Toward the development of a one-dose classical swine fever subunit vaccine: antigen titration, immunity onset, and duration of immunity

Highly contagious classical swine fever (CSF) remains a major trade and health problem in the pig industry, resulting in large economic losses worldwide. In CSF-endemic countries, attenuated CSF virus (CSFV) vaccines have been routinely used to control the disease. However, eradication of CSFV in a geographical area would require permanent reduction to zero presence of the virus. It is therefore of paramount importance to develop a safe, potent, and non-infectious CSF vaccine. We have previously reported on a cost-effective CSF E2 subunit vaccine, KNB-E2, which can protect against CSF symptoms in a single dose containing 75 µg of recombinant CSFV glycoprotein E2. In this study, we report on a series of animal studies undertaken to elucidate further the efficacy of KNB-E2. We found that pigs vaccinated with a single KNB-E2 dose containing 25 µg of recombinant CSFV glycoprotein E2 were protected from clinical symptoms of CSF. In addition, KNB-E2-mediated reduction of CSF symptoms was observed at two weeks post-vaccination and the vaccinated pigs continued to exhibit reduced CSF clinical signs when virus challenged at two months and four months post-vaccination. These results suggest that KNB-E2 effectively reduces CSF clinical signs, indicating the potential of this vaccine for safely minimizing CSF-related losses.

Genetic Diversity of Echinococcus granulosus Genotype G1 in Xinjiang, Northwest of China

Cystic echinococcosis (CE) caused by E. granulosus is a serious helminthic zoonosis in humans, livestock and wildlife. Xinjiang is one of high endemic province for CE in China. A total of 55 sheep and cattle livers containing echinococcal cysts were collected from slaughterhouses in Changji and Yining City, northern region of Xinjiang. PCR was employed for cloning 2 gene fragments, 12S rRNA and CO1 for analysis of phylogenetic diversity of E. granulosus. The results showed that all the samples collected were identified as G1 genotype of E. granulosus. Interestingly, YL5 and CJ75 strains were the older branches compared to those strains from France, Argentina, Australia. CO1 gene fragment showed 20 new genotype haploids and 5 new genotype haplogroups (H1-H5) by the analysis of Network 5.0 software, and the YLY17 strain was identified as the most ancestral haplotype. The major haplotypes, such as CJ75 and YL5 strains, showed identical to the isolates from Middle East. The international and domestic trade of livestock might contribute to the dispersal of different haplotypes for E. granulosus evolution.

Generation and characterization of monoclonal antibody against glycoprotein VP1 encoded by caprine enterovirus CEV-JL14 / 中国兽医学报

VP1 gene encoded by the newly identified caprine enterovirus CEV-JL 14 was amplified and cloned to prokaryotic expression vector pGEX-4T-1.Recombinant GST-VP1 fusion protein was expressed,purified,emulsified with Freund's complete adjuvant and used to immunize the BALB/c mice following a standard procedure.The spleen cells from immunized mice were collected and fused with myeloma cells after its antibody titer reached over 104 times detected by indirect ELISA.Hybridoma cell clones secreting monoclonal antibodies against VP1 were screened and their stability and specificity were further determined.The identified hybridoma cells were injected to mice intraperitoneally and ascites were collected at 7 DPI.Isotypes of the monoclonal antibodies against the recombinant VP1 protein were characterized to be either IgG1 or IgG2b,which showed a high specificity for detection of caprine enterovirus antigens by immunoperoxidase monolayer assay,thus laying a solid basis for future study related to viral pathogenesis,detection and diagnostics for caprine enterovirus infection.

Detection and identification of a new bat bocavirus species in Yunnan / 军事医学

Objective To discover more novel bat viruses and molecularly characterize bat-borne bocavirus diversity in Yunnan.Methods Twenty-six Aselliscus stoliczkanus were sampled in Jinghong , Yunnan, and subjected to viral metagenomic analysis.Specific PCR was used to detect any bocavirus in these samples based on the metagenomic result , while full genome was amplified and compared with other bocaviruses .Results and Conclusion Totally, 3 of the 26 (11.5%) bats were positive for bocavirus, the full genome of which contained 5203 nucleotides and could encode NS1, NP and VP1/VP2 proteins.Phylogenetic analysis showed that this virus shared up to 58.7% and 53.3% amino acid identities with canine bocavirus 1 and canine minute virus .According to ICTV criteria (85%amino acid sequence identity ) on a new species of bocavirus , this virus could be a novel species within genus Bocaparvovirus .This study provides important data to better understand viral diversity in bats and to uncover the relationship between bocavirus and its hosts .

Isolation and identification of the Akabane virus from mosquitoes in Yunnan Province, China / 病毒学报

To evaluate the prevalence of mosquito-borne viruses in Manshi and Ruili (Yunnan Province, China), we collected 2 149 mosquitoes (17 species) in August 2010. Virus isolation was undertaken by the cul- ture of baby hamster kidney cells (BHK-21 cells). Two virus-like isolates were obtained: DHL10M117 was isolated from collected in Mangshi; DHL10M110 was obtained from Anopheles vagus collected in Rui- li. Both isolates caused cytopathic effects,illness and death in suckling mice inoculated with these isolates via the intracerebral route. Two positive amplicons, 702-bp from the S segment and 456-bp from the M segment,were obtained using reverse transcription-polymerase chain reaction using primers specific for the Akabane virus (AKV). Phylogenetic analysis suggested that these two virus stains had a distant relation- ship with AKVs from Kenya and Australia,but were genetically close to those from Japan,South Korea, and Taiwan. However,they were separate from other Asian strains and grouped into a small branch. The highest nucleotide and amino-acid sequence identity of the S segment was found with the CY-77 strain from Taiwan (96.6% and 99.6% for DHL10M117 and 96.7% and 100% for DHL10M110,respectively). Com- parison of the M segment showed they shared the highest amino acid identity with CY-77 (99.6% and 100%, respectively), whereas the highest nucleotide identity was found with the Iriki strain from Japan (99.6% and 100%, respectively). Compared with the MP496 strain from Kenya,they displayed lower lev- els of sequence homology, at 69.7% and 70.0% for nucleotide sequences of the two loci,and 91. 0% for a- mino acids. Our results identified that DHL10M117 and DHL10M110 were strains of AKV,and provided molecular biological evidence for the existence of AKV in Yunnan Province. These AKV strains that are circulating in Yunnan Province share a close genetic relationship with strains from the rest of Asia. Culex tritaeniorhynchus and Anopheles vagus may serve as transmission vectors.

Establishment of a One-Step Real-Time RT-PCR Method for the Detection of Venezuelan Equine Encephalitis Virus / 病毒学报

Venezuelan equine encephalitis (VEE) is a zoonotic disease caused by the Venezuelan equine encephalitis virus (VEEV) complex. This disease has not yet been reported in China, and it is therefore essential to establish a rapid and accurate method for detection of the virus in order to prevent and control this disease. In this study, a one-step real-time quantitative RT-PCR method was developed for the detection of the VEEV complex. A pair of specific primers and a Taqman probe were designed corresponding to a conserved region of the VEEV gene nspl, allowing the detection of all known strains of different sub- types of the virus. Using RNA synthesized by in vitro transcription as template, the sensitivity of this method was measured at 3.27 x 10(2) copies/microL. No signal was generated in response to RNA from Chikungunya virus (CHIKV), nor to RNA encoding the nsp1 fragment of Eastern equine encephalitis virus (EE-EV) or Western equine encephalitis virus (WEEV), all of which belong to the same genus as VEEV. This indicates that the method has excellent specificity. These results show that this one-step real-time quantitative RT-PCR method may provide an effective tool for the detection of VEEV in China.

Proteomic Analyses of Purified Particles of the Rabies Virus / 病毒学报

The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication.

Metagenomic analysis of bat virome in several Chinese regions / 生物工程学报

Bats are important reservoir animals and more than 60 viruses have been identified in bats with many of them highly pathogenic to human. In order to understand the natural background, genetic diversity of bat viruses in China and discover potential viral pathogens, Solexa sequencing based viral metagenomics focusing on bats tissues was established and to analyze the virome of bats collected from Jilin, Yunnan and Hunan province. By Solexa sequencing, 116 442 324 useful reads were obtained and assembled into 4 872 contigs, of which 8.2% (4 002/4 4872) were annotated to 36 viral families, including 19 vertebrate virus families, 6 plant virus families, 4 insect virus families and 4 phages. Further contigs analyses showed that some adenovirus, bocavirus, picobirnavirus, parvovirus contigs sequences were similar with known viruses. However, part of them shared limited identities to these viruses implying the discovery of new viruses. Moreover, PCR validation of adenovirus and bocavirus confirmed the results obtained by viral metagenomics. This study aimed to understand bat virome in China by viral metagenomics and could be helpful to establish effective surveillance on wildlife-associate zoonoses.

Quantitative detection of anti-classical swine fever virus siRNA expression in cells by stem-loop RT-qPCR / 生物工程学报

RNA interference (RNAi) is a promising technology in development of specific antiviral therapy, but the quantitative detection of small interfering RNA (siRNA) expressed in vivo is the main challenge to assess its antiviral effect. In order to detect the siRNA molecules (siN1 and SiN2) particularly expressed in cells to inhibit the replication of classical swine fever virus (CSFV), serial specific stem-loop primers were designed and synthesized. Two of them (SLP-N1-6 and SLP-N2-8) were selected by screening in cross combination and successfully used in establishment of an optimal stem-loop RT-qPCR, which showed high specificity and sensitivity in detection of anti-CSFV siRNA expressed in PK-15 cells. The method was capable of detecting 10(2) to 10(8) copies of siRNA molecule with good parallel relationship (R(sq) = 0.999) and high amplification efficiency (Eff. = 98.2%). Therefore, the established stem-loop RT-qPCR can be used as an ideal tool in quantitative assessment of the anti-CSFV effects of RNAi in combination with detection of viral antigens using indirect immunofluorescent assay and TCID50, providing a novel technique for evaluating the antiviral effects of the siRNA expressed in anti-CSFV transgenic pigs to be established in future.

Prediction,synthesis and characteristics of B-cell epitopes of CSFV envelope glycoprotein E2 / 中国免疫学杂志

Objective:Study on characteristics of two synthesizd peptides based on CSFV E2 protein. Methods:B cell epitopes of CSFV E2 antigen were predicted using accessibility and flexibility schemes, associated with antigenicity , secondary structure and multiple sites prediction. Two antigen peptides (Pep1 and Pep2) have been designed and synthesized and their reactivety were detected with 8 McAbs and antiserum against mE2 protein, then the peptides were conjugated with BSA and immunized rabbits respectively. Results:Both Pep1 and Pep2 could react with antiserum and McAb A11, Pep2 could interact with McAbD5 and McAbD8. Only Pep1 BSA conjugate can stimulate high level and specific antibodies.Conclusion: The peptide1 has good antigenicity.