Effect of intestinal microbiota on duck short-beak and dwarf syndrome caused by novel goose parvovirus.

Short-beak and dwarf syndrome (SBDS) is caused by infection with novel goose parvovirus (NGPV), which leads to intestinal dysbiosis, developmental delay, short beak, lameness, and paralysis in ducks and is the cause of skeletal health problems. NGPV infection can cause intestinal microbial disturbances, but it is still unclear whether the intestinal microbiota affects the pathogenicity of NGPV. Here, the effects of intestinal microbiota on NGPV-induced SBDS in Cherry Valley ducks were assessed by establishing a duck model for gut microflora depletion/reestablishment through antibiotics (ABX) treatment/fecal microbiota transplanted (FMT). By measuring body weight, beak length, beak width and tarsal length, we found that SBDS clinical symptoms were alleviated in ducks treated with ABX, but not in FMT ducks. Next, we conducted a comprehensive analysis of bone metabolism, gut barrier integrity, and inflammation levels using quantitative real-time PCR (qPCR), enzyme linked immunosorbent assay (ELISA), biochemical analysis and histological analysis. The results showed that ABX treatment improved bone quality reduced bone resorption, mitigated tissue lesions, protected intestinal barrier integrity, and inhibited systemic inflammation in NGPV-infected ducks. Moreover, cecal microflora composition and short-chain fatty acids (SCFAs) production were examined by bacterial 16S rRNA sequencing and gas chromatography. The results revealed that ABX treatment mitigated the decreased abundance of Firmicutes and Bacteroidota in NGPV-infected ducks, as well as increased SCFAs production. Furthermore, ABX treatment reduced the mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and nuclear factor κB (NF-κB) expression, which are correlated with systemic inflammation in SBDS ducks. These findings suggested that intestinal microflora depletion alleviated NGPV-induced SBDS by maintaining intestinal homeostasis, inhibiting inflammatory response and alleviating bone resorption. These results provide evidence for the pivotal role of intestinal microbiota in the process of SBDS and contribute a theoretical basis for the feasibility of microecological preparation as a method to control SBDS.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Nanoparticulate chitosan-TNF-α-VLPs activate mast cells and enhance adaptive immunity induced by foot-and-mouth disease virus-like particles in mice.

Chitosan nanoparticulate vaccines have attracted considerable attention to potentiate immune responses. A chitosan-TNF-α-VLPs nanoparticle vaccine against foot-and-mouth disease virus (FMDV) prepared though inotropic gelation method and whether this nanoparticulate vaccine can activate mast cells and enhance immune responses induced by FMDV virus-like particles (VLPs) in mice was investigated. The nanoparticle was approximately spherical, and its size was approximately 200-300 nm. Following immunization via subcutaneous injection, the chitosan-TNF-α-VLPs nanoparticles could induce higher levels of FMDV-specific antibodies and stimulation index value than VLPs only (P < 0.01) and had similar levels to commercial vaccine group and VLPs+adjuvant group (P > 0.05). No significant differences were observed in the concentrations of IL-4, IFN-γ and IL-10 among the chitosan-TNF-α-VLPs group, VLPs+adjuvant group and commercial vaccine group (P > 0.05). Of note, the chitosan-TNF-α-VLPs nanoparticles can effectively activate mast cells in lymph nodes. These results indicated that the chitosan-TNF-α-VLPs nanoparticles can enhance both humoral and cell-mediated immunity, and both Th1 and Th2 responses, even activate mast cells, demonstrating that chitosan-TNF-α nanoparticles are potential as a vaccine adjuvant to enhance immune responses induced by FMDV-VLPs.

Development of a novel method for diagnosis of fasciolosis based on cathepsin L7 in ruminants.

Fasciolosis is a widely distributed zoonosis reported over 81 countries around the world. Good and early diagnostic method is critical in controlling this disease and prevention of injury to the liver and bile ducts. In this study, we identified a novel member (cathepsin L7) of cathepsin family from Fasciola spp.. Firstly, the biological character of CL7 was analyzed according to the information of cathepsin L family, and then rCL7 was expressed and purified, a new iELISA based on CL7 was developed. The results exhibited CL7 iELISA had 100% sensitivity 100% specificity in sheep (cut-off 1.329) and 100% sensitivity 93.75% specificity in cattle (cut-off 0.756). Moreover, anti-Fasciola CL7 antibodies could be detected in early Fasciola gigantica infected buffaloes, as early as 3 week-post-infection (WPI). In conclusion, it is suggested that CL7 with low cost, early detection, good specificity and sensitivity could be used as a candidate antigen for detection of ruminant fasciolosis.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

Histone acetylation regulates BMMCs recognition of foot-and-mouth disease virus-like particles.

Foot-and-mouth disease (FMD) is one of the most economically and socially devastating diseases affecting animal agriculture worldwide. Foot-and-mouth disease virus (FMDV) virus-like particles (VLPs) have been widely studied as a candidate vaccine. Mast cells (MCs) are highly versatile innate immunity cells that perform various functions in regulating innate and adaptive immune responses. Recently, we found that MCs can recognize recombinant FMDV VP1-VP4 protein to produce various cytokines with differential expression, suggesting that this may be epigenetically regulated. In this study, we evaluated the effect of trichostatin A (TSA), a histone deacetylase inhibitor, on bone marrow-derived mast cells (BMMCs) recognition of FMDV-VLPs in vitro. BMMCs can recognize FMDV-VLPs via mannose receptors (MRs) and resulted in enhanced expression and secretion of tumour necrosis factor α (TNF-α) and interleukin (IL)-13. Nevertheless, BMMCs recognition of FMDV-VLPs to secrete IL-6 was irrelevant to MRs, and MRs may play a negative regulation for IL-10 secretion. Pre-treatment with TSA caused decreased expression of IL-6, TNF-α and IL-13, and increased expression of IL-10. Furthermore, the expression of nuclear factor-kappa B (NF-κB) was supressed in TSA treated BMMCs, suggesting histone acetylation may alter NF-κB expression to influence the TNF-α and IL-13 secretion. Pre-treatment with TSA had no influence on the expression of microphthalmia-associated transcription factor (MITF) and GATA-2. These data therefore suggest that altered histone acetylation regulates the immune responses induced by BMMCs recognition of FMDV-VLPs, providing an understanding and theory basis for the prevention and control of FMD based MCs.

Recombinant porcine Interferon-α and Interleukin-2 fusion protein (rPoIFNα+IL-2) shows potent anti-pseudorabies virus activity in vitro and in vivo.

Pseudorabies virus (PRV) variants have been widely prevalent since 2011, leading to substantial losses to the swine industry. Although PRV can cause cross-species transmission and induce human infection, no drugs can currently prevent PRV infection. Interferons (IFNs) and interleukin-2 (IL-2) are important cytokines that mediate several biological functions including antiviral activity and immune regulation. In this study, we expressed and purified a recombinant porcine IFN-α and IL-2 fusion protein (rPoIFNα+IL-2), which did not show a cytotoxic effect on PK-15 cells. The antiviral activity was evaluated in PK-15 cells using the cytopathic effect inhibition method, and the results indicated that rPoIFNα+IL-2 can inhibit the replication of PRV, with an antiviral activity of approximately 104 U/mL. Moreover, the proliferation of peripheral blood mononuclear cells was enhanced by rPoIFNα+IL-2. Additionally, rPoIFNα+IL-2 substantially increased the expression of IFN-stimulated genes, including IFIT1, ISG15, MX1, and OAS, which are critical for antiviral activity. Furthermore, rPoIFNα+IL-2 alleviated the clinical symptoms and reduced mortality in mice infected with PRV. Simultaneously, rPoIFNα+IL-2 increased the expression levels of IFN-γ and IL-10 and inhibited the expression of IL-1ß and IL-6. Additionally, the viral DNA copies in different tissues in the rPoIFNα+IL-2-treated group were lower than those in the untreated group. These findings indicate that rPoIFNα+IL-2 may serve as an antiviral agent for the prevention and treatment of PRV infection and may expand the potential function of IFN antiviral drugs in the future.

Pathogenicity and Transmissibility of Goose-Origin H5N6 Avian Influenza Virus Clade 2.3.4.4h in Mammals.

Throughout the last decade, H5N6 avian influenza viruses (AIVs) circulating in poultry and infecting humans have caused increasing global concerns that they might become a pandemic threat to global health. Since AIVs could occasionally cause asymptomatic infections in geese, virus monitoring in such a host should be critical to the control of cross-species infection. In addition, previous studies showed that clade 2.3.4.4h H5N6 AIVs could infect mammals without adaptation. However, the pathogenicity and transmissibility of goose-origin clade 2.3.4.4h H5N6 AIVs in mammals remain unknown. In this study, two H5N6 AIVs were isolated from a domestic chicken (A/chicken/Hebei CK05/2019 (H5N6)) and a goose (A/goose/Hebei/GD07/2019(H5N6)). This study is the first to evaluate the pathogenicity and transmissibility of goose-origin clade 2.3.4.4h H5N6 AIVs in mammals by comparison with chicken-origin 2.3.4.4h H5N6 AIVs. The CK05 virus had an affinity for α-2,3-receptors, while the GD07 virus had an affinity for both α-2,3-and α-2,6-receptors. The GD07 virus had a higher replication capacity in vitro and more severe pathogenicity in mice than the CK05 virus. The CK05 virus could not be transmitted effectively among guinea pigs, whereas the GD07 virus could be transmitted through direct contact among guinea pigs. The results of this study indicated the potential health threat of clade 2.3.4.4h H5N6 AIVs to mammals and emphasized the importance of continuous monitoring of H5N6 AIVs, especially in waterfowl.

Rapid detection of bovine rotavirus a by isothermal reverse transcription recombinase polymerase amplification assays.

BACKGROUND: Bovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals. RESULTS: An RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 102 copies per reaction and 1.4 × 101 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively. CONCLUSIONS: In this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.

2B and 3C Proteins of Senecavirus A Antagonize the Antiviral Activity of DDX21 via the Caspase-Dependent Degradation of DDX21.

Senecavirus A (SVA), also known as Seneca Valley virus, is a recently discovered picornavirus that can cause swine vesicular disease, posing a great threat to the global swine industry. It can replicate efficiently in cells, but the molecular mechanism remains poorly understood. This study determined the host's differentially expressed proteins (DEPs) during SVA infection using dimethyl labeling based on quantitative proteomics. Among the DE proteins, DDX21, a member of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase (DDX) family, was downregulated and demonstrated inhibiting SVA replication by overexpression and knockdown experiment. To antagonize this antiviral effect of DDX21, SVA infection induces the degradation of DDX21 by 2B and 3C proteins. The Co-IP results showed that 2B and 3C did not interact with DDX21, suggesting that the degradation of DDX21 did not depend on their interaction. Moreover, the 3C protein protease activity was necessary for the degradation of DDX21. Furthermore, our study revealed that the degradation of DDX21 by 2B and 3C proteins of SVA was achieved through the caspase pathway. These findings suggest that DDX21 was an effective antiviral factor for suppressing SVA infection and that SVA antagonized its antiviral effect by degrading DDX21, which will be useful to guide further studies into the mechanism of mutual regulation between SVA and the host.

Development of a new TaqMan-based real-time RT-PCR assay for the specific detection of bovine kobuvirus.

Bovine kobuvirus (BKV) is a novel kobuvirus considered to be closely related to calf diarrhea and has become a worldwide epidemic. Currently, the BKV lacks an efficient and convenient detection method to assist the research on BKV prevalence. In this study, a new and specific TaqMan-based real-time RT-PCR for the detection of BKV was developed using the conserved region of the 3D gene. The assay was highly specific for BKV, without cross-amplification with other non-targeted pathogens. The limit of detection of this assay was 102 copies. Standard curves showed a strong linear correlation from 102 to 106 copies of BKV standard RNA per reaction, and the parameters revealed as a slope of -3.54, efficiency of 91.64%, and regression coefficients (R2) of 0.998. The assay was also reproducible, with the intra-assay and inter-assay coefficient of variation <1.0%. The newly developed real-time RT-PCR was validated using 243 fecal samples collected from diarrheic or non-diarrheic cattle from nine regions in Hebei province and revealed the positive detection of BKV at a ratio of 19.34% (47/243). Sequencing of partial 3D genes from 13 positive samples and the following phylogenetic analysis demonstrated the reliability of the assay. In conclusion, the newly developed TaqMan-based real-time RT-PCR could be used for the screening and epidemic monitoring of BKV.

The Cellular and Viral circRNAs Induced by Fowl Adenovirus Serotype 4 Infection.

Circular RNAs (circRNAs) are a new class of noncoding RNAs that play vital roles in many biological processes. Virus infection induces modifications in cellular circRNA transcriptomes and expresses viral circRNAs. The outbreaks of Hydropericardium-hepatitis syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry worldwide. To investigate the expression of circRNAs during FAdV-4 infection, we performed transcriptome analysis of FAdV-4-infected leghorn male hepatoma (LMH) cells. In total, 19,154 cellular circRNAs and 135 differentially expressed (DE) cellular circRNAs were identified. The characteristics of the DE cellular circRNAs were analyzed and most of them were related to multiple biological processes according to GO and KEGG enrichment analysis. The accuracy of 10 cellular circRNAs were verified by semiquantitative RT-PCR and sequencing. The change trend was consistent with the RNA sequencing results. Moreover, 2014 viral circRNAs were identified and 10 circRNAs were verified by the same methods. Our analysis showed that seven circRNAs with the same 3' terminal and variable 5' terminal regions were located at pTP protein and DNA pol protein of FAdV-4, which may be generated via alternative splicing events. Moreover, the expression level of viral circRNAs was closely related to the replication efficiency of the virus and partial of the viral circRNAs promoted the replication of FAdV-4. Competing endogenous RNA analysis further showed that the effects of cellular and viral circRNAs on host or viral genes may act via miRNAs. Collectively, our findings first indicate that FAdV-4 infection induced the differential expression of cellular circRNAs and FAdV-4 also expressed viral circRNAs, some of which affected FAdV-4 replication. These findings will provide new clues for further understanding FAdV-4 and provide a basis for investigating host-virus interactions.

Emergence and genomic analysis of a novel ostrich-origin GPV-related parvovirus in China.

In recent years, ostrich disease characterized by paralysis and diarrhea has been circulating in some regions of China, causing huge economic losses to the ostrich breeding industry. In our study, clinical samples from diseased ostriches were collected, and only parvovirus was detected. The virus distribution analysis by histopathology and quantitative real-time PCR assays indicated that the virus had a wide range of tissue tropisms. The full-length genome of the ostrich parvovirus (OsPV) was sequenced and comprehensively analyzed. Interestingly, the phylogenetic and alignment results indicated that the OsPV and the goose parvovirus (GPV) form a separate branch. In contrast to GPV strains, OsPV showed 2 new 14 nucleotide deletions in the inverted terminal repeat (ITR) region. Furthermore, recombination analysis indicated that OsPV was a recombination strain between the vaccine strain SYG61v and the virulent strain B strain, with the major parent of OsPV as the SYG61v strain and the minor parent as the B strain. The 14 nucleotide deletions in the ITR region as well as recombination may be some of the reasons for the cross-species transmission of parvovirus from goose to ostrich. The above data will contribute to a better understanding of the molecular biology of the novel OsPV and help to develop the vaccine candidate strain.

cGAS Restricts PRRSV Replication by Sensing the mtDNA to Increase the cGAMP Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes great economic losses globally to the swine industry. Innate immune RNA receptors mainly sense it during infection. As a DNA sensor, cyclic GMP-AMP synthase (cGAS) plays an important role in sensing cytosolic DNA and activating innate immunity to induce IFN-I and establish an antiviral cellular state. In contrast, the role of innate immune DNA sensors during PRRSV infection has not been elucidated. In this study, we found that cGAS facilitates the production of IFN-ß during PRRSV infection. Western blot and virus titer assays suggested that cGAS overexpression suppressed the replication of multiple PRRSV strains, while knockout of cGAS increased viral titer and nucleocapsid protein expression. Besides, our results indicated that the mitochondria were damaged during PRRSV infection and leaked mitochondrial DNA (mtDNA) into the cytoplasm. The mtDNA in the cytoplasm co-localizes with the cGAS, and the cGAMP activity was increased when the cGAS was overexpressed during PRRSV infection. Furthermore, the cGAMP also possesses an anti-PRRSV effect. These results indicate for the first time that cGAS restricts PRRSV replication by sensing the mtDNA in the cytoplasm to increase cGAMP activity, which not only explains the molecular mechanism by which cGAS inhibits PRRSV replication but also provides research ideas for studying the role of the cGAS-STING signaling pathway in the process of RNA virus infection.

Rapid detection of porcine encephalomyocarditis virus (EMCV) by isothermal reverse transcription recombinase polymerase amplification assays.

In this study, we combined reverse transcription recombinase polymerase amplification assay with the fluorescence detection platform (qRT-RPA) and lateral flow biosensor (LFB RT-RPA) to allow for rapid detection of porcine encephalomyocarditis virus (EMCV). Primers and probes were designed to target the highly conserved region of 3D gene of porcine EMCV. The optimal reaction condition of qRT-RPA and LFB RT-RPA was set as 42 °C for 20 min. The assays were highly specific to EMCV and no cross-reactions were observed with seven other porcine viruses. With a 10-fold serially diluted EMCV genomic RNA as template, the limit of detection was 1.0 × 102 and 1.0 × 101 copies for qRT-RPA assay and LFB RT-RPA assay, respectively. A total of 92 samples from different sources were examined using qRT-RPA, LFB RT-RPA and qRT-PCR. We found 100% diagnostic agreement between qRT-RPA (23/92) and qRT-PCR (23/92), and 97.83% diagnostic agreement between LFB RT-RPA (25/92) and qRT-PCR (23/92). There was no significant difference in performance between the RT-RPA assays developed in this study and a previously described qRT-PCR. However, RT-RPA assays were rapid and easy to perform while LFB RT-RPA exhibited higher sensitivity for EMCV than qRT-PCR. Therefore, the developed EMCV RT-RPA assays provide an attractive and promising tool for effective detection of EMCV in low-resource settings.

The game between host antiviral innate immunity and immune evasion strategies of senecavirus A - A cell biological perspective.

Innate immunity is the first line of the cellular host to defend against viral infection. Upon infection, viruses can be sensed by the cellular host's pattern recognition receptors (PRRs), leading to the activation of the signaling cascade and the robust production of interferons (IFNs) to restrict the infection and replication of the viruses. However, numerous cunning viruses have evolved strategies to evade host innate immunity. The senecavirus A (SVA) is a newly identified member of the Picornaviridae family, causing severe vesicular or ulcerative lesions on the oral mucosa, snout, coronary bands, and hooves of pigs of different ages. During SVA infection, the cellular host will launch the innate immune response and various physiological processes to restrict SVA. In contrast, SVA has evolved several strategies to evade the porcine innate immune responses. This review focus on the underlying mechanisms employed by SVA to evade pattern recognition receptor signaling pathways, type I interferon (IFN-α/ß) receptor (IFNAR) signaling pathway, interferon-stimulated genes (ISGs) and autophagy, and stress granules. Deciphering the antiviral immune evasion mechanisms by SVA will enhance our understanding of SVA's pathogenesis and provide insights into developing antiviral strategies and improving vaccines.

Construction and Generation of a Recombinant Senecavirus a Stably Expressing the NanoLuc Luciferase for Quantitative Antiviral Assay.

Senecavirus A (SVA), also known as Seneca Valley virus, is a recently emerged picornavirus that can cause swine vesicular disease, posing a great threat to the global swine industry. A recombinant reporter virus (rSVA-Nluc) stably expressing the nanoluciferase (Nluc) gene between SVA 2A and 2B was developed to rapidly detect anti-SVA neutralizing antibodies and establish a high-throughput screen for antiviral agents. This recombinant virus displayed similar growth kinetics as the parental virus and remained stable for more than 10 passages in BHK-21 cells. As a proof-of-concept for its utility for rapid antiviral screening, this reporter virus was used to rapidly quantify anti-SVA neutralizing antibodies in 13 swine sera samples and screen for antiviral agents, including interferons ribavirin and interferon-stimulated genes (ISGs). Subsequently, interfering RNAs targeting different regions of the SVA genome were screened using the reporter virus. This reporter virus (rSVA-Nluc) represents a useful tool for rapid and quantitative screening and evaluation of antivirals against SVA.

Complete genome and molecular characterization of genotype VII velogenic Newcastle disease virus isolated in China.

Circulation of dominant genotype VII of Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in China. Although most of genotype VII NDV has frequently been isolated in China to date, the genome sequence difference between duck-origin and chicken-origin NDVs remains largely unknown. In this study, a NDV strain of Chicken/China/HB/2017 (HB), isolated during an outbreak in China, was subjected to genetic, biological, phylogenetic and the pathogenicity characterization. The complete genome of HB strain is 15,192 nucleotides (nt) long and consisting of six genes in the order of 3'-NP-P-M-F-HN-L-5'. Several amino acid mutations were identified in the functional domains of F and HN proteins, including fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on the F gene revealed that the HB strain and three other duck-origin NDV strains in China were grouped under subgenotype VII.1.1 and shared 99.1~99.2% nucleotide identity. Additionally, the challenge experiment results showed that the strain was highly pathogenic with 100% morbidity and mortality. Virus shedding was detected from 2 days post-infection until the fifth day. In conclusion, this study offers our understanding of circulating strains of NDV and genes involved in virulence and evolution between different hosts. Keywords: Newcastle disease virus; China; complete genome; genotype VII; mutations.

Direct and Rapid Detection of Mycoplasma bovis in Bovine Milk Samples by Recombinase Polymerase Amplification Assays.

This study aimed to detetct Mycoplasma bovis (M. bovis) in bovine milk quickly and directly by developing and validating isothermal recombinase polymerase amplification (RPA) assays. Targeting the uvrC gene of M. bovis, an RPA assay based on the fluorescence monitoring (real-time RPA) and an RPA assay combined with a lateral flow strip (LFS RPA) were conducted. It took 20 min for the real-time RPA to finish in a Genie III at 39°C, and 15 min were required to perform the LFS RPA in an incubator block at 39°C, followed by the visualization of the products on the lateral flow strip within 5 min. Both of the two assays showed high specificity for M. bovis without any cross-reaction with the other tested pathogens. With the standard recombinant plasmid pMbovis-uvrC serving as a template, both RPA assays had a limit of detcion of 1.0 × 101 copies per reaction, equivalent to that of a real-time PCR assay. In the 65 milk samples collected from cattle with mastitis, the M. bovis genomic DNA was detected in 24 samples by both the real-time RPA and the LFS RPA assays. The developed RPA assays could detect M. bovis in bovine milk in an efficient, convenient, and credible manner as attractive and promising tools, and the assays would be helpful in the rapid response to M. bovis infection causing bovine mastitis.

Latent pseudorabies virus infection in medulla oblongata from quarantined pigs.

Pseudorabies virus (PRV) is a major pathogen in pig husbandry and is also a risk to human well-being. Pigs with latent PRV infection carry the virus lifelong, and it can be activated under conducive conditions. This poses a very important challenge to the control of the virus and may even prevent its elimination. To investigate latent infection with wild-type (wt) PRV, and also infection due to the use of live attenuated vaccines on farms, 80 pigs from two large-scale swine operations were traced. At 6 months old, the quarantined pigs were slaughtered and brain samples were collected. A PCR assay targeting the gB and gE genes was developed to detect PRV DNA fragments in medulla oblongata. Five of the samples (6.3%) were gB and gE gene fragment double-positive, 60 of the samples (75%) were gB single-positive, and 15 samples (18.7%) showed double-negative. A portion of latency-associated transcripts (LATs), EP0 mRNA, were found to be present in the gB gene fragment positive samples. Furthermore, the five double-positive samples were transmitted blindly, and apparent cytopathic effects were found in three of the five samples in the fourth generation. By means of Western blotting, PCR and sequencing, two of the isolated viruses were found to be related to vaccine strain Bartha-K61. Another was closely related to domestic epidemic strains HN1201 and LA and relatively unrelated to other Asian isolates. These results suggest that the live vaccines are latently present in brains, in a manner similar to wt PRV, and this poses potential safety issues in the pig husbandry industry. Wt PRV and live vaccine viruses were found to co-exist in pigs, demonstrating that the live vaccines were unable to confer complete sterilizing immunity, which may explain outbreaks of pseudorabies on vaccinated farms.