Mammalian orthoreoviruses (MRVs) are widespread and infect virtually all mammals. We report here the first case of a natural mutant and reassortant serotype 3 reovirus from mink in China, known as MRV3 SD-14. Whole-genome sequence analysis showed that the MRV3 SD-14 may have resulted from a reassortment involving MRVs that infected swine, humans and mink. Interestingly, the S1 segment, which encodes the viral attachment protein σ1, which influences viral virulence and cell tropism in the host, had a stop codon mutation at amino acid 246. Surveillance of the virulence and evolution of MRVs in humans and other animals deserves more attention.

To the Editor: Parainfluenza virus 5 (PIV5), family Paramyxoviridae, genus Rubulavirus, was previously known as simian virus 5 because of its discovery in primary monkey kidney cells in 1954 (1). PIV5 was later isolated from various hosts, including humans, dogs, pigs, cats, and rodents. The neutralizing antibody for PIV5 is detectable in symptomatic and asymptomatic humans; thus, its association with human disease remains controversial (2). In addition, previous studies have not documented illness in infected animals, except kennel cough in dogs (1,3). Isolation of PIV5 from cattle has not previously been reported. Since 2012, an infectious respiratory disease has been prevalent in weaning calves (≈10 d to 2 mo of age) in Baicheng City, Jilin Province, China. Initial clinical signs included secretion of clear nasal mucus, anorexia, sluggishness, and loss of bodyweight. After 10–20 d, ≈10% of the sick calves died of dyspnea and interstitial pneumonia. Farmers observed that 80%–90% of calves in the affected farms demonstrated clinical signs, but most recovered. All attempts of local veterinarians to treat the animals with various chemical compounds and antimicrobial drugs failed. The disease persists throughout the year but occurs mainly during spring (from February through March), which has resulted in substantial economic losses in the cattle industry. To identify the causative agent of the disease, we tested 15 lung samples from calves that had died and 10 lung samples from healthy calves that were slaughtered for serum products (all from 1 farm). The samples were homogenized in phosphate-buffered saline and analyzed by using electron microscopy. Paramyxovirus-like particles were identified in the tissues of sick calves. Reverse transcription PCR with the generic primers for the paramyxovirus polymerase gene was performed (4). Of the 25 calf specimens, the 15 samples from the sick calves were positive by reverse transcription PCR, and amplicons of the expected size were obtained and sequenced. The generated sequences were closely related to the PIV5 sequences available from GenBank, particularly to sequences of the recently identified KNU-11 and SER viruses in pigs (5,6). The suspensions of lung tissue from sick calves were purified by centrifugation at 12,000 × g for 5 min, and 0.2 mL of the supernatant was added to a Vero cell monolayer in a 25-cm2 cell culture flask (EasyFlasks; Thermo Fisher Scientific, Odense, Denmark). After virus adsorption for 1 h at 37°C, the cell monolayer was rinsed with phosphate-buffered saline (pH 7.4) and then incubated in Dulbecco minimal essential medium/2% newborn calf serum at 37°C in a 5% CO2- humidified incubator. The infected cells were serially passaged every 3 days at 37°C and detected by using monoclonal antibody against SV5 (AbD Serotec; Bio-Rad, Kidlington, UK) by indirect fluorescent antibody test (7). A PIV5 strain was isolated in the cell culture and designated PIV5-BC14 (BC14 stands for Baicheng City 2014). For amplification and analysis of the full-length viral genome, 13 pairs of primers covering overlapping fragments of the genome were designed on the basis of the sequence of the PIV5 isolate KNU-11 (8). The 3′ and 5′ termini of the genome were resolved by using the 3′ and 5′ Full RACE Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China). The PIV5-BC14 genome (GenBank accession no. {type:entrez-nucleotide,attrs:{text:KM067467,term_id:689594763,term_text:KM067467}}KM067467) was 15,246 nt with coding and untranslated regions at the same positions as in other PIV5 isolates (9). However, comparison of this genome with 15 available full-length genomes of PIVs revealed that 18 nt substitutions, resulting in 9 aa changes, are found only in PIV5-BC14. Among these 9 aa changes, 5 (at positions 303, 634, 1054, 1722, and 1773) are present in an RNA-dependent RNA polymerase protein, 2 in a phosphoprotein (at positions 43 and 332), 1 in a nucleoprotein (at position 75), and 1 in a hemagglutinin–neuraminidase (at position 322). The highest nucleotide identity (99.72%/99.52%) was observed between PIV5-BC14 and porcine PIV, particularly with the SER virus isolate. This observation was confirmed after construction of a phylogenetic tree based on the 15 available nucleotide sequences of the full-length genomes (Figure). The analysis was carried out by using the maximum-likelihood method in MEGA 5.0 (10), and the reliability of tree topology was evaluated through bootstrapping with 1,000 replicates. Figure Maximum-likelihood phylogenetic tree based on the complete genome sequences of parainfluenza virus 5 (PIV5). The black triangle indicates isolate PIV5-BC14 (Baicheng City 2014). Scale bar indicates nucleotide substitutions per site. Although the pathogenic role of PIV5 infections in cattle remains unknown, no PIV5 RNA was found in any apparently healthy cattle from the same farm. This result suggests a strong relationship between the identified virus and the disease.

为了解我国规模化养貂场的貂圆环病毒感染情况,利用PCR方法对2013年9月到2014年9月采集自山东、河北和辽宁地区185份水貂样品进行检测,共得到101份阳性样品,阳性率为54.6％,并在每个地区选取1份阳性样品进行序列分析.结果表明,我国规模化养貂场水貂圆环病毒感染率较高,序列分析表明,山东地区与辽宁地区毒株序列一致,河北地区毒株略有差别.系统演化分析显示,分离获得的貂圆环病毒基因与蝙蝠圆环病毒亲缘关系一致性最高为73％,与其他圆环病毒亲缘关系较远.

为研制水貂圆环病毒(MiCV)亚单位疫苗或建立相关诊断技术,利用常规PCR扩增出优化后的貂圆环病毒Cap蛋白基因序列,并插入毕赤酵母分泌型表达载体pPICZαA中.获得的重组表达载体pPICZαA-Cap以Sac Ⅰ进行线性化后,经电击转化整合入毕赤酵母GS115菌株中,用含Zeocin的YPD平板筛选出重组子pPICZαA-Cap-GS115.PCR、SDS-PAGE和Western-blot分析鉴定的结果显示,貂圆环病毒Cap蛋白成功在毕赤酵母中分泌表达.

野生狐和貉狂犬病流行于新疆、内蒙古和黑龙江地区,是当地牛、羊和骆驼等家畜狂犬病的主要传染源.在国内尚无野生动物口服疫苗的前提下,对家畜进行狂犬病灭活疫苗接种,具有重要的公共卫生学意义.本研究利用国产犬用狂犬病灭活疫苗(CVS-11株)对牛进行了免疫效果和安全性评价.以不同剂量疫苗肌内注射免疫成年牛350头,通过比较免疫后狂犬病病毒中和抗体水平和持续期,确定犬用狂犬病灭活疫苗(CVS-11株)在牛的最佳免疫剂量为1次性注射2剂量疫苗,免疫持续期为1年.结果表明,犬用狂犬病灭活疫苗(CVS-11株)免疫牛时,具有较高的免疫原性和安全性,适用于牛等大型动物的狂犬病注射免疫.

Dear Editor,Rabies is a generally fatal disease caused by the rabies virus(RABV),and is transmitted mainly by Carnivora and Chiroptera(Fooks A R,et al.,2014;Tao X,et al.,2013).In China,stray dogs and some wild animals(e.g.,Chinese ferret badgers,foxes,and raccoon dogs)are the principal reservoirs for RABV(Hu R L,et al.,2009).Historically,rabies in wild foxes and raccoon dogs(Nyctereutes procyonoides)was recorded in the early

本试验旨在利用毕赤酵母系统表达狂犬病病毒(Rv)糖蛋白(GP)基因,并对重组蛋白进行反应原性研究.构建重组表达质粒pPiczαA-G,线性化后电转入酵母GS115中,经平板初筛及PCR鉴定正确后命名为pPiczαA-G-GS115.重组菌经1.0％甲醇诱导表达,对目的蛋白进行双抗体夹心ELISA检测、SDS-PAGE电泳、Western blot分析.结果表明,ELISA检测诱导72h重组蛋白表达量最高;SDS-PAGE分析显示目的蛋白相对分子量约为65 kD,与理论值相符.Western blot结果表明,重组蛋白可与RV阳性血清发生特异性反应.本试验为进一步研制狂犬病病毒ELISA抗体诊断试剂盒奠定基础.

<正>自2006年至今,我们连续7年在江西、浙江和安徽等省份的环千岛湖和环鄱阳湖周围地区开展了国家二类保护野生动物鼬獾的狂犬病监测,并通过文献调阅和实际检测进行了该种动物的回顾性及现行流行病学调查。结果显示,1995年以来,在上述

The genome of Irkut virus, isolate IRKV-THChina12, the first non-rabies lyssavirus from China (of bat origin), has been completely sequenced. In general, coding and non-coding regions of this viral genome are similar to those of other lyssaviruses. However, alignment of the deduced amino acid sequences of the structural proteins of IRKV-THChina12 with those of other lyssavirus representatives revealed significant variability between viral species. The nucleoprotein and matrix protein were found to be the most conserved, followed by the large protein, glycoprotein and phosphoprotein. Differences in the antigenic sites in glycoprotein may result in only partial protection of the available rabies biologics against Irkut virus, which is of particular concern for pre- and post-exposure rabies prophylaxis.

ABSTRACT An Irkut virus (IRKV) was recently isolated from a bat in China. The protective ability of rabies biologics available in the Chinese market and experimental biologics against the rabies virus (RABV) and IRKV were assessed in a hamster model via preexposure prophylaxis (PrEP) and postexposure prophylaxis (PEP) experiments. The results demonstrated that a single dose of rabies vaccine did not induce adequate protection against IRKV infection. However, routine PrEP with three doses of vaccine induced complete protection against IRKV infection. Higher doses of RABV immunoglobulins and alpha interferon were required during PEP to protect hamsters against IRKV versus RABV infection. Experimental recombinant vaccines containing IRKV glycoproteins induced more-reliable protection against IRKV than against RABV infection. Those findings may be explained by limited cross-neutralization of these viruses (confirmed via in vitro tests) in conjunction with antigenic distances between RABV and IRKV. These results indicate that the development and evaluation of new biologics for PrEP and PEP are required to ensure sufficient protection against IRKV infection in China and other territories where this virus is present.

We identified a novel mink orthoreovirus, MRV1HB-A, which seems to be closely related to human strain MRV2tou05, which was isolated from 2 children with acute necrotizing encephalopathy in 2005. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent.

Background: Emerging evidence suggests that microRNAs play a critical role in the pathogenesis of breast cancer. Several molecular epidemiological studies were conducted in recent years to evaluate the association between has-miR-146a rs2910164 polymorphism and breast cancer risk in diverse populations. However, the results remain conflicting rather than conclusive.Methodology/Principal findings: We performed a meta-analysis of 6 case-control studies that included 4238 breast-cancer cases and 4469 case-free controls. We assessed the strength of the association, using odds ratios (ORs) with 95% confidence intervals (CIs). Overall, this meta-analysis showed that the rs2910164 polymorphism was not associated with a significantly increased risk of breast cancer in all genetic models (for GC vs GG: OR = 1.00, 95% CI = 0.9021.09, P-heterpgeneity = 0.364; for CC vs GG: OR = 1.16, 95% CI = 0.9821.36, P-heterpgeneity = 0.757; for GC+CC vs GG: OR = 1.02, 95% CI = 0.9321.12, P-heterpgeneity = 0.562; for CC vs GC+GG: OR = 1.10, 95% CI = 0.9621.26, P-heterpgeneity = 0.441). However, in the stratified analysis by ethnicity, we found the rs2910164 polymorphism was associated with increased breast cancer risk among Europeans in homozygote comparison (CC vs. GG: OR = 1.29, 95% CI = 1.0221.63, P-heterpgeneity = 0.950, P = 0.032) and recessive model (CC vs. GC+GG: OR = 1.31, 95% CI = 1.0521.65, P-heterpgeneity = 0.839, P = 0.019). No publication bias was found in the present study.Conclusions/Significance: This meta-analysis suggests, for the first time, that the CC homozygote of rs2910164 may contribute to breast cancer susceptibility in Europeans.

AIM: To investigate the apoptosis-inducing effect of a laboratory-attenuated vesicular stomatitis virus (VSV) strain on HepG2 cells and to explore the underlying mechanisms. METHODS: After HepG2 cells were infected with VSV at a multiplicity of infection (MOI) of 1.0, cell viability was determined by MTT assay; morphological assessment of apoptosis was performed by acridine orange (AO)/ethidium bromide (EB) and Hoechst/PI staining; apoptotic cells were quantified by annexin V/PI double-staining and cell cycle analysis; mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining; and activation of caspase proteolytic cascade was measured with caspase-9, cas-pase-8 and -3 colorimetric assay kits. RESULTS: The attenuated VSV strain could markedly inhibit HepG2 cell proliferation in a time-dependent manner. After HepG2 cells were exposed to VSV at an MOI of 1.0 for 24 h, the percentages of early apoptotic cells (26.46% ± 6.01% vs 4.86% ± 2. 28%, t = -5.817, P < 0.01) and cells in sub-G1 phase (14.07% ± 3.83% vs 3.99% ± 1.36%, t = -4.293, P < 0.05) were increased compared with mock-infected cells. VSV infection signifcantly decreased mitochondria membrane potential (ΔΨm) (t = -4.586, P < 0.05) and increased the activity of caspase-9 and caspase-3 (both P < 0.05). CONCLUSION: Human hepatoma cell line HepG2 is highly susceptible to infection with oncolytic VSV. VSV can inhibit the proliferation of HepG2 cell and promote apoptosis through the intrinsic mitochondria pathway. VSV-induced collapse of the mitochondrial trans-membrane potential could exert a feedback effect to elicit caspase-9, and then lead to the activation of the key downstream factor caspase-3.

Zoonotic visceral leishmaniasis is an important vector-borne infectious disease in western China. In this study, an epidemiological study was carried out on the vector of zoonotic visceral leishmaniasis in rural areas from Sichuan Province, southwestern China. In the 1263 phlebotomine sandflies captured, 859 (68.01%) were females and 404 (31.99%) males, belonging to Phlebotomus chinensis (83.37%), Sergentomyia koloshanensis (6.57%), Sergentomyia squamirostris (4.04%), and Sergentomyia barraudi (6.02%), respectively. The average prevalence of Leishmania parasites in P. chinensis females was 1.98%, which was detected by real-time quantitative PCR. Phylogenetic analysis based on ITS2-rDNA revealed that Leishmania parasites detected in sandflies belonged to the L. donovani group and formed a novel haplotype. This was the first report on molecular detection of L. donovani in naturally infected P. chinensi from China.

Objective: To investigate the apoptosis-inducing effect of an attenuated vesicular stomatitis virus in cervical cancer HeLa cells,and to explore the possible mechanism.Methods: HeLa cells were infected with VSV(MOI=1.0) and the cell proliferation was determined by MTT assay at 6,12,18,24,and 30 h after infection.Morphological changes of apoptosis were observed by acridine orange(AO)/ethidium bromide(EB) staining.Annexin V/PI double-staining was performed to detect early apoptosis rate of HeLa cells and the sub-G1 apoptotic peak was examined by flow cytometry.The mitochondrial membrane potential of HeLa cells was measured by the JC-1 staining.The activities of caspase-9,-8 and-3 were measured by caspase assay kit.Results: After HeLa cells were exposed to attenuated VSV for 12 h and 24 h,the viabilities of cells were reduced to(78.4±1.9)% and(63.1±5.6)%(P0.01);the early apoptosis rates were(16.88±2.48)% and(31.9±4.24)%(P0.01);the Sub-G1 apoptotic peaks were(14.85±1.48)% and(21.05±2.28)%(P0.01),respectively.Attenuated VSV significantly decreased mitochondrial membrane potential in HeLa cells with the increase of infection time(P0.05).The activities of caspase-9 and caspase-3 of HeLa cells were significantly increased after VSV infection(all P0.05).Conclusion: The attenuated VSV can inhibit the proliferation of HeLa cells and trigger apoptosis via caspase-9 and caspase-3-dependent pathway.

In the present study, we characterized the pathways by which a laboratory-attenuated vesicular stomatitis virus (La-VSV) induces apoptosis in BHK cells. It was found that La-VSV induced a loss of mitochondrial membrane potential (ΔΨm) and activated caspase-9 and -3, but not caspase-8, indicating that the induction of apoptosis by La-VSV may involve an intrinsic apoptotic pathway. Although aberrant expression of microRNAs (miRNAs) has been linked to viral infection, little is known about changes in the cellular miRNA expression profile following VSV infection. Here, we attempted to identify miRNA expression profiles in VSV-infected BHK cells using miRNA microarray. Data analysis revealed that 28 miRNAs consistently responded to VSV-infection, 12 of which were down-regulated and 16 of which were up-regulated. miR-146a of these miRNAs has been found to be up-regulated in LPS-stimulated monocytes and VSV-infected macrophages, suggesting that VSV-induced miR-146a expression occurs not only in immune cells but also in other host cells. We further found that miR-706 inhibited VSV-induced apoptosis by decreasing caspase-3 and -9 activation, suggesting that induction of miR-706 expression may be a novel strategy for survival of VSV, allowing it to escape the apoptosis response of the host. In summary, our results indicate that miRNAs might play important roles in VSV infection and that their aberrant expression could be involved in VSV pathogenesis.

The Escherichia coli(E.coli) strain from intestinal tract of died newborn swine was isolated and cultured.Preliminary identification of the isolated strain was conducted by conventional biochemical tests,and the molecular biology detections of toxicity gene and typing gene were completed by multi-PCR.Stable toxin and heat-labile enterotoxin genes of E.coli were detected from the isolated strain.By amplifying and sequencing bacterial 16s rDNA and fliC gene,the isolated strain was identified as H10 by Blast analysis.The homology of strain H10 was 99% with bacterial 16s rDNA gene and 98% with fliC gene.

MicroRNAs (miRNAs) are small regulatory RNA molecules functioning to modulate gene expression at the post-transcriptional level, and playing an important role in many developmental and physiological processes. Ten thousand miRNAs have been discovered in various organisms. Although considerable progress has been made in computational methodology to identify miRNA targets, most predicted miRNA targets may be false positive. Due to the lack of effective tools to identify miRNA targets, the study of miRNAs is seriously retarded. In recent years, some molecular cloning strategies of miRNA targets have been developed, including RT-PCR using miRNAs as endogenous primers, labeled miRNA pull-down assay (LAMP) and RNA ligase-mediated amplification of cDNA end (RLM-RACE). The identified miRNA targets should be further validated via effects of miRNA alteration on the target protein levels and bioactivity. This review summarizes advances in strategies to identify miRNA targets and methods by which miRNA targets are validated.

To isolate avian influenza virus from suspective dead chicken caused by AIV,and detect its subtype,the SPF chicken embryo was used.After electron microscope observation of the virus from allantoi fluid,HA and HI experiments and RT-PCR were used to detect its subtype.The result showed that the AIV was confirmed as H5 subtype.RT-PCR was used to detect its HA and NA gene,its subtype was defined as H5N1.The analysis of the isolated AIV NA gene showed that this NA gene hold 97% homology with GenBank strain(EU429747),but had obvious mutation at 3′end.

The pathohistology and PCR methods were applied for the detection of PRRSV and PCV2 of sick swine tissues from a pig farm in Jilin province.The results showed there was co-infection of PRRSV and PCV2 among the sick swines.The cloning analysis of PCR amplification of PRRSV demonstrated higher identity to the base sequence of the template(EF112445.1).