An improved system to generate recombinant canine distemper virus.

BACKGROUND: Canine distemper virus (CDV) is a pathogen with the capability of cross-species transmission. It has crossed the species barrier to infect many other species, and its host range is expanding. The reverse genetic platform, a useful tool for scientific research, allows the generation of recombinant viruses from genomic cDNA clones in vitro. METHODS: To improve the reverse genetic system of CDV, a plasmid containing three independent expression cassettes was constructed for co-expression of the N, P, and L genes and then transfected with a full-length cDNA clone of CDV into Vero cells. RESULTS: The results indicated that the established rescue system has the advantages of being more convenient, easy to control the transfection ratio, and high rescue efficiency compared with the conventional reverse genetics system. CONCLUSION: This method not only reduces the number of transfection plasmids, but also improves the rescue efficiency of CDV, which could provide a reference for the recovery of other morbilliviruses.

Comparison of heterologous prime-boost immunization strategies with DNA and recombinant vaccinia virus co-expressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus in pigs.

Vaccination is principally used to control and treat porcine reproductive and respiratory syndrome virus (PRRSV) infection. This study investigated immunogenicity and protective efficacy of heterologous prime-boost regimens in pigs, including recombinant DNA and vaccinia virus vectors coexpressing PRRSV European genotype (EU) isolate GP3 and GP5: group A, pVAX1-EU-GP3-GP5 prime and rddVTT-EU-GP3-GP5 boost; group B, rddVTT-EU-GP3-GP5 prime and pVAX1-EU-GP3-GP5 boost; group C, empty vector pVAX1; group D, E3L gene-deleted vaccinia virus E3L- VTT. Vaccine efficacy was tested in an EU-type PRRSV (Lelystad virus strain) challenge pig model based on evaluating PRRSV-specific antibody responses, neutralizing antibodies, cytokines, T lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, clinical symptoms, viremia and tissue virus loads. Plasmid DNA was delivered as chitosan-DNA nanoparticles, and Quil A (Quillaja) was used to increase vaccine efficiency. All piglets were boosted 21 days post the initial inoculation (dpi) and then challenged 14 days later. At 14, 21, 28 and 35 dpi, groups A and B developed significantly higher PRRSV-specific antibody responses compared with control groups C and D. Two weeks after the boost, significant differences in neutralizing antibody and IFN-γ levels were observed between groups A, C, D and B. At 49 dpi, groups A and B had markedly increased peripheral blood CD3+CD4+ T cell levels. Following virus challenge, group A showed viremia, but organ virus loads were lower than those in other groups. Thus, a heterologous prime-boost vaccine regimen (rddVTT-EU-GP3-GP5 prime, pVAX1-EU-GP3-GP5 boost) can improve humoral- and cell-mediated immune responses to provide resistance to EU-type PRRSV infection in vivo.

Progress in PRRSV Infection and Adaptive Immune Response Mechanisms.

Since its discovery, Porcine reproductive and respiratory syndrome (PRRS) has had a huge impact on the farming industry. The virus that causes PRRS is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and because of its genetic diversity and the complexity of the immune response, the eradication of PRRS has been a challenge. To provide scientific references for PRRSV control and vaccine development, this study describes the processes of PRRSV-induced infection and escape, as well as the host adaptive immune response to PRRSV. It also discusses the relationship between PRRSV and the adaptive immune response.

An easy method to generate recombinant pseudorabies virus expressing the capsid protein of Porcine circovirus type 2d.

Introduction: Homologous recombination is an effective way to generate recombinant viruses for vaccine research such as pseudorabies virus (PRV) and adenovirus. Its efficiency can be affected by the integrity of viral genome and the linearization sites. Methods: In the study, we described a simple approach to isolate the viral DNA with high genomic integrity for large DNA viruses and a time-saving method to generate recombinant PRVs. Several cleavage sites in the PRV genome were investigated by using the EGFP as a reporter gene for identification of PRV recombination. Results: Our study showed that cleavage sites of XbaI and AvrII are ideal for PRV recombination which showed higher recombinant efficiency than others. The recombinant PRV-EGFP virus can be easily plaque purified in 1-2 weeks after the transfection. By using PRV-EGFP virus as the template and XbaI as the linearizing enzyme, we successfully constructed the PRV-PCV2d_ORF2 recombiant virus within a short period by simply transfecting the linearized PRV-EGFP genome and PCV2d_ORF2 donor vector into BHK-21 cells. This easy and efficient method for producing recombinant PRV might be adapted in other DNA viruses for the generation of recombinant viruses.

Current progress on innate immune evasion mediated by Npro protein of pestiviruses.

Interferon (IFN), the most effective antiviral cytokine, is involved in innate and adaptive immune responses and is essential to the host defense against virus invasion. Once the host was infected by pathogens, the pathogen-associated molecular patterns (PAMPs) were recognized by the host pattern recognition receptors (PRRs), which activates interferon regulatory transcription factors (IRFs) and nuclear factor-kappa B (NF-κB) signal transduction pathway to induce IFN expression. Pathogens have acquired many strategies to escape the IFN-mediated antiviral immune response. Pestiviruses cause massive economic losses in the livestock industry worldwide every year. The immune escape strategies acquired by pestiviruses during evolution are among the major difficulties in its control. Previous experiments indicated that Erns, as an envelope glycoprotein unique to pestiviruses with RNase activity, could cleave viral ss- and dsRNAs, therefore inhibiting the host IFN production induced by viral ss- and dsRNAs. In contrast, Npro, the other envelope glycoprotein unique to pestiviruses, mainly stimulates the degradation of transcription factor IRF-3 to confront the IFN response. This review mainly summarized the current progress on mechanisms mediated by Npro of pestiviruses to antagonize IFN production.

Molecular biology of canine parainfluenza virus V protein and its potential applications in tumor immunotherapy.

Canine parainfluenza virus (CPIV) is a zoonotic virus that is widely distributed and is the main pathogen causing canine infectious respiratory disease (CIRD), also known as "kennel cough," in dogs. The CPIV-V protein is the only nonstructural protein of the virus and plays an important role in multiple stages of the virus life cycle by inhibiting apoptosis, altering the host cell cycle and interfering with the interferon response. In addition, studies have shown that the V protein has potential applications in the field of immunotherapy in oncolytic virus therapy or self-amplifying RNA vaccines. In this review, the biosynthesis, structural characteristics and functions of the CPIV-V protein are reviewed with an emphasis on how it facilitates viral immune escape and its potential applications in the field of immunotherapy. Therefore, this review provides a scientific basis for research into the CPIV-V protein and its potential applications.

Isolation and pathogenicity analysis of mink orthoreoviruses.

Mammalian orthoreoviruses (MRVs) can infect many mammals including human, and numerous higher virulent MRVs have been reported in recent years. The first mink orthoreovirus was reported in China in 2011. In the present study, three new strains of mammalian orthoreoviruses were isolated from mink and found to be most closely related to human strain MRV2Tou05 and other human strains. Mink experiments demonstrated that the isolated mink reoviruses did not lead to severe pathogenicity. Viruses were eliminated within 2 weeks after infection, but they may cause viral enteritis disease in puppies.

A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post-vaccination monitoring.

BACKGROUND: Virus neutralization test (VNT) is widely used for serological survey of classical swine fever (CSF) and efficacy evaluation of CSF vaccines. However, VNT is a time consuming procedure that requires cell culture and live virus manipulation. C-strain CSF vaccine is the most frequently used vaccine for CSF control and prevention. In this study, we presented a neutralizing monoclonal antibody (mAb) based competitive enzyme-linked immunosorbent assay (cELISA) with the emphasis on the replacement of VNT for C-strain post-vaccination monitoring. RESULTS: One monoclonal antibody (6B211) which has potent neutralizing activity against C-strain was generated. A novel cELISA was established and optimized based on the strategy that 6B211 can compete with C-strain induced neutralizing antibodies in pig serum to bind capture antigen C-strain E2. By testing C-strain VNT negative pig sera (n = 445) and C-strain VNT positive pig sera (n = 70), the 6B211 based cELISA showed 100% sensitivity (95% confidence interval: 94.87 to 100%) and 100% specificity (95% confidence interval: 100 to 100%). The C-strain antibody can be tested in pigs as early as 7 days post vaccination with the cELISA. By testing pig sera (n = 139) in parallel, the cELISA showed excellent agreement (Kappa = 0.957) with VNT. The inhibition rate of serum samples in the cELISA is highly correlated with their titers in VNT (r2 = 0.903, p < 0.001). In addition, intra- and inter-assays of the cELISA exhibited acceptable repeatability with low coefficient of variations (CVs). CONCLUSIONS: This novel cELISA demonstrated excellent agreement and high level correlation with VNT. It is a reliable tool for sero-monitoring of C-strain vaccination campaign because it is a rapid, simple, safe and cost effective assay that can be used to monitor vaccination-induced immune response at the population level.

Metagenomic Analysis of Flaviviridae in Mosquito Viromes Isolated From Yunnan Province in China Reveals Genes From Dengue and Zika Viruses.

More than 6,000 mosquitoes of six species from six sites were collected and tested for their virome using metagenomics sequencing and bioinformatic analysis. The identified viral sequences belonged to more than 50 viral families. The results were verified by PCR of selected viruses in all mosquitoes, followed by phylogenetic analysis. In the present study, we identified the partial dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) sequences in mosquitoes. Metagenomic analysis and the PCR amplification revealed three DENV sequences, one of which encodes a partial envelope protein. Two ZIKV sequences both encoding partial nonstructural protein 3 and one JEV sequence encoding the complete envelope protein were identified. There was variability in the viral titers of the newly isolated virus JEV-China/YN2016-1 of different passage viruses. The newly identified Zika virus gene from ZIKV-China/YN2016-1 was an Asian genotype and shared the highest nucleotide sequence identity (97.1%) with a ZIKV sequence from Thailand isolated in 2004. Phylogenetic analysis of ZIKV-China/YN2016-1 and ZIKV-China/YN2016-2 with known Flavivirus genes indicated that ZIKV has propagated in Yunnan province, China.

Metagenomic Sequencing From Mosquitoes in China Reveals a Variety of Insect and Human Viruses.

We collected 8,700 mosquitoes in three sites in China, which belonged to seven species. Their viromes were tested using metagenomic sequencing and bioinformatic analysis. The abundant viral sequences were detected and annotated belonging to more than 50 viral taxonomic families. The results were verified by PCR, followed by phylogenetic analysis. In the present study, we identified partial viral genes of dengue virus (DENV), a novel circovirus (CCV), densovirus (DNV), Japanese encephalitis virus (JEV), and Wuhan mosquito virus (WMV) in mosquitoes. Metagenomic analysis and PCR amplification revealed three DENV sequences, which were as homologous to the NS3 gene of DENV from Singapore isolated in 2005, with at least 91% nucleotide (nt) identity. Seven fragments of JEV encoding structural proteins were identified belonging to genotype I. They all shared high homology with structural protein genes of JEV isolated from Laos in 2009. The production of infectious virus particles of the newly isolated virus YunnanJEV2017-4 increased after passage from the BHK-21 cell line to the Vero cell line. Novel circovirus-related genes were identified and as being related to an unnamed gene of a mosquito circovirus (MCCV) sequence from the USA isolated in 2011, with at least 41% nt identity: this distant relationship suggests that the parent virus might belong to a novel circovirus genus. Additionally, numerous known viruses and some unknown viruses were also detected in mosquitoes from Yunnan province, China, which will be tested for propagation.

Molecular detection and genomic characterization of Torque teno canis virus in domestic dogs in Guangxi Province, China.

The Torque teno canis virus (TTCaV) is a small virus with circular single-stranded DNA that has been reported to cause infections in dogs. The present study aimed to identify the presence of TTCaV in blood samples obtained from domestic dogs, and examine its diversity and evolution of the genomes. Five strains of TTCaV were detected, and the overall prevalence was found to be 7% (28/400). Phylogenetic analysis showed that the five genomes were closely clustered with the previously known Cf-TTV10 and LDL strains and formed a Thetatorque virus. Homology analysis of the whole genome showed a sequence identity of 94.6%-96.8% among the five genomes. The percent sequence similarity among the five complete genomes ranged from 95.3% to 97.4% and from 95.1% to 97% compared to the Cf-TTV10 and LDL strains respectively. The ORF1-encoded amino acid sequences showed 94.4%-97.2% identity among the five isolates. Our findings suggest that the TTCaV has a large genetic diversity and showed that TTCaV and canine parvovirus (CPV) co-infection exists in China. Further studies on the pathogenicity of TTCaV are required.

Development of monoclonal antibody for differentiating porcine reproductive and respiratory syndrome virus and identification of a novel non-structural protein 2 epitope peptide.

Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (NP) is the immunodominant region of PRRSV viral proteins. Non-structural protein 2 (Nsp2) and its hypervariable region play an essential role in the differential diagnosis of PRRSV. Western blot and immunofluorescence assay (IFA) analyses found that 2 out of 18 monoclonal antibodies (MAbs) recognized the NP and that 5 of 11 MAbs recognized Nsp2-120aa. IFA data demonstrated that 2 MAbs raised against the NP have a positive reaction to PRRSV; either HP-PRRSV, classic PRRSV or the vaccine strain at 1:100 dilution. Two MAbs raise against Nsp2-120aa also react positively with the classic PRRSV nor HP-PRRSV, but not with the PRRSV vaccine strain TJM-F92. Epitope mapping using truncated proteins identified a novel Nsp2-120aa epitope. In addition, we show that MAb BR/PNsp2-2A20 recognizes a 20 amino acid peptide (707) GRFEFLPKMILETPPPHPCG (727) of Nsp2. Based on our findings, we propose that MAb BR/PNsp2-2A20, raised against Nsp2-120aa of PRRSV, as a candidate specific diagnostic MAb for differentiation of the PRRSV virulent strains infected pig from vaccine strain TJM-F92 inoculated ones. The MAbs developed here have potential for use in diagnostic and research tools, including immunofluorescence assay, enzyme-linked immunosorbent assay and Western blotting.

Profiling of Host Cell Response to Successive Canine Parvovirus Infection Based on Kinetic Proteomic Change Identification.

Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the Metacore(TM) database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection.

Novel Nsp2 deletion based on molecular epidemiology and evolution of porcine reproductive and respiratory syndrome virus in Shandong Province from 2013 to 2014.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease affecting swine worldwide. In this study, a total of 385 samples were collected from Shandong pig farms during 2013 and 2014, when pigs were not inoculated with any vaccine. Results indicated that, out of 385 samples, 47 (12.21%) were PRRSV-RNA-positive. The gene sequence analysis of 12 ORF5, 12 ORF7, and 8 Nsp2 of these samples was used to determine the molecular epidemiology of PRRSV in different parts of China's Shandong Province. The phylogenetic tree based on these 3 genes indicated that the Chinese PRRSV strains could be divided into five subgroups and two large groups. The 8 study strains were clustered into subgroup IV, another 4 strains into subgroup I. The first 8 strains shared considerable homology with VR-2332 in ORF5 (96-97.5%), the other 4 strains shared considerable homology with JXA1 (94-98%). Phylogenetic tree of GP5 showed that the eight isolates formed a tightly novel clustered branch, subgroup V, which resembled but differed from isolate VR-2332. When examined using Nsp2 alone, the first 8 strains showed considerable homology with a U.S. vaccine strain, Ingelvac MLV (89.6-98.4%). One novel pattern of deletion was observed in Nsp2. The genetic diversity of genotype 2 PRRSV tended to vary in the field. The emergence of novel variants will probably be the next significant branch of PRRSV study.

Characterization of a virulent dog-originated rabies virus affecting more than twenty fallow deer (Dama dama) in Inner Mongolia, China.

Rabies has emerged as a serious problem in the most recent years in northern China. A rabies virus (RABV) isolate, IMDRV-13, was recovered from brain samples of dog-bitten rabid fallow deer (Dama dama) in a farm in Hohhot, Inner Mongolia. We tested the susceptibility of mouse neuroblastoma (MNA) cells and BSR cells as well as that of adult mice to IMDRV-13. The isolate was found to be a virulent isolate with an equivalent pathogenicity index (0.12) and a slight lower neurotropism index (1.07) compared with those of challenge virus standard, CVS-24, which was 0.13 and 1.23, respectively. The complete genome of IMDRV-13 was determined subsequently and found to be 11,924 nucleotides (nt) in length with the same genomic organization as other RABVs. Phylogenetic tree based on complete genome sequences of 43 RABV isolates and strains indicated that IMDRV-13, along with other two isolates in Inner Mongolia, CNM1101C and CNM1104D, clustered within the dog-associated China I clade, which is also the dominant lineage in the current rabies epidemic in China. In addition, sequence analysis of the glycoprotein G identified an amino acid substitution (I338→T338) unique to the IMDRV-13 within antigenic sites III (330-338), this mutation also leads to an additional potential N-glycosylation site (N336), which may represent a useful model to study relationship of N-glycosylation in G protein and specific properties such as pathogenicity or host adaption of RABV.

Enhanced immune responses in pigs by DNA vaccine coexpressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus.

The European (EU) type of porcine reproductive and respiratory syndrome virus (PRRSV) has recently emerged in China. In this study, three recombinant DNA vaccines, pVAX1-EU-ORF3-ORF5 (coexpressing EU type PRRSV GP3 and GP5), pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed and evaluated for their abilities to induce humoral and cellular responses as well as to protect piglets against homologous virus challenge. All piglets were given booster vaccinations at 21 days after the initial inoculation and then challenged 14 days later. Pigs inoculated with pVAX1-EU-ORF3-ORF5 developed significantly higher (P<0.05) PRRSV-specific antibody responses, neutralizing antibodies and levels of IL-4 and IL-10 than those given pVAX1-EU-ORF3, pVAX1-EU-ORF5 or pVAX1. Moreover, pigs immunized with pVAX1-EU-ORF3-ORF5 had markedly increased levels of IFN-γ and IL-2 in serum and T-lymphocytes (CD3(+)CD4(+) and CD3(+)CD8(+) T cells) in peripheral blood. Thus, EU-type PRRSV GP3 and GP5 proteins demonstrated good immunogenicity and reactogenicity and could induce cellular immunity in pigs. Following challenge with the Lelystad virus (LV) strain, piglets inoculated with pVAX1-EU-ORF3-ORF5 showed viremia and virus load distributed in organ tissues that were significantly lower (P<0.05) than those in the pVAX1-EU-ORF3 group and control group, and slightly lower than those in the pVAX1-EU-ORF5 group (P>0.05). As GP3 could enhance humoral- and cell-mediated immune responses to GP5, the results of this study suggested that these two proteins delivered by a vaccine can synergistically induce immunity against PRRSV.

Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus.

BACKGROUND: The European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) has recently emerged in China. The coexistence of Genotype-I and -II PRRSV strains could cause seriously affect PRRSV diagnosis and management. Current vaccines are not able to protect against PRRSV infection completely and have inherent drawbacks. Thus, genetically engineered vaccines, including DNA vaccine and live vector engineered vaccines, have been developed. This study aimed to determine the enhanced immune responses of mice inoculated with a DNA vaccine coexpressing GP3 and GP5 of a Genotype-I PRRSV. RESULTS: To evaluate the immunogenicity of GP3 and GP5 proteins from European-type PRRSV, three DNA vaccines, pVAX1-EU-ORF3-ORF5, pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed, which were based on a Genotype-I LV strain (GenBank ID: M96262). BALB/c mice were immunized with the DNA vaccines; delivered in the form of chitosan-DNA nanoparticles. To increase the efficiency of the vaccine, Quil A (Quillaja) was used as an adjuvant. GP3 and GP5-specific antibodies, neutralizing antibodies and cytokines (IL-2, IL-4, IL-10 and IFN gamma) from the immunized mice sera, and other immune parameters, were examined, including T-cell proliferation responses and subgroups of spleen T-lymphocytes. The results showed that ORF3 and ORF5 proteins of Genotype-I PRRSV induced GP3 and GP5-specific antibodies that could neutralize the virus. The levels of Cytokines IL-2, IL-4, IL-10, and IFN-γ of the experimental groups were significantly higher than those of control groups after booster vaccination (P < 0.05). The production of CD3+CD4+ and CD3+CD8+ T lymphocyte was also induced. T lymphocyte proliferation assays showed that the PRRSV LV strain virus could stimulate the proliferation of T lymphocytes in mice in the experimental group. CONCLUSIONS: Using Quil A as adjuvant, Genotype-I PRRSV GP3 and GP5 proteins produced good immunogenicity and reactivity. More importantly, better PRRSV-specific neutralizing antibody titers and cell-mediated immune responses were observed in mice immunized with the DNA vaccine co-expressing GP3 and GP5 proteins than in mice immunized with a DNA vaccine expressing either protein singly. The results of this study demonstrated that co-immunization with GP3 and GP5 produced a better immune response in mice.

Immunogenicity analysis following human immunodeficiency virus recombinant DNA and recombinant vaccinia virus Tian Tan prime-boost immunization.

This study assessed and compared the immunogenicity of various immunization strategies in mice using combinations of recombinant DNA (pCCMp24) and recombinant attenuated vaccinia virus Tian Tan (rddVTT-CCMp24). Intramuscular immunization was performed on days 0 (prime) and 21 (boost). The immunogenicity of the vaccine schedules was determined by measuring human immunodeficiency virus (HIV)-specific binding antibody levels and cytokine (interleukin-2 and interleukin-4) concentrations in peripheral blood, analyzing lymphocyte proliferation capacity against HIV epitopes and CD4(+)/CD8(+) cell ratio, and monitoring interferon-gamma levels at different times post-immunization. The results showed that pCCMp24, rddVTT-CCMp24 and their prime-boost immunization induced humoral and cellular immune responses. The pCCMp24/rddVTT-CCMp24 immunization strategy increased CD8(+) T cells and induced more IFN-γ-secreting cells compared with single-shot rDNA. The prime-boost immunization strategy also induced the generation of cellular immunological memory to HIV epitope peptides. These results demonstrated that prime-boost immunization with rDNA and rddVTT-CCMp24 had a tendency to induce greater cellular immune response than single-shot vaccinations, especially IFN-γ response, providing a basis for further studies.

Japanese encephalitis virus in mosquitoes and swine in Yunnan province, China 2009-2010.

The residential regions of Yunnan province, canton of Jing Hong, in China were surveyed for Japanese encephalitis virus (JEV) infection in mosquito and swine vectors to determine the frequency of JEV-carrying zoonotic vectors in 2009-2010. A total of 21,500 mosquitoes were collected and divided by species, and brain tissue was collected from 108 stillborn piglets. The infection rates for the different JEV species were 13.2% for Culex tritaeniorhynchus, 2.7% for Anopheles sinensis, 0.7% for Armigeres subalbatus, and 18.5% for stillborn piglets. The complete genomes of two JEV samples that were collected in different seasons and different regions, Yunnan 0901 and Yunnan 0902, were sequenced from a pool of Culex mosquitoes and stillborn piglets that had been collected randomly from several piggeries. Multiple sequence alignment with 24 fully-sequenced genes and 93 complete sequences of the JEV-encoded E gene revealed nucleotide homologies ranging from 97.2-99.6% and 94.5-99.7% in mosquitoes and piglets, respectively, and deduced amino acid homologies ranging from 97.4-98.1% and 96.0-98.2%, respectively. Phylogenetic analyses of the Yunnan 0901 and Yunnan 0902 strains' full-length genomes and E gene sequences indicated that these strains are most closely related to six Chinese SA14-derived viruses, and distantly related to the Australian FU, vellore P20778, and Japanese Ishikawa strains, and the previously isolated YN86-B8639 strains. The phylogenetic relationships based on the full-length genome were similar to those found for the E gene, indicating that phylogenetic analysis of the E gene will be a useful approach for genotyping of JEV, but not to better understand the potential changes in the biological characteristics and genetic relationship of JEV isolates.

Reverse transcription loop-mediated isothermal amplification for rapid detection of Japanese encephalitis virus in swine and mosquitoes.

Japanese encephalitis (JE) can infect many agriculturally important animals and humans, and has a high incidence in Asia. One of the natural hosts of the mosquito-borne JE virus (JEV) is domestic pigs, which act as amplifier hosts. Porcine infection results in fatal encephalitis, abortion, and stillbirth in pregnant sows, and hypospermia in boars. In this study, a rapid JEV detection method for swine and mosquitoes was developed based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting the nucleocapsid (E) genes of JEV genotype I (lineage K94PO5), and genotype III (lineage SA14-14-2). Fifty-six swine blood samples and 20,000 mosquitoes were used to evaluate the method, compared to conventional RT-polymerase chain reaction (PCR) and real-time RT-PCR. RT-LAMP had detection limits of 2.57 and 2.34 copies/µL for JEV I and III, respectively. Assay sensitivity was similar to real-time RT-PCR, but was 10-fold higher than conventional RT-PCR. Assay specificity was high, showing no cross-reactivity to other flaviviruses. The results of virus isolation and identification of swine blood samples and mosquito samples were fully consistent with RT-LAMP. Finally, the JEV RT-LAMP assay was simpler and less time consuming than conventional RT-PCR or real-time RT-PCR, since the amplification step could be completed in a single tube within 50 min at 63°C. In conclusion, the newly-developed RT-LAMP assay is an accurate and convenient method for rapid and sensitive diagnosis of JEV in swine and mosquitoes, and may prove to be a practical molecular tool for surveillance and epidemiological investigations.