In contrast to dog-associated human rabies cases decline year by year due to the rabies vaccination coverage rates increase in China, ferret badger (FB, Melogale moschata)-associated human rabies cases emerged in the 1990s, and are now an increasingly recognized problem in southeast China. To investigate epidemiology, temporal evolution dynamics, transmission characterization, and pathogenicity of FB-associated rabies viruses (RABVs), from 2008 to 2018, we collected 3,622 FB brain samples in Jiangxi and Zhejiang Province, and detected 112 RABV isolates. Four FB-related lineages were identified by phylogenetic analysis (lineages A-D), the estimated Times to Most Recent Common Ancestor were 1941, 1990, 1937, and 1997 for lineages A-D, respectively. Furthermore, although no FB-associated human rabies case has been reported there apart from Wuyuan area, FB-RABV isolates are mainly distributed in Jiangxi Province. Pathogenicity of FB-RABVs was assessed using peripheral inoculation in mice and in beagles with masseter muscles, mortality-rates ranging from 20 to 100% in mice and 0 to 20% in beagles in the groups infected with the various isolates. Screening of sera from humans with FB bites and no post-exposure prophylaxis to rabies revealed that five of nine were positive for neutralizing antibodies of RABV. All the results above indicated that FB-RABV variants caused a lesser pathogenicity in mice, beagles, and even humans. Vaccination in mice suggests that inactivated vaccine or recombinant subunit vaccine products can be used to control FB-associated rabies, however, oral vaccines for stray dogs and wildlife need to be developed and licensed in China urgently.

Complex rabies transmission dynamics, including in dogs, wildlife livestock, and human-acquired rabies, can be observed in China. A temporary decrease in human rabies deaths with a simultaneous increase in animal rabies transmission is a typical example of “sectoral management separation” but not of the recommended “one-health” concept. In contrast to reliance on mass dog vaccination, reliance on postexposure prophylaxis to reduce human rabies burden is costly and ineffective in the prevention of rabies transmission from dogs to humans and other susceptible animal species. To answer the WHO call for the “elimination of dog-mediated human rabies by 2030,” China faces the challenge of a lack of a strong political commitment and a workable plan and must act now before the rabies transmission dynamics become increasingly complicated by spreading to other species, such as ferret badgers in the Southeast and raccoon dogs and foxes in the North.

Mink refractory diarrhea is a seasonal disease that occurs in many mink farms in China. Mink circovirus (MiCV) has been recognized as the causative agent of the disease. The aim of the study was to develop a subunit vaccine against mink refractory diarrhea. A recombinant baculovirus strain expressing the capsid protein was constructed using the baculovirus expression vector system (BEVS). A subunit vaccine was developed based on the capsid protein with appropriate adjuvant. Then, a field trial was carried out in two districts in order to evaluate the efficiency of the subunit vaccine. The field trial indicated that in total, only 1.8% of the minks developed typical diarrhea in the vaccinated group compared with 74.5% in the control group. The vaccination could significantly reduce the infection rate of MiCV among the mink herds and could restrain the virus’ shedding from feces. Furthermore, the vaccinated group had a higher average litter size in the following year compared to the control group. Collectively, the results indicated that the subunit vaccine based on the capsid protein can provide reliable protection against MiCV infection.

In China, cats cause about 5% of human rabies cases. Rabies control in cats plays a role in achieving the ultimate goal of elimination of dog rabies-mediated human deaths. However, there is no cat-specific rabies vaccine in China yet. In this study, we constructed a recombinant rabies vaccine by using a felid herpesvirus 1 (FHV-1) isolate, and deleted the gI/E in the FHV-1 and replaced the region with a glycoprotein (G) of rabies virus (RABV) strain BD06 through homologous recombination. The recombinant virus FHV-RVG was recovered and purified, and the expression of RABV glycoprotein was verified by indirect immunofluorescent assay. For potency in cats, each animal was inoculated intranasally with 1 ml FHV-RVG at 106.5 TCID50. Blood samples were collected at defined intervals for antibody titration. The animals were challenged by herpes and rabies after completion of vaccination on day 180 and day 194, respectively. Our results demonstrated all vaccinated cats generated antibodies against both FHV-1 and RABV, and reached an arbitrary protective titer > 0.5 IU/ml for rabies viral neutralizing antibody (VNA) by day 14 post inoculation (dpi) and titer peaked on 30 dpi with VNA at 24.5 ± 10.23 IU/ml. All vaccinated cats presented no clinical signs of FHV-1 infection and survived rabies challenge, while the control cats had severe rhinotracheitis and died from rabies after challenge. All this demonstrated that the FHV-based recombinant vaccine is effective in protection against both FHV-1 and RABV infections.

Lyssaviruses, including Rabies virus, Duvenhage virus, European bat lyssavirus 1, European bat lyssavirus 2, Australian bat lyssavirus, and Irkut virus (IRKV), have caused human fatalities, but infection of IRKV in dogs has not been previously reported. In China, a dead dog that previously bit a human was determined to be infected with IRKV. Pathogenicity tests revealed that IRKVs can cause rabies-like disease in dogs and cats after laboratory infection. The close relationship between humans and pets, such as dogs and cats, may generate a new spillover-spreading route for IRKV infection. Therefore, additional attention should be paid to trans-species infection of IRKV between bats and dogs or dogs and humans through investigation of the prevalence and circulation patterns of IRKV in China.

Chinese ferret badger (FB)-transmitted rabies is a serious threat to public health in southeast China. Although mostly associated with dogs, the rabies virus (RABV) presents genetic diversity and has a significantly wide host range in China. Instead of the dog- and wildlife-associated China II lineage in the past decades, the China I lineage has become the main epidemic group hosted and transmitted by dogs. In this study, four new lineages, including 43 RABVs from FBs, have been classified within the dog-dominated China I lineage since 2014. FB RABVs have been previously categorized in the China II lineage. Moreover, FB-hosted viruses seem to have become the main independent FB-associated clade in the phylogenetic tree. This claim suggests that the increasing genetic diversity of RABVs in FBs is a result of the selective pressure from coexisting dog rabies. FB transmission has become complicated and serious with the coexistence of dog rabies. Therefore, apart from targeting FB rabies, priority should be provided by the appropriate state agencies to perform mass immunization of dog against rabies.

Rabies features encephalitis and neurological symptoms. Although its clinical course can be prolonged by life support measures, its fatal outcome is virtually inevitable. Coronary artery disease has become so common that anyone complaining of chest discomfort is suspected. Despite the fact that rabies encephalitis is often complicated with heart impairment or myocarditis, it can still be ignored when cardiac symptoms like chest discomfort or shortness of breath act as chief complaints. This case report demonstrates a rabies patient presenting with conditions similar to acute coronary syndrome; he even underwent coronary angiography. He was the only rabies patient admitted by cardiologists in our hospital. This case reminded us that some unusual cause of cardiac manifestations needs to be considered during daily management of chest discomfort.

Rabies is a lethal neurological disease caused by the neurotropic rabies virus (RABV). To investigate the innate immune response in the brain during rabies infection, key gene transcripts indicative of innate immunity in a mouse model system were measured using real-time RT-PCR. Mice were infected via the intracerebral or intramuscular route with either attenuated rabies virus (SRV9) or pathogenic rabies virus (BD06). Infection with SRV9 resulted in the early detection of viral replication and the rapid induction of innate immune response gene expression in the brain. BD06 infection elicited innate immune response gene expression during only the late stage of infection. We measured Na-fluorescein uptake to assess blood-brain barrier (BBB) permeability, which was enhanced during the early stage of SRV9 infection and significantly enhanced during the late stage of BD06 infection. Furthermore, early SRV9 replication increased the maturation and differentiation of dendritic cells (DCs) and B cells in the inguinal lymph nodes and initiated the generation of virus-neutralizing antibodies (VNAs), which cooperate with the innate immune response to eliminate virus from the CNS. However, BD06 infection did not stimulate VNA production; thus, the virus was able to evade the host immune response and cause encephalitis. The rabies virus phosphoprotein has been reported to counteract IFN activation. In an in vitro study of the relationship between IFN antagonism and RABV pathogenicity, we demonstrated that SRV9 more strongly antagonized IFN activity than did BD06. Therefore, there is no positive relationship between the IFN antagonist activity of the virus and its pathogenicity.

Objective To express the N protein of Peste des petits ruminants virus (PPRV) in prokaryotic cells,purify the expressed product and develop an indirect ELISA for its antibody.Methods The N gene sequence of PPRV was synthesized,based on which a recombinant plasmid pSumo-mut-N was constructed and transformed to E.coli Arctic Express for expression under induction of IPTG.The IPTG concentration and time for induction were optimized.The expressed fusion protein was purified by Ni column affinity chromatography,and used as a coating antigen for development of an indirect ELISA method for PPRV antibody.Clinical samples were determined by the developed method.Results Restriction analysis and sequencing proved that recombinant plasmid pSumo-mut-N was constructed correctly.The condition for induction of expression of N protein was optimized as induction with 0.5 mmol/L IPTG at is 11 ℃ for 10 h.The expressed recombinant protein,with a relative molecular mass of about 71 900,reached a purity of more than 80％ after purification and a concentration of 120 μg/ml,which showed good reactogenicity with PPRV positive antibody.The coincidence rate of determination result by the indirect ELISA developed with the N protein and that of known serum samples was 100％.Conclusion The N protein of PPRV was expressed in prokaryotic cells and purified,with which the developed ELISA method laid a foundation of preparation and application of indirect ELISA kit for PPRV antibody,and of an important significance in epidemiological monitoring and control of epidemic of PPRV.

Rabies remains a continuous threat to public health in Beijing.In this study,a total of 224 brain tissues were collected from suspected infected stray dogs within Beijing between January 2015 and December 2016.Among them,total of 67 samples were diagnosed positive for rabies.In the phylogenetic analysis,rabies in Beijing is currently a relatively independent public health issue originating from local rabid dogs apart from the imported cases from elsewhere in the country.Because vaccination of unregistered dogs against rabies is still neglected in Beijing and other regions of China,national and local authorities should play central roles in all related aspects,such as development of policies,engagement of stakeholders for public and professional education,entire vaccination process,and animal management.

Rabies, a zoonotic disease, causes > 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and hPAB, were coated with adjuvant canine-Gp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water (O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5–6 IU/mL in mice and 7–9 IU/mL in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%–80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.

A pair of specific primers were designed according to the whole genomic sequence of porcine circovirus type2( PCV2 ) published on GenBank. The suspicious samples were collected from large scale pig farms in different areas of Guangdong province and examined in PCR and resulting amplicons were cloned and sequenced. The results showed that nucleotide sequences of 11 PCV2 isolates were stable and their similarities were 93.7% to 99.8% with the reference strain published in the GenBank. The complete sequences of 10 PCV2 isolates were1767 bp and belonged to PCV2b subtype. However, the complete sequence of one PCV2 isolate was 1768 bp and belonged to PCV2a subtype.

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.

In contrast to many countries where rabies has been well controlled in humans and livestock, even in wildlife, rabies is still endemic in almost regions of China. In Northwest China, rabies transmitted by stray dogs and wild foxes has caused heavy economic losses to local herdsmen, as well as causing numbers of human cases. In this study, as part of an investigation of ways to prevent rabies epidemics in livestock, we report an analysis of domestic cattle and camel rabies cases in Ningxia Hui (NHAR) and Inner Mongolia Autonomous Region (IMAR) and the immune efficacy of canine inactivated rabies vaccines in these animals. We found that rabies viruses from these animals are closely related to dog-hosted China I and fox-associated China III lineages, respectively, indicating that the infections originated from two different sources (dogs and wild foxes). As well as the previously reported Arctic and Arctic-related China IV lineage in IMAR, at least three separate phylogenetic groups of rabies virus consistently exist and spread throughout Northwest China. Since there is no licensed oral vaccine for wild foxes and no inactivated vaccine for large livestock, local canine inactivated vaccine products were used for emergency immunization of beef and milk cattle and bactrian (two-humped) camels in local farms. Compared with a single injection with one (low-efficacy) or three doses (high-cost), a single injection of a double dose of canine vaccine provided low-price and convenience for local veterinarians while inducing levels of virus neutralizing antibodies indicative of protection against rabies for at least 1 year in the cattle and camels. However, licensed vaccines for wildlife and large domestic animals are still needed in China.

为评价水貂出血性肺炎、多杀性巴氏杆菌病、肺炎克雷伯杆菌病三联灭活疫苗的质量,本研究对该三联灭活疫苗进行了疫苗的单剂量、单剂量重复和超剂量免疫的安全性试验,最小免疫剂量、免疫期等效力试验以及疫苗保存期试验.结果表明,该三联灭活疫苗对2月龄以上水貂安全,最小免疫保护剂量为o.5 mL(绿脓杆菌、多杀性巴氏杆菌和肺炎克雷伯杆菌含量分别为3.0×109 c fu/mL、3.0×109 cfu/mL和2.5×109 c fu/mL),免疫剂量为1.0 mL/只时即可为水貂提供8个月的完全免疫保护,疫苗在2℃～8℃贮藏15个月仍保持原有的免疫保护效率.本研究研制的该三联灭活疫苗使用安全,对受试动物能够提供100％的免疫保护.本研究为水貂出血性肺炎、多杀性巴氏杆菌病和肺炎克雷伯杆菌病的预防和该三联灭活疫苗的正确使用提供了参考依据.

目的 探讨被动免疫的狂犬病抗血清和免疫球蛋白(IgG)对小鼠的攻毒保护作用及其可能的作用机制.方法 单独使用IgG,按照125、250、1 000 IU/kg体重的剂量,分别在攻击致死剂量的强毒前后注射平均体重(20±1)g的昆明小鼠,观察14 d(脑内攻毒)或28 d(外周攻毒)后,记录小鼠发病及死亡情况,计算存活率.同时以类似的方法使用不同效价的抗血清进行试验.结果 在攻毒前先注射IgG或抗血清的条件下,小鼠的存活率与被动免疫的IgG剂量呈正比,当IgG剂量达1 000 IU/kg时,小鼠可得到完全保护;在脑内攻毒后注射IgG或抗血清的条件下,无论被动免疫的IgG剂量高低,均不能提供任何保护;但在外周攻毒3d后注射IgG,高剂量的IgG可提供部分保护.结论 血脑屏障可能允许部分IgG进入脑内,并在脑内病毒感染早期阻断病毒复制,但病毒在脑内开始复制后,给予任何剂量的IgG均不能对病毒感染提供保护.

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.

目的 构建表达狂犬病病毒(rabies virus, RABV)BD06株磷蛋白(phosphoprotein,P)的重组人5型腺病毒,并进行鉴定.方法 质粒pMD 18-T-BD06P经双酶切后,获得完整的P蛋白基因,将其克隆至腺病毒表达系统穿梭质粒pacAd5CMVK-NpA,构建重组穿梭质粒pacAd5CMV-BD06P,与骨架质粒pacAd5 9.2-100经Pac Ⅰ酶切线性化后,共转染HEK293AD细胞,经同源重组,获得表达RABV P蛋白的重组人5型腺病毒rAd5-BP.采用Kaber法计算rAd5-BP滴度,RT-PCR、Western blot、直接免疫荧光方法(direct immunofluorescence assay,dFA)检测P蛋白基因在HEK-293AD细胞的转录及表达水平.以107 TCID50 rAd5-BP肌肉注射免疫昆明小鼠,14 d后,经尾静脉采血,分离血清,IFA法检测抗RABVP蛋白抗体及其反应活性.结果 经酶切及测序鉴定证明重组穿梭质粒pacAd5CMV-P构建正确;重组腺病毒rAd5-BP滴度可达108 TCID50/ml,能够介导P蛋白在HEK293AD细胞中的转录及表达,接种小鼠后可刺激机体产生针对RABVP蛋白抗体,且与RABV具有良好的反应活性.结论 成功构建了RABVBD06株P蛋白重组人5型腺病毒,该病毒能够介导P蛋白在小鼠体内有效表达,刺激机体产生针对RABVP蛋白抗体,且与RABV具有良好的反应活性,为进一步研究RABV P蛋白在病毒感染过程中的作用奠定了基础.

Pseudorabies (PR, Aujeszky's disease) is an acute, highly contagious viral disease resulting in major economic losses to the swine industry. PR is endemic in wild and domestic animals, although its natural host is the pig. Here, we report an outbreak of PR in foxes on a fur-producing farm in Yuncheng county, Shandong, China, that were fed pig offal. The diagnosis of PR was based on nervous signs and standard PCR methods and by isolation of PRV from fox brain tissue in Vero cells. The diagnosis was confirmed by an indirect immunofluorescence assay and electron microscopy. Phylogenetic analysis of a partial (804 nt) viral glycoprotein gC gene sequence indicated that it was likely to be a field strain closely related to a cluster of PRV previously identified in China.