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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.

Background Aleutian mink disease parvovirus (AMDV) causes Aleutian mink disease (AMD), which is a serious infectious disease of mink. The aim of this study was to get a better understanding of the molecular epidemiology of AMDV in northeast China to control and prevent AMD from further spreading. This study for the first time isolated AMDV from fecal swab samples of mink in China. Results A total of 157/291 (54.0%) of the fecal swab samples were positive for AMDV. Of these, 23 AMDV positive samples were randomly selected for sequence alignment and phylogenetic analysis based on the acquired partial fragments of VP2 gene with the hypervariable region. Comparative DNA sequence analysis of 23 AMDV isolates with a reference nonpathogenic (AMDV-G) strain revealed 8.3% difference in partial VP2 nucleotide sequences. Amino acid alignment indicated the presence of several genetic variants, as well as one single amino acid residue deletion. The most concentrated area of variation was located in the hypervariable region of VP2 protein. According to phylogenetic analysis, the Chinese AMDV strains and the other reference AMDV strains from different countries clustered into three groups (clades A, B and C). Most of the newly sequenced strains were found to form a Chinese-specific group, which solely consisted of Chinese AMDV strains. Conclusion These findings indicated that a high genetic diversity was found in Chinese AMDV strains and the virus distribution were not dependent on geographical origin. Both local and imported AMDV positive species were prevalent in the Chinese mink farming population. The genetic evidence of AMDV variety and epidemic isolates have importance in mink farming practice.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease that represents a serious problem in the deer-rearing industry. To better understand an MCF-like disease that has emerged in northern China since 2015, we investigated ten cases by documenting clinical and epidemiological data and analysing causative agents and histopathological changes. In addition, a retrospective screen for Macavirus DNA and a questionnaire-based survey were conducted. Epizootic MCF in Chinese sika deer herds has emerged with a low morbidity of 3.8% (95% CI: 2.5%-5.1%) and a high mortality of 93.2% (95% CI: 86.6%-99.9%). The disease course varied from 3 to 12 days. Aetiologically, OvHV-2 was predominant in the MCFV, accounting for most MCF cases (21/23). In contrast, only two CpHV-2 isolates were phylogenetically closely related to CpHV-2. Diarrhoea and nasal discharges were the most frequent manifestations, although clinical signs varied in some cases. Pathologically typical lesions of haemorrhage, necrosis and lymphoid cell infiltration were readily observed in a variety of organs. Vasculitis caused by vascular and perivascular lymphoid cell infiltration was common. The retrospective survey suggested a low positive rate (3/275) of MCFV DNA in peripheral blood lymphocytes (PBL). The questionnaire-based survey suggested the disease was neglected by local veterinarians, who did not acknowledge the risk of co-rearing deer with reservoir species. Collectively, the emerging epizootic MCF in Chinese sika deer herds remains neglected, emphasizing the urgency of initiating full-field diagnoses and control strategies.

After antigen stimulation, T cells preferentially increase aerobic glycolysis to meet the bioenergetic and biosynthetic demands of T cell activation, proliferation, and effector functions. Lactate, a by-product of glycolysis, has been reported to function as an important energy source and signaling molecule. Here, we found that lactate anions are involved in cytokine production in T cells after TCR activation. During ex vivo T cell activation, the addition of excess sodium lactate (NaL) increased the production of cytokines (such as IFNγ/IL-2/TNFα) more than the addition of sodium chloride (NaCl). This enhanced cytokine production was dependent on TCR/CD3 activation but not CD28 activation. In vivo, NaL treatment inhibited tumour growth in subcutaneously transplanted tumour models in a T cell-dependent manner, which was consistent with increased T cell cytokine production in the NaL treatment group compared to the NaCl treatment group. Furthermore, a mechanistic experiment showed that this enhanced cytokine production was regulated by GAPDH-mediated post-transcriptional regulation. Taken together, our findings indicate a new regulatory mechanism involved in glycolysis that promotes T cell function.

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophage-specific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype after lipopolysaccharide (LPS) treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the granulocyte-macrophage colony-stimulating factor/STAT5 signaling pathway. Pathological changes in mice with dextran sodium sulfate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine.

Broad coverage of mink enteritis virus (MEV) vaccination program in northeast of China has provided effective protection from mink viral enteritis. Nevertheless, MEV vaccine failures were reported due to continually evolving and changing virulence of field variants or wild-type MEV. In this study, a combined loop-mediated isothermal amplification (LAMP) and single nucleotide polymorphism (SNP) method, named LAMP-SNP assay, was developed for detection and differentiation of wild-type and vaccine strains of MEV. Four primers in MEV-VP2-LAMP were used to detect both wild-type and vaccine strains of MEV in our previous publication, and other four primers in LAMP-SNP were designed to amplify the NS1 gene in wild-type MEV and only used to detect wild-type viruses. The LAMP-SNP assay was performed in a water bath held at a constant temperature of 65 °C for 60 min. LAMP-SNP amplification can be judged by both electrophoresis and visual assessment with the unaided eyes. In comparison with virus isolation as the gold standard in testing 171 mink samples, the percentage of agreement and relative sensitivity and specificity of the LAMP-SNP assay were 97.1, 100%, and 94.0%, respectively. There were no cross-reactions with other mink viruses. The LAMP-SNP assay was found to be a rapid, reliable and low-cost method to differentiate MEV vaccine and field variant strains.

PEDV remains one of the most important swine diseases that infects pigs of all ages. It causes devastating viral enteric disease in piglets with a high mortality rate, leading to significant threats and huge economic loss to the pork industry. In this study, a transcriptomic shotgun sequencing (RNA-Seq) procedure was used to study gene responses against PEDV infection. Genome-wide analysis of differentially expressed genes (DEGs) was performed in Vero E6 cells post-PEDV infection. mTOR signaling pathway activator-MHY1485, and inhibitor-PP242 were used to study the antiviral function. Results revealed that the IRF3 was significantly up-regulated post-PEDV infection. Although most of the IFN-regulatory and –related genes evaluated in this study were either down-regulated or remained unchanged, IL11 behaved significantly up-regulated, with the peak at 16 hpi. Nearly 90% of PEDV infections were suppressed in the PP242 pretreated cells whereas the reverse effect was observed in the MYH1485 pretreated cells. Results indicated that the mTOR signaling pathway played a vital role in the PEDV antiviral regulation in the Vero E6 cells. Future studies will contribute to better understand the cellular antiviral mechanism against PEDV., Highlights • RNA-Seq was used to study gene responses against PEDV infection. • Genome-wide analysis of DEGs was performed in Vero E6 cells post-PEDV infection. • The mTOR signaling pathway activator and inhibitor can affect the PEDV infection rate.

Canine parvovirus (CPV) is an important pathogen in domestic dogs, and the original antigenic types CPV-2 and its variants, CPV-2a, 2b and 2c, are prevalent worldwide. A multiplex TaqMan real-time PCR method was developed for the detection and differentiation of four antigenic types of CPV. A set of primers and probes, CPV-305F/CPV-305R and CPV-2-305P (for CPV-2)/CPV-2a-305P (for CPV-2a, 2b and 2c), was able to differentiate CPV-2 and its variants (CPV-2a, 2b and 2c). Another set of primers and probes, CPV-426F/CPV-426R and CPV-2-426P (for CPV-2 and 2a)/CPV-2b-426P (for CPV-2b)/CPV-2c-426P (for CPV-2c), was able to differentiate CPV-2a (2), CPV-2b, and CPV-2c. With these primers and probes, the multiplex TaqMan real-time PCR assay detected effectively and differentiated CPV-2, 2a, 2b and 2c by two separate real-time PCRs. No cross reactivity was observed with canine distemper virus, canine adenovirus, and canine coronavirus. The detection limit of the assay is 101 genome copies/μL for CPV-2, CPV-2a, CPV-2b, and 102 copies/μL for CPV-2c. The multiplex real-time PCR has 100% agreement with DNA sequencing. We provide a sensitive assay that simultaneously detects and differentiate four antigenic types of CPV and the method was also used for quantification of CPVs viral genome., Highlights • The Multiplex TaqMan real-time PCR can simultaneously detect and differentiate four antigenic types of CPV. • The method is suit for using in detection of CPVs in China. • The method showed a high specificity and sensitivity. • The method was also used for quantification of CPVs viral genome.

Background Caprine herpesvirus 2 (CpHV-2) infection usually induces chronic malignant catarrhal fever (MCF) in sika deer (Cervus nippon), with the primary signs of weight loss, dermatitis and alopecia. Case presentation Here, we report a case of CpHV-2-associated acute MCF in a sika deer herd raised in an intensive management system distant to the reservoir goats. Affected deer developed clinical signs of high fever (41 °C) followed by nasal discharge and lameness. Severe lesions of hemorrhage, necrosis and infiltration of lymphoid cells could readily be observed in the lung, kidney, heart valves and subcutaneous tissue surrounding a tendon. Etiologically, identical CpHV-2 specific DNA sequences were detected in peripheral blood lymphocyte (PBL) from the affected deer and reservoir goats. Conclusion In summary, domestic goats were the reservoir of the CpHV-2, which is the causative agent of the outbreak of MCF in the three hinds. The disease was probably transmitted via aerosol infection. In addition, necrosis and inflammation in subcutaneous tissue surrounding a tendon was the reason for lameness. Therefore, MCF should be put into a differential diagnostic list when similar disease occurs in sika deer herds. Electronic supplementary material The online version of this article (10.1186/s12917-018-1365-8) contains supplementary material, which is available to authorized users.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of porcine intestinal disease, which causes vomiting, diarrhea, and dehydration in piglets. PEDV is associated with the most severe pathogenesis in one-week-old piglets, with mortality rates reaching 100%. A PEDV strain was isolated from the intestinal tract of diarrheic piglets from a pig farm in Jiangsu Province in March 2016, termed the JS201603 isolate. The isolated virus was confirmed to be PEDV via RT-PCR, electron microscopy, a cytopathic effect assay and sequence analysis. The S and ORF3 genes of the JS201603 isolate were sequenced, revealing that the S gene was associated with a 15-base insertion at 167 nt, 176 - 186 nt, and 427 - 429 nt, as well as a six-base deletion in 487 - 492 nt, indicating that it was a current epidemic variant compared with the classical strain, CV777. No deletion occurred between 245 - 293 nt of the ORF3 gene in the JS201603 isolate compared with the vaccine isolates YY2013 and SQ2014. An experimental infection model indicated that the piglets in the challenge group successively developed diarrhea, exhibiting yellow-colored loose stools with a foul odor. The piglets in the JS201603 isolate challenge group displayed reduced food consumption, lost weight, and in severe cases even died. No abnormalities were observed in the control group. The JS201603 variant isolated in this study contributes to the evolutionary analysis of diarrhea virus. The experimental infection model has established a foundation for further studies on vaccine development.

Mink enteritis virus (MEV), an autonomous parvovirus, causes acute hemorrhagic enteritis in minks. The molecular pathogenesis of MEV infection has not been fully understood. In this study, we observed significantly increased apoptosis in the esophagus, small intestine, mesenteric lymph nodes, and kidney in minks experimentally infected with strain MEVB. In vitro infection of feline F81 cells with MEVB decreased cell viability and induced cell cycle arrest at G(1) phase and apoptosis. By screening MEV nonstructural proteins (NS1 and NS2) and structural proteins (VP1 and VP2), we demonstrated that the MEV NS1 induced apoptosis in both F81 and human embryonic kidney 293T (HEK293T) cells, similar to that induced during MEV infection in minks. We found that the NS1 protein-induced apoptosis in HEK293T cells was mediated not by the death receptor but by the mitochondrial pathway, as demonstrated by mitochondrial depolarization, opening of mitochondrial transition pore, release of cytochrome c, and activation of caspase-9 and -3. Moreover, in NS1-transfected cells, we observed an increase of Bax expression and its translocation to the mitochondria, as well as an increased ratio of the Bax/Bcl-2, reactive oxygen species (ROS) production, and activated p38 mitogen-activated protein kinase (MAPK) and p53. Taken together, our results demonstrated that MEV induces apoptosis through activation of p38 MAPK and the p53-mediated mitochondrial apoptotic pathway induced by NS1 protein, which sheds light on the molecular pathogenesis of MEV infection. IMPORTANCE MEV causes fatal hemorrhagic enteritis in minks. Apoptosis is a cellular mechanism that effectively sacrifices virus-infected cells to maintain homeostasis between the virus and host. In this study, we demonstrated that MEV induces apoptosis both in vivo and in vitro. Mechanistically, the viral large nonstructural protein NS1 activates p38 MAPK, which leads p53 phosphorylation to mediate the mitochondrial apoptotic pathway but not the death receptor-mediated apoptotic pathway. This is the first report to uncover the mechanism underlying MEV-induced apoptosis.

Several biological processes as well as infectious agents, physiological or environmental stress, and perturbed antioxidant response can promote oxidative stress. Oxidative stress usually happens when cells are exposed to more electrically charged reactive oxygen species (ROS) such as H2O2 or O2-. ROS are well known for being both beneficial and deleterious. Recent studies have indicated that ROS are deleterious to cells, leading to programmed cell death (PCD) at high concentrations. At low concentrations, however, ROS can act as signaling molecules in a variety of cellular processes. In this review, we present an update of our current understanding of the role and regulation of reactive oxygen species in various viral infections, cellular signaling pathways and immune responses. We then discuss how the antioxidant defense system acts as an antiviral effector to limit cell damage.

TLR8 is an important sensor of single-stranded RNA (ssRNA) from the viral genome and plays an essential role in innate antiviral responses via the recognition of conserved viral molecular patterns. In this report, TLR8 in the Chinese raccoon dog was characterized and analyzed for the first time. The full-length sequence of raccoon dog TLR8 (RdTLR8) cDNA was cloned by rapid amplification of cDNA ends (RACE) and is 3191bp with a 3117-bp open reading frame (ORF) encoding 1038 amino acids. The putative protein exhibits typical features of the TLR families, with 19 leucine-rich repeats (LRRs) in the extracellular domain and a cytoplasmic TIR domain. Comparative analyses of the RdTLR8 amino acid sequence indicated a 73.6-99.4% sequence identity with dog, horse, pig, sheep, cattle, human and mouse TLR8. Phylogenetic analysis grouped 71 mammalian TLR proteins into five sub-families, wherein RdTLR8 was clustered into a monophyletic TLR8 clade in the TLR9 family, which was completely coincident with the evolutionary relationship among mammals. Quantitative real-time PCR analysis revealed extensive expression of RdTLR8 in tissues from healthy Chinese raccoon dogs with the highest expression in the peripheral blood mononuclear cells (PBMCs) and the lowest expression in the skeletal muscle. HEK293 cells cotransfected with a RdTLR8 expression plasmid and an NF-κB-luciferase reporter plasmid significantly responded to the agonist 3M-002, indicating a functional TLR8 homolog. In addition, raccoon dog PBMCs exposed to the canine distemper virus (CDV) wild strain CDV-PS and the TLR8 agonist 3M-002 showed significant upregulation of RdTLR8 mRNA and proinflammatory cytokines TNF-α and IFN-α, suggesting that RdTLR8 might play an important role in the immune response to viral infections in the Chinese raccoon dog.

Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses.

The highly contagious canine distemper virus (CDV) is a non-segmented single-stranded negative-sense RNA virus, which belongs to the Morbillivirus genus of the Paramyxoviridae family. The phosphoprotein (P) of CDV plays the important role in the virus replication and pathogenesis. In this study, we characterized four monoclonal antibodies (MAbs), designated as Pc7, Pc8, Pc11 and Pc25 MAbs against the P protein of CDV-PS strain. A series of overlapping P protein-derived peptides representing the CDV-PS phosphoprotein (aa232-507) were screened to identify linear peptide epitopes recognized by each MAb. Finally, four epitopes, 238SHGMGIVAGSTN249 (E2-9), 264GPSVSAENVRQ274 (E6-2), 390INPELRPIIGR400 (E27-2) and 252TQSALKSTG260 (E4-9), are minimal linear epitopes recognized by the Pc7, Pc8, Pc11 and Pc25 MAbs, respectively. Each identified B-cell epitope was able to be recognized by CDV positive dog serum. Alignment analysis of the amino acid sequences indicated that the linear B-cell epitope of the Pc11 MAb is relatively conserved among different CDV strains, but the linear B-cell epitopes recognized by Pc7, Pc8 and Pc25 MAbs are not conserved among CDV strains. Our results revealed that the E27-2 peptide might be a common B-cell binding epitope of CDV antibodies. These findings may provide a useful basis for the development of new diagnostic assays for CDV.

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.

Background The Nucleoprotein (NP) is the most abundant and highly immunogenic protein in canine distemper virus (CDV), playing an important role in CDV viral replication and assembly. Results In this study, a specific monoclonal antibody, named C8, was produced against the NP protein C terminal (amino acids 401–523). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis.The results indicated that 444GDKYPIHFNDER455 was the minimal linear epitope that could be recognized by mAb C8. Sequence alignments demonstrated that this linear epitope is less conserved among three CDV genotypes. We next analyzed the level of conservation of the defined epitope in19 Chinese CDV clinical isolates, and it has one site variation in amino acid among these CDV isolations. 2 isolates have the amino acid mutations F451L, while one has P448Ssubstitution.Phylogenetic analysis showed the two isolates with F451Lsubstitution had a closer relationship in a virulent strain ZJ-7, so the epitope may be a significant tag associated with virus virulence. Conclusion This collection of mAb along with defined linear epitope may provide useful reagents for investigations of NP protein function and the development of CDV specific diagnostics.

Bovine viral diarrhea virus (BVDV) is often detected in commercial bovine serum. BVDV genetic diversity was investigated in commercial bovine serum of Chinese origin. Twenty-two batches of bovine serum were obtained from 10 suppliers with different geographic origins in China, and 20 batches of bovine serum were positive by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. Phylogenetic reconstructions of partial 5'UTR sequences indicated that the samples examined in this work clustered within the BVDV type 1 and BVDV type 2 genotypes. Interestingly, 3 sample sequences clustered into CSFV. These results suggest a high genetic diversity in Chinese BVDV field isolates. This study will benefit epidemiological surveys of BVDV detected in China.

Bovine viral diarrhea virus (BVDV) can contaminate biological products produced in bovine or porcine cells or manufactured using bovine sera. A rapid, specific, sensitive, and practical method of detecting BVDV in bio-products is needed. The purpose of this study was to compare three assays with respect to their ability to accurately detect BVDV in biological samples, namely reverse-transcription loop-mediated isothermal amplification (RT-LAMP), SYBR green I-based real-time RT-PCR, and conventional RT-PCR. All assays detected BVDV nucleotide and differentiated between BVDV-free and -contaminated bovine sera successfully. In addition, the results were specific to BVDV: the amplification of samples containing the closely related classical swine fever virus or other pathogenic bovine viruses yielded negative results. The lowest detection threshold, 10(1) copies, was displayed by the SYBR green I-based real-time RT-PCR and RT-LAMP assay. This assay was also the most effective in the detection of BVDV contamination in a set of commercially available bovine sera. The field conditions suggest that RT-LAMP is specific and sensitive to detecting BVDV in biological samples and may be used for quality control of biomaterials.

In this study, a total of 37 samples (58.0%) were found through PCR assay to be positive for canine parvovirus (CPV) of 66 suspected faecal samples of dogs collected from various cities throughout China. Eight CPV isolates could be obtained in the CRFK cell line. The sequencing of the VP2 gene of CPV identified the predominant CPV strain as CPV-2a (Ser297Ala), with two CPV-2b (Ser297Ala). Sequence comparison revealed homologies of 99.3-99.9%, 99.9% and 99.3-99.7% within the CPV 2a isolates, within the CPV 2b isolates and between the CPV 2a and 2b isolates, respectively. In addition, several non-synonymous and synonymous mutations were also recorded. The phylogenetic tree revealed that most of the CPV strains from different areas in China were located in the formation of a large branch, which were grouped together along with the KU143-09 strain from Thailand and followed the same evolution. In this study, we provide an updated molecular characterization of CPV 2 circulation in China.

Canine distemper (CD) is a highly contagious, systemic, viral disease of dogs seen worldwide. The nucleocapsid protein (NP) of canine distemper virus encloses virus assembly and has some regulatory functions in viral transcription and replication. Here, we describe a procedure to generate a monoclonal antibody (MAb) against CDV N protein and investigate its characteristics. Western blot analysis showed that the MAbs produced in this study were against CDV N specifically. Indirect immunofluorescence assay demonstrated that they could recognize native N protein in CDV-infected Vero cells. The MAbs reported here may provide valuable tools for the further exploration of biological properties and functions of N protein and may also be developed for potential clinical applications.

Toll-like receptors (TLRs) can recognize conserved molecular patterns and initiate a wide range of innate and adaptive immune responses against invading infectious agents. The aim of this study was to assess the transcript profile of mink TLRs (mTLRs) in mink peripheral blood mononuclear cells (PBMCs) and a range of tissues, and to explore the potential role of mTLRs in the antiviral immune response process. The results indicated that the mTLR partial nucleotide sequences had a high degree of nucleotide identity with ferret sequences (95-98%). Phylogenetic analysis showed that mammalian TLRs grouped into five TLR families, with a closer relationship of the mTLRs with those of ferret than the other mammalian sequences. Moreover, all the mTLRs were ubiquitously expressed in lymphoid organs (spleen and lymph nodes) and PBMCs. Interestingly, the mTLR expression patterns in lung, uterus, and heart showed quite a lot of similarity. Another remarkable observation was the wide expression of mTLR1-3 mRNAs in all tissues. Among the analyzed tissues, skeletal muscle was revealed to being the lowest repertoire of mTLR expression. Additionally, mink PBMCs exposed to the canine distemper virus revealed significant upregulation of mTLR2, mTLR4, mTLR7, and mTLR8 mRNAs, indicating that mTLRs have a role in innate immunity in the mink. Collectively, our results are the first to establish the basic expression patterns of mTLRs and the relationship between mTLRs and a virus, which will contribute to better understanding of the evolution and the functions of mTLRs in the innate immune system in minks.

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 MetacoreTM 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.

Canine parvovirus (CPV) type 2c is a new antigenic variant of CPV-2. Since the year 2000 it has spread to several countries, causing severe hemorrhagic enteritis in dogs. In 2014 and 2015, 58 fecal samples were collected from dogs in Beijing with suspected CPV infection. Regardless of the vaccination status of the dogs, 43 samples were found positive for CPV according to PCR results; i.e., 18, 7, and 18 respectively belonged to antigenic types new CPV-2a, new CPV-2b, and CPV-2c. A phylogenetic tree based on their VP2 gene sequences indicated that the Chinese CPV-2c strains form a separate cluster. In addition to synonymous mutations, the CPV-2c strains also contain a unique coding mutation in VP2 that leads to glycine at residue 5, instead of the highly conserved alanine at this position in all other CPV-2c strains sequenced to date. Using F81 cells, several novel isolates of CPV-2c, each with the Ala5Gly mutation, were obtained. One of these was used to infect experimentally beagle dogs, which subsequently developed the typical clinical symptoms of CPV infection. Hence, it appears that CPV-2c is still evolving in China, a finding that warrants continuous surveying and the eventual adaptation of current vaccines.

Background Porcine reproductive and respiratory syndrome (PRRS) remains a major threat to swine industry all over the world. The aim of this study was to investigate the mechanism of pathogenesis and immune responses caused by a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Results All piglets experimentally infected with a HP-PRRSV TJ strain virus developed typical clinical signs of PRRS. The percentages of CD3+, CD4+, and CD8+ lymphocytes significantly decreased in the infected group as compared to the uninfected control animals (p

The variable regions of the heavy chain (VH) and light chain (VL) were amplified by RT-PCR from the hybridoma 1N8, which secretes the monoclonal antibody against CDV N protein (aa 277-471). The VL and VH amplicons were combined using SOE-PCR by a 12 amino acid flexible linker (SSGGGGSGGGGS), which produced the scFv gene (named scFv/1N8). After sequence analysis, the scFv/1N8 gene was cloned into the prokaryotic expression vector PET32a with a His-tag. The recombinant scFv/1N8 protein was successfully expressed in recombinant Escherichia coli by IPTG induction. Moreover, the binding activity and specificity of the scFv were determined by indirect ELISA (His-tag) and competitive ELISA. The recombinant scFv/1N8 protein reported here will provide some basis for further antiviral drug research based on the scFv molecule.

Background: Highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) causes prolonged high fever, red discoloration of the body, blue ears and a high mortality. Previously, we found that the PRRSV vaccine strain TJM contained a deletion of 120 amino acids (aa 628-747) in nonstructural protein 2 (Nsp2). We aimed to explore the replication features of PRRSV after adding the transiently expressed product of these 120 aa in vitro. Methods: We constructed seven eukaryotic expression plasmids containing different parts of the 120-aa sequence, transfected them into Marc-145 cells and then inoculated the cells with 103 TCID50 TJM per well. We detected virus replication at mRNA and protein level by real-time RT-PCR and Western blotting, respectively, and determined the virus titer. Results: The transiently expressed 120 aa and one of its truncated polypeptides inhibited PRRSV TJM propagation on Marc-145 cells. The complete 120-aa sequence induced a remarkable decrease in PRRSV replication, causing a reduction in structural protein levels between 36 and 48 h after infection. Additionally, aa 628-727 partly reduced the replication of PRRSV on Marc-145 cells. Conclusions: The 120 aa from Nsp2, especially aa 628-727, play a negative role in PRRSV TJM proliferation.

Canine parvovirus (CPV) is highly contagious and is a major cause of haemorrhagic enteritis and myocarditis in dogs. We investigated the genetic variation of emerging CPV strains by sequencing 64 CPV VP2 genes from 216 clinical samples of dogs from Heilongjiang, Jilin, Liaoning, Shandong and Hebei in 2014. Genetic analysis revealed that CPV-2b was predominant in Hebei and CPV-2a was predominant in the other four provinces. In addition, a CPV-2c strain has emerged in Shandong province. All samples had a Ser-Ala substitution at residue 297 and an Ile-Arg substitution at residue 324. Interestingly, in five separate canine samples, we found a mutation of Gln370 to Arg, until now detected only in isolates from pandas. The phylogenetic analysis showed clear distinctions between epidemic isolates and vaccine strains and between Chinese CPV-2c strains and CPV-2c strains found in other countries. Monitoring recent incidence of CPV strains enables evaluation and implementation of disease control strategies.

Background Mink enteritis virus (MEV) causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality. Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a recently developed technique for the rapid detection of bacterial and viral DNA. Here we describe a novel nanoPCR assay for the clinical detection and epidemiological characterization of MEV. Results This assay is based upon primers specific for the conserved region of the MEV NS1 gene, which encodes nonstructural protein 1. Under optimized conditions, the MEV nanoPCR assay had a detection limit of 8.75 × 101 copies recombinant plasmids per reaction, compared with 8.75 × 103 copies for conventional PCR analysis. Moreover, of 246 clinical mink samples collected from five provinces in North-Eastern China, 50.8% were scored MEV positive by our nanoPCR assay, compared with 32.5% for conventional PCR. Furthermore no cross reactivity was observed for the nanoPCR assay with respect to related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). Phylogenetic analysis of four Chinese wild type MEV isolates using the nanoPCR assay indicated that they belonged to a small MEV clade, named “China type”, in the MEV/FPLV cluster, and were closely clustered in the same location. Conclusions Our results indicate that the MEV China type clade is currently circulating in domestic minks in China. We anticipate that the nanoPCR assay we have described here will be useful for the detection and epidemiological and pathological characterization of MEV. Electronic supplementary material The online version of this article (doi:10.1186/s12917-014-0312-6) contains supplementary material, which is available to authorized users.

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of significant economic importance that causes reproductive and respiratory problems in pigs. The replicase non-structural protein 2 (Nsp2) of the porcine reproductive and respiratory syndrome virus (PRRSV) is recognized as the most variable region within the PRRSV genome. This review discusses the molecular characteristics and biological and immunological functions of the PRRSV Nsp2 and its involvement in the virus's pathogenesis. The role of Nsp2 in cell and tissue tropism, replication and growth, and variation and pathogenicity of PRRSV and the differences in virulence among different strains are described in the present review. Nsp2 is an ideal marker for monitoring genetic variation and for developing differential diagnostic tests.

A virus was isolated from mink showing clinical and pathological signs of enteritis in China. This virus, designated MEV/LN-10, was identified as mink enteritis virus (MEV) based on its cytopathic effect in the feline F81 cell line, the hemagglutination (HA) and hemagglutination inhibition (HI) assay, electron microscopy (EM) and animal infection experiments. The complete viral genome was cloned and sequenced. Phylogenetic and recombination analyses on the complete MEV/LN-10 genome showed evidence of recombination between MEV and canine parvovirus (CPV). The genome was composed of the NS1 gene originating from CPV while the VP1 gene was of MEV origin. This is the first demonstration of recombination between a CPV and MEV in nature. Our findings not only provide valuable evidence indicating that recombination is an important genetic mechanism contributing to the variation and evolution of MEV, but also that heterogeneous recombination can occur in the feline parvovirus subspecies.

Highlights ► The method established in the study can be used to detect and differentiate between local CDV field isolates and vaccines effectively and successfully. ► Based on the molecular sequence investigation of wild-type local CDV strain, this study suggests a potential link at the phylogenetic relationship between strains and their geographical source. ► The method is rapid and affordable, and can be used for clinical detection and epidemiological surveillance., A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335 bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555 bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China.

NM1 is a highly pathogenic North American-type porcine reproductive and respiratory syndrome virus (PRRSV). The complete genome sequence shows that NM1 shares high sequence identity (99.2 to 99.4%) to other HP-PRRSV isolates, containing two discontinuous deletions, a 1-amino-acid deletion at position 481 and a 29-amino-acid deletion at positions 533 to 651, in nonstructural protein 2.

Background Bovine viral diarrhea virus (BVDV) is a pathogen found worldwide in calves. It can cause significant economic losses in agriculture. Many BVDV strains have been isolated in China. However, the pathogenesis and complete gene characteristics of BVDV isolate have yet not been reported in China. Here, a BVDV isolate was isolated and its pathogenesis and complete genome were studied. Results A new isolate of bovine viral diarrhea virus, named JL-1, was isolated from the spleen of a sick cow with diarrhea using MDBK cell culture. The complete genome of JL-1 is 12,276 nucleotides and contains a 5′-UTR of 382 nucleotides, a 3′-UTR of 188 nucleotides, and a large ORF encoding a polyprotein consisting of 3,901 amino acids. Genomic comparison and phylogenetic analyses of complete genomic sequence clearly showed that JL-1 fell into the BVDV-1b subtype. The result of pathogenesis of JL-1 strain showed that all infected calves developed clinical signs of elevated rectal temperatures, decreased leucopenia, and viral discharge. Viral antigen was detected in infected animal tissues using immunohistochemistry. Animals in the mock were normal. These results demonstrated that BVDV JL-1 was a virulent strain. Conclusions This is the first study to report the pathogenesis and complete gene characterization of the BVDV strain in China. This report may set a good foundation for further study of BVDV in China.