strongly enhanced IRES-mediated translation efficiency but failed to regulate DHAV-1 replication in a duck embryo epithelial (DEE) cell line. The viral propagation of DHAV-1 strongly enhanced IGF2BP1 expression level, and viral protein accumulation was identified as the key point to this increment. Collectively, our data demonstrated the positive role of IGF2BP1 in DHAV-1 viral proteins translation and provided data support for the replication mechanism of DHAV-1.

Identification of a functional nuclear localization signal in 3Dpol/3CD of duck hepatitis A virus 1.

The nuclear localization signals (NLS) were usually composed of basic residues (K and R) and played an important role in delivery of genomes and structural protein into nucleus. In this research, we identified that 3Dpol/3CD entered into nucleus during viral propagation of duck hepatitis A virus type 1 (DHAV-1). To investigate the reason that 3Dpol/3CD entered into nucleus, the amino acid sequence of 3CD was analyzed through NLS Mapper program. The basic region 17PRKTAYMRS25 was subsequently proved to be a functional NLS to guide 3Dpol/3CD into nucleus. 18R, 19K and 24R were found essential for maintaining the nuclear targeting activity, and exchange between 24R and 24K had no impact on cellular localization of 3Dpol. Since the entry of 3Dpol/3CD into nucleus was essential for shutoff of host cell transcription and maintaining the viral propagation of picornavirus numbers, our study provided new insights into the mechanism of DHAV-1 propagation.

C-terminal 20 residues of ORF3 protein of duck circovirus genotype 2 regulates the nuclear localization and inhibits apoptotic activity of ORF3 protein.

Duck circovirus (DuCV) is divided into genotypes 1 and 2. The DuCV ORF3 protein is a newly identified viral protein with apoptotic activity. In this study, the differences in the gene sequences, subcellular localization, and apoptotic activities of the ORF3 proteins of DuCV genotypes 1 and 2 were analyzed. A T-to-A point mutation at nucleotide 236 (T236A) in the ORF3 gene sequence of DuCV genotype 1 was observed, which generates a premature stop codon (TAG) and resulted in a truncated ORF3 protein. The ORF3 protein of DuCV genotype 2 is 20 amino acids longer at its C-terminus than the truncated ORF3 protein of genotype 1. A variant monopartite-type nuclear localization signal (RRLRTCNCRACRTLK) was identified within the C-terminal region of the ORF3 protein of DuCV genotype 2, which is essential for the nuclear localization of the protein. The 20 C-terminal residues of the DuCV genotype 2 ORF3 protein also inhibits the apoptotic activity of the protein. Our findings provide insight into the biological and functional characteristics of the DuCV ORF3 protein.

The Functional Role of the 3' Untranslated Region and Poly(A) Tail of Duck Hepatitis A Virus Type 1 in Viral Replication and Regulation of IRES-Mediated Translation.

The duck hepatitis A virus type 1 (DHAV-1) is a member of Picornaviridae family, the genome of the virus contains a 5' untranslated region (5' UTR), a large open reading frame that encodes a polyprotein precursor and a 3' UTR followed by a poly(A) tail. The translation initiation of virus proteins depends on the internal ribosome-entry site (IRES) element within the 5' UTR. So far, little information is known about the role of the 3' UTR and poly(A) tail during the virus proliferation. In this study, the function of the 3' UTR and poly(A) tail of DHAV-1 in viral replication and IRES-mediated translation was investigated. The results showed that both 3' UTR and poly(A) tail are important for maintaining viral genome RNA stability and viral genome replication. During DHAV-1 proliferation, at least 20 adenines were required for the optimal genome replication and the virus replication could be severely impaired when the poly (A) tail was curtailed to 10 adenines. In addition to facilitating viral genome replication, the presence of 3' UTR and poly(A) tail significantly enhance IRES-mediated translation efficiency. Furthermore, 3' UTR or poly(A) tail could function as an individual element to enhance the DHAV-1 IRES-mediated translation, during which process, the 3' UTR exerts a greater initiation efficiency than the poly(A)25 tail.

[Study on immune efficacy of Newcastle disease chitosan microsphere vaccine].

Newcastle disease is an acute and highly contagious disease caused by Newcastle disease virus (NDV), one of which does great harms to the poultry industry. The most basic measure of controlling New Castle disease is to alid vaccine, now we usually use La Sota live vaccine and inactivated NDV vaccine, but these two vaccines both have more or less limitation. It can produce higher mucosal immunity titers by taking vaccine orally, meanwhile it can induce humoral and cell-mediated immune response and mucosal immunity strongly. Therefore, it becomes the focus of the research, which prepare new pattern vaccines taking orally. NDV chitosan microsphere vaccine was prepared using chitosan as capsule wall material, NDV as core material, glutaraldehyde as cross-linking material, and its even particle diameter was 5.83um, and its surface was smooth and glossy, no obviously pore space, yellow brown pykno-ball, and its safety and potency were evaluated. The SPF chickens were immunized with NDV chitosan microsphere vaccine, La Sota live vaccine and inactivated NDV vaccine respectively. To evaluate vaccine's immune efficacy, using MTT to measure lymphocytes proliferation in vitro, using HI to measure serum special IgG and using ELISA tests to detect mucosal sIgA titers. The results show that NDV chitosan microsphere vaccine was safe, could induce humoral and cell-mediated immune response and mucosal immunity strongly. The results of the potency tests conformed that the vaccine could produce good protective effect.

Intraspecies and interspecies transmission of mink H9N2 influenza virus.

H9N2 influenza A virus (IAV) causes low pathogenic respiratory disease and infects a wide range of hosts. In this study, six IAVs were isolated from mink and identified as H9N2 IAV. Sequence analysis revealed that the six isolates continued to evolve, and their PB2 genes shared high nucleotide sequence identity with H7N9 IAV. The six isolates contained an amino acid motif PSRSSR↓GL at the hemagglutinin cleavage site, which is a characteristic of low pathogenic influenza viruses. A serosurvey demonstrated that H9N2 IAV had spread widely in mink and was prevalent in foxes and raccoon dogs. Transmission experiments showed that close contact between H9N2-infected mink and naive mink, foxes and raccoon dogs resulted in spread of the virus to the contact animals. Furthermore, H9N2 challenge experiments in foxes and raccoon dogs showed that H9N2 IAV could infect these hosts. Virological and epidemiological surveillance of H9N2 IAV should be strengthened for the fur animal industry.

[Immunogenicity of envelope glycoprotein gene of reticuloendotheliosis virus expressed in insect cell].

A recombinant baculovirus expressing reticuloendotheliosis virus env gene was constructed with Bac-to-Bac Baculovirus Expression Systems. After transfecting the recombinant virus into Sf9 cells for 3 days, REV env can be detected by indirect immunofluorescence antibody assay (IFA) and Western blot with specific monoclonal antibodies of REV. The oil-water emulsion vaccine was then produced using this infected Sf9 cells lycates and inoculated SPF chickens to validate the immunogenicity for REV. The results show that special anti-REV antibody can maintain more than 45 days and resist the infection of REV viruses. This is the first success to induce anti-REV antibody in chickens by none-live viruses.

[Study on the characterization of the bi-directional promoter between pp38 gene and 1.8kb mRNA transcripts of Marek's disease viruses].

The bi-directional promoter between pp38 gene and 1.8kb mRNA transcripts of Marek's disease viruses (MDV) was divided into two single-direction promoters from the replication of MDV genomic DNA. The pp38 gene was cloned into pUC18 vector for plasmid pUC-pp38. Then the complete bi-directional promoter was cloned into pUC-pp38 in two directions to form plasmids pPro(f)pp38 and pPro(r)pp38, and the divided two single directional promoters were cloned in pUC-pp38 for plasmids pdPro(f)pp38 and pdPro(r)pp38. 24 to 48 hours after transfection to chicken embryo fibroblast (CEF) cells, the expression of pp38 could be detected in above 4 samples with Indirect Immuno-fluorescent Assay (IFA). In order to analysis the activity of the promoter quantificationally, CAT was used as the report gene. The complete or divided promoters were cloned into pCAT-Basic vector for plasmids pPro(f)CAT, pPro(r)CAT, pdPro(f)CAT and pdPro(r)CAT. The activity of CAT was measured from the lysed CEF cells, when they were transfected for 48 hours by the above four plasmids, respectively. The results showed the activity of the divided promoters reduce on both directions, especially for the direction of 1.8kb mRNA transcript, nearly down to 1/41.

[PCR detection of PCV-2 and PRRSV in porcine pleuropneumonia samples].

PCV-2 (Porcine circovirus type-2, PCV-2) and PRRSV (Porcine reproductive and respiratory syndrome virus, PRRSV) were detected by PCR from 253 porcine pleuropneumonia samples and 125 clinically healthy lung samples were collected from different districts of Shandong province. The results showed that 171 samples for PCV-2 and 101 samples for PRRSV were positive. The positive ratio were 67.5% and 40%, respectively. The co-infection number of PCV-2 and PRRSV was 68 in 253 samples, the positive ratio was 26.8%. While in the clinically healthy samples, only 21 samples for PCV-2 and 12 samples for PRRSV were detected positive, the positive ratio were 16.8% and 9.6%, respectively, no co-infection samples were found. Statistical result showed significant difference between positive ratio for PCV-2 and PRRSV in porcine pleuropneumonia samples and that of in clinically healthy samples. The above results demonstrated that there maybe some relationship between the infection of porcine pleuropneumonia and PCV-2 and PRRSV in pigs.

[Construction of infectious clone of subgroup J avian leukosis virus strain NX0101 and its pathogenicity].

By using PCR, 3 fragments of provirus cDNA of avian leukosis virus (ALV-J) strain NXO101 were amplified from the genomic DNA of ALV-J infected cells,and then combined in the right direction and sequences into recombinant plasmid pALV-J-NX, containing the whole genome of NX0101. After transfection of chicken embryo fibroblast (CEF) cells with plasmid pALV-J-NX DNA, the rescued virus was identified in CEF by indirect fluorescence antibody test with ALV-J specific monoclonal antibody JE9. The rescued virus could replicate in CEF at a titer of 10(5.6)/mL. The chicken experiment demonstrated that the rescued virus was still able to induce tumors in commercial meat-type broilers.

Dog to dog transmission of a novel influenza virus (H5N2) isolated from a canine.

In 2009, an influenza virus (IV), A/canine/Shandong/JT01/2009 (CA/SD/JT01/09), was isolated from the dog exhibiting respiratory signs in China, and was a novel H5N2. Intraspecies transmission of the virus in dog population had thus far remained unclear. To determine whether the novel H5N2 was transmitted among dogs, we conducted contact exposure and inoculation experiments. Susceptible dogs were housed in the room which the novel H5N2 infected dogs were housed in. As a result, the direct contact resulted in intraspecies transmission. Most of the infected dogs and the sentinel animals developed mild respiratory syndrome, including transient increased body temperatures, conjunctivitis, sneezing, nasal discharge and mild coughing, virus shedding and seroconversion, but no fatal disease. These data suggest that dogs may play a role in transmission and spread of influenza virus.

[Sequence analysis of VP1 gene of the duck hepatitis A virus type 3 strains isolated from Shandong Province of China in 2012].

To reveal the genetic variation of the viral protein 1 (VP1) gene of the duck hepatitis A virus type 3 (DHAV-3), the VP1 gene of 13 virulent DHAV-3 strains isolated from Shandong province of China in 2012 were amplified by RT-PCR, sequenced and analyzed. The results showed that all the VP1 genes of the 13 isolates contained 720 nucleotides encoding 240 amino acids, and shared with nucleotide identities of 94. 6%-99.9% and amino acid identities of 95.0%-100%. The nucleotide and amino acid sequence homologies between the 13 DHAV-3 isolates and other 31 DHAV-3 reference strains were 92.5%-100% and 90. 8%-100%, respectively. Phylogenetic analysis showed that the VP1 gene of DHAV-3 had distinct geographical characteristics. Distribution of genotypes of the 44 DHAV-3 strains was as follows: except the vaccine strain B63, all the other Chinese isolates belonged to genotype I (GI), Vietnamese wild isolates mainly belonged to subtype 1 (S1) of genotype II (GII), and all Korean isolates belonged to subtype 2 (S2) of GII.

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus.

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

Improved duplex RT-PCR assay for differential diagnosis of mixed infection of duck hepatitis A virus type 1 and type 3 in ducklings.

Infection with duck hepatitis A virus (DHAV) causes an acute, rapidly spreading, and fatal disease of young ducklings. DHAV type 1 (DHAV-1) and type 3 (DHAV-3) have been identified in China. In this study, a duplex RT-PCR assay was developed to identify DHAV-1 and DHAV-3 with mixed infection. The method was shown to be high specificity and sensitivity. The minimum detection limit of the method has been determined to be 10pg total RNA templates extracted from duck liver samples or 10² copies viral RNA of DHAV-1 and DHAV-3 respectively. Using the method, from 60 clinical liver samples of 26 duckling flocks in Shandong, Guangdong, Sichuan and Henan provinces of China, 15 (57.7%) flocks were identified as mixed infection of DHAV-1 and DHAV-3, and 9 (34.6%) flocks were DHAV-1 or DHAV-3 single infection. Among them, 38.3% (23/60) of duckling samples were detected as mixed infection of DHAV-1 and DHAV-3, and 48.3% (29/60) of samples were DHAV-1 or DHAV-3 single infection. These results indicated that the improved duplex RT-PCR method provides a rapid and cost-effective laboratory differential diagnosis for mixed infection of DHAV-1 and DHAV-3 in ducklings.