Search

Foot-and-mouth disease (FMD) is caused by FMD virus (FMDV) is a highly contagious disease of ruminants, which is primarily controlled by vaccination. The monitoring of antisera after vaccination is currently depending on liquid-phase blocking ELISA (LPBE). Recently, bacterium-original FMD virus-like particle (VLP) showed the potential as vaccine candidates. In this study, to minimize the risk of live virus involvement, the

In order to analyse the prevalence of cat viral diseases in China, including feline parvovirus (FPV), feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV) and feline infectious peritonitis virus (FIPV), a total of 1,326 samples of cats from 16 cities were investigated from 2016 to 2019. Collectively, 1,060 (79.9%) cats were tested positive for at least one virus in nucleotide detection, and the positive rates of cat exposure to FeLV, FPV, FHV-1, FCV, FIV and FIPV were 59.6%, 19.2%, 16.3%, 14.2%, 1.5% and 0.5%, respectively. The prevalence of FHV-1 and FPV was dominant in winter and spring. Cats from north China showed a higher positive rate of viral infection than that of cats from south China. The virus infection is not highly correlated with age, except that FPV is prone to occur within the age of 12 months. In the serological survey, the seroprevalences of 267 vaccinated cats to FPV, FCV and FHV-1 were 83.9%, 58.3% and 44.0%, respectively. Meanwhile, the seroprevalences of 39 unvaccinated cats to FPV, FCV and FHV-1 were 76.9% (30/39), 82.4% (28/34) and 58.6% (17/29), respectively. This study demonstrated that a high prevalence of the six viral diseases in China and the insufficient serological potency of FCV and FHV-1 remind the urgency for more effective vaccines.

Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:216). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.

Emergent coronaviruses (CoVs) such as SARS-CoV and MERS-CoV have posed great threats to public health worldwide over the past two decades. Currently, the emergence of SARS-CoV-2 as a pandemic causes greater public health concern. CoV diversity is due to the large size and replication mechanisms of the genomes together with having bats as their optimum natural hosts. The ecological behavior and unique immune characteristics of bats are optimal for the homologous recombination of CoVs. The relationship of spatial structural characteristics of the spike protein, a protein that is critical for recognition by host receptors, in different CoVs may provide evidence in explaining the coevolution of CoVs and their hosts. This information may help to enhance our understanding of CoV evolution and thus provide part of the basis of preparations for any future outbreaks.

The pandemic SARS‐CoV‐2 has been reported in 123 countries with more than 5,000 patients died from it. However, the original and intermediate hosts of the virus remain unknown. In this study, 1,914 serum samples from 35 animal species were used for detection of SARS‐CoV‐2‐specific antibodies using double‐antigen sandwich ELISA after validating its specificity and sensitivity. The results showed that no SARS‐CoV‐2‐specific antibodies were detected in above samples which excluded the possibility of 35 animal species as intermediate host for SARS‐CoV‐2. More importantly, companion animals including pet dogs (including one dog the SARS‐CoV‐2 patient kept and two dogs which had close contact with it) and cats, street dogs and cats also showed serological negative to SARS‐CoV‐2, which relieved the public concerns for the pets as SARS‐CoV‐2 carriers.

One Gram-negative Bacillus was isolated from a brain sample of a pig with neurological symptoms. Pathological examination showed meningitis at necropsy. Ochrobactrum anthropi (O. anthropi) was successfully isolated from the brain sample and was confirmed by biochemical reaction results (API 20 NE) and gene sequencing. The strain was highly resistant to β-lactam antibiotics. Mice were experimentally infected with O. anthropi and showed typical meningitis. This is the first report on O. anthropi isolated from a pig, and indicates that O. anthropi may have a broader host spectrum of infection. Keywords: Ochrobactrum anthropi, Pig brain

Feline herpesvirus type 1 (FHV-1) is a common causative agent of domestic cats rhinotracheitis and gradually threatens the wild felid worldwide. The endangered snow leopard belongs to the family Felidae and is also the top predator on the Tibetan Plateau. Herein, FHV-1 was identified and isolated from three dead snow leopards with symptom of sneezing and rhinorrhea. To explore the relationship between FHV-1 and their death, histopathology and molecular biology was performed. The organs and nasal swabs were collected for examinations of histopathology, the nucleic acid of the pathogen, viral isolation, and sequence analysis. The results reveal that all three snow leopards were infected with FHV-1. The first case primarily died of old cerebral infarction and secondary non-suppurative meningoencephalitis probably caused by FHV-1. The second case mainly died of renal failure accompanied by interstitial pneumonia caused by FHV-1. The third case was doubted to be related to the reactivation of latency of FHV-1. The gD and gE gene sequence alignment of the FHV-1 isolate strain revealed that the isolated strain originated from a domestic cat. Therefore, FHV-1 infection can cause different lesions of snow leopards and shows a high risk for the wild felid. We should focus more on protecting felid against threatening of FHV-1 infection originating from domestic cats.

Feline calicivirus (FCV) is a highly contagious viral pathogen of upper respiratory infections and oral disease in cats. To investigate the prevalence and gene characteristic of FCV in China, a total of 1739 clinical swabs of cat eyes and nasal were collected from 19 cities in China from 2019 to 2020. The FCV from clinical samples were isolated in F81 cells, and the gene sequences of the isolated FCV’s capsid proteins were phylogenetically analyzed by constructing the phylogenetic tree with the FCV vaccine strain F9 and reference strains of other countries. Results revealed a prevalence of 13.0% (226/1739) for FCV in China in this study, and samples from Langfang showed the highest prevalence in the cities. The 74 FCV strains isolated from clinical samples shared the nucleotide identity of 73.4%-79.1% and the amino acid identity of 83%-90% comparing with the F9 strain. Phylogenetic analysis reveals two branches of these FCV strains from China, which distinct from the vaccine strains of F9 and 255, and other reference strains. Structurally, the highly variable sites of capsid protein were exposed on the protein surface between circulating strains in China and the vaccine strain F9. Overall, this study would promote the understanding of the FCV prevalence and gene characteristics in China.

The large-scale outbreaks of Pseudorabies in china since 2011 in vaccine-immunized pig farms were caused by the highly virulent Pseudorabies virus (PRV). To investigate the factors involved in the virulence enhancement of the variant PRV, four recombinant viruses with the virulence gene’s replacement, gI/gE/11K and TK/gI/gE/11K, between the variant strain HN1201 and the classical strain Fa were constructed based on bacterial artificial chromosome infectious clones. Compared with their parental strain, the viral titers of the recombinant PRV strains are not strongly influenced by the replacement of TK/gI/gE/11K, while as previously reported, the strain HN1201 and its derived viruses showed the higher mortality, the severer clinical symptoms and tissues damage than that of strain Fa and its derived strains. In summary, the TK/gI/gE/11K genes of variant strain HN1201 showed no contribute to its virulence enhancement compares with the classical strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) has varied constantly and circulated in the pig industry worldwide. The prevention and control of porcine reproductive and respiratory syndrome (PRRS) is complicated. A visual, sensitive and specific diagnostic method is advantageous to the control of PRRS. The collateral cleavage activity of LwCas13a is activated to degrade non-targeted RNA, when crRNA of LwCas13a bond to target RNA. The enhanced Cas13a detection is the combination of collateral cleavage activity of LwCas13a and recombinase polymerase amplification (RPA). In this study, the enhanced Cas13a detection for PRRSV was established. The novel method was an isothermal detection at 37°C, and the detection can be used for real-time analysis or visual readout. The detection limit of the enhanced Cas13a detection was 172 copies/μl, and there were no cross-reactions with porcine circovirus 2, porcine parvovirus, classical swine fever virus and pseudorabies virus. The enhanced Cas13a detection can work well in clinical samples. In summary, a visual, sensitive and specific nucleic acid detection method based on CRISPR-Cas13a was developed for PRRSV.

We isolated Japanese encephalitis virus (JEV) from brain samples of 2 seals with lethal encephalitis at Weihai Aquarium, Weihai, China, in 2017. We confirmed our findings by immunohistochemical staining and electron microscopy. Phylogenetic analysis showed this virus was genotype I. Our findings suggest that JEV might disseminate though infected zoo animals.

Eighteen canine distemper virus (CDV) isolates were obtained from clinical samples in Henan province, China, between 2012 and 2016. These viruses could not be recognized by 1A4, a monoclonal antibody specific for the H protein of CDV vaccine strains. The complete haemagglutinin (H) genes of all 18 isolates were sequenced, and phylogenetic analysis showed that they segregated into two clusters within the Asia-1 genotype. Moreover, the H genes of four viruses were found to lack a potential N-glycosylation site at position 309, which is the most conserved site within the Asia-1 genotype of CDV, and a novel potential N-glycosylation site (amino acids 517–519) was found in strain HL013, which has not been reported previously. These results will help in achieving a better understanding of the evolution of CDV in China. Electronic supplementary material The online version of this article (10.1007/s00705-019-04298-7) contains supplementary material, which is available to authorized users.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease and the most economically important disease of the swine industry worldwide. Highly pathogenic-PRRS virus (HP-PRRSV) is a variant of PRRSV, which caused high morbidity and mortality. Scavenger receptor CD163, which contains nine scavenger receptor cysteine-rich (SRCR) domains, is a key entry mediator for PRRSV. A previous study demonstrated that SRCR domain 5 (SRCR5), encoded by exon 7, was essential for PRRSV infection in vitro. Here, we substituted exon 7 of porcine CD163 with the corresponding exon of human CD163-like 1 (hCD163L1) using a CRISPR/Cas9 system combined with a donor vector. In CD163Mut/Mut pigs, modifying CD163 gene had no adverse effects on hemoglobin-haptoglobin (Hb-Hp) complex clearance or erythroblast growth. In vitro infection experiments showed that the CD163 mutant strongly inhibited HP-PRRSV replication by inhibiting virus uncoating and genome release. Compared to wild-type (WT) pigs in vivo, HP-PRRSV-infected CD163Mut/Mut pigs showed a substantially decreased viral load in blood and relief from PRRSV-induced fever. While all WT pigs were dead, there of four CD163Mut/Mut pigs survived and recovered at the termination of the experiment. Our data demonstrated that modifying CD163 remarkably inhibited PRRSV replication and protected pigs from HP-PRRSV infection, thus establishing a good foundation for breeding PRRSV-resistant pigs via gene editing technology.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is a severe infectious disease in the swine industry. PRRSV infection is mediated by porcine CD163 (pCD163). Scavenger receptor cysteine-rich domain 5 coded by exon 7 of pCD163 is essential for PRRSV infection. In this study, we generated CD163 exon 7 deleted (CD163E7D) pigs using CRISPR/Cas9 mediated homologous recombination and somatic cell nuclear transfer (SCNT). The deletion of exon 7 had no adverse effects on CD163-associated functions. Pigs were further challenged with a highly pathogenic PRRSV (HP-PRRSV) strain. The CD163E7D pigs exhibited mild clinical symptoms and had decreased viral loads in blood. All CD163E7D pigs survived the viral challenge, while all the WT pigs displayed severe symptoms, and 2 out of 6 WT pigs died during the challenge. Our results demonstrated that CD163 exon 7 deletion confers resistance to HP-PRRSV infection without impairing the biological functions of CD163.

Porcine reproductive and respiratory syndrome (PRRS) continues to be one of the most important swine diseases worldwide. Interferon-γ (IFNγ)-mediated type I cell-mediated immune response plays an important role in protection from, and clearance of, PRRS virus (PRRSV). Several lymphocyte subsets including T-helper, CTLs, Th/memory cells, and γδ T lymphocytes were previously reported to produce IFNγ during PRRSV infection. However, the proportion and phenotypic characterization of these IFNγ-secreting lymphocytes have not been explored. In this study, IFNγ producted by different lymphocyte subsets was assessed by multi-color flow cytometry after vaccination with PRRSV modified live vaccine (PRRSV-MLV) and challenge with homogeneous or heterogeneous PRRSV. The results showed that T-helper cells were the major IFNγ-secreting cell population after PRRSV-MLV vaccination and PRRSV challenge. Additionally, the proportion of IFNγ producing Th/memory cells and γδ T cells increased after PRRSV challenge. This difference was accounted for an enhanced ability to produce IFNγ in Th/memory cells and an enlarged quantity of γδ T cells. The results presented here could contribute to our understanding of the roles of IFNγ in protective immunity against PRRSV infection and may be useful for assessment of cell-mediated immunity in vaccine tests. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-018-0073-7) contains supplementary material, which is available to authorized users.

An outbreak of canine distemper in 2017 in mink breeding farms (Shandong province, China) caused severe pneumonia, hardened footpads, and death in more than 5000 vaccinated animals. Sequencing of the hemagglutinin and fusion protein genes from the WH2 canine distemper virus (CDV) strain we isolated from the infected minks were clustered into the recently isolated CDV Asia-1 genotype group. The WH2 strain was distinct from the current vaccine strains, containing a novel potential N-glycosylation site in its hemagglutinin protein. It also contained amino acid mutations in the fusion protein gene (I87N, T110P and L386I), and the T110P mutation results in N-glycosylation site silencing. WH2 was highly virulent in both unvaccinated and vaccinated animals in our pathogenesis experiments. Immunohistochemistry results revealed positive staining of different organs in unvaccinated and vaccinated animals. The serum in vitro neutralizing antibody titers for the vaccinated mink group and a dog were higher for the WH2 strain than those of the HNly150520B strain (isolated from a dog). These findings indicate that the current commercial vaccines provide incomplete protection against WH2 challenge infections. Thus, a new vaccine strain is urgently needed to protect against variant CDV strains.

Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection. Supplementary Information The online version contains supplementary material available at 10.1186/s13567-021-00944-8.

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious and economically devastating viral disease of cloven-hoofed animals worldwide. In this study, the coexpression of small ubiquitin-like modifier (SUMO)–fused capsid proteins of FMDV serotype O by single plasmid in Escherichia coli was achieved with an optimal tandem permutation (VP0–VP3–VP1), showing a protein yield close to 1:1:1. After SUMO removal at a low level of protease activity (5 units), the assembled FMDV virus-like particles (VLPs) could expose multiple epitopes and have a size similar to the naive FMDV. Immunization of pigs with the FMDV VLPs could induce FMDV-specific humoral and cellular immune responses effectively, in a dose-dependent manner. These data suggested that the stable FMDV VLPs with multiple epitope exposure were effective for the induction of an immune response in pigs, which laid a foundation for the further development of the FMDV subunit vaccine.

Minks are raised in many countries and have transmitted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans. However, the biologic properties of SARS-CoV-2 in minks are largely unknown. Here, we investigated and found that SARS-CoV-2 replicates efficiently in both the upper and lower respiratory tracts, and transmits efficiently in minks via respiratory droplets; pulmonary lesions caused by SARS-CoV-2 in minks are similar to those seen in humans with COVID-19. We further found that a spike protein-based subunit vaccine largely prevented SARS-CoV-2 replication and lung damage caused by SARS-CoV-2 infection in minks. Our study indicates that minks are a useful animal model for evaluating the efficacy of drugs or vaccines against COVID-19 and that vaccination is a potential strategy to prevent minks from transmitting SARS-CoV-2.

The recently identified pathogenic Porcine circovirus type 3 (PCV3) may threaten to reduce the pig population dramatically worldwide. In our previous study, a PCV3-specific monoclonal antibody (mAb-1H11) was successfully applied in immune-histochemistry staining and ELISA, which specifically recognize PCV3 capsid protein in PCV3-positive pig tissues. In the present study, we expressed and purified the soluble sole capsid protein of PCV3. The purified capsid protein was capable of self-assembly into virus-like-particles (VLPs), which is validated by transmission electron microscopy and dynamic light scattering assays. Moreover, the epitope of mAb-1H11 was identified in the CD-loop region (a.a. 72-79) on the VLP surface, which is confirmed by PCV2-PCV3 epitope swapping assay. For the first time, we determined the cryo-EM structure of PCV3-VLP at 8.5 Å resolution that reveals the detailed structural information of PCV3-VLP. In our cryo-EM structure, PCV3-VLP is composed of 60 capsid protein subunits assembled with T = 1 icosahedral symmetry. Consistent to our bio-dot Western blot assay, the structural comparison between PCV3 and PCV2 revealed significant structural differences in the surface-exposed loops, including the CD-loop (a.a. 72-79) and the EF-loop (a.a. 109-131). Our work provides a structural framework for engineering future PCV3 vaccine and diagnosis kits development.

Foot-and-mouth disease (FMD), caused by FMD virus (FMDV), is a highly contagious epidemic disease, which is controlled primarily by prophylactic vaccination and serological monitoring after vaccination. Here, we have developed a solid-phase competition ELISA (SPCE) method based on virus-like particles (VLPs) of FMDV serotype A. The use of VLPs in the SPCE assay as a replacement for inactivated FMDV provides a high level of biosafety. The SPCE showed high concordance rates when compared with the virus neutralization test and liquid-phase blocking ELISA for testing clinical serum samples and successive serological monitoring (kappa = 0.925). Thus, this SPCE is an alternative method for post-immunization detection of antibodies against FMDV serotype A, with high specificity and sensitivity.

Co-infection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs) is commonly observed under field conditions and elicits more severe diseases than any singular infection. In this study, the co-infection of PRRSV, PCV2 and PCV3 was analyzed in tissue samples collected from 150 pigs from April 2016 to April 2018. PRRSV, PCV2 and PCV3 was detected in 55 (36.67%), 43 (28.67%) and 3 (2%) of 150 pigs respectively. Remarkably, one lung sample (SD17-36) collected from a diseased pig was co-infected with PRRSV, PCV2 and PCV3. The complete genomes of SD17-36 viruses of PRRSV, PCV2 and PCV3 were determined, which belong to the subgroups of NADC30-like PRRSV, PCV2d and PCV3a respectively. Sequence comparison showed that PRRSV SD17-36 isolate contains a N33 deletion in GP5. Animal challenge study showed that the novel NADC30-like PRRSV SD17-36 isolate is low pathogenic. Our results indicate that the co-infection of PRRSV and PCVs might cause diseases even when PRRSV plays a limited role in the pathogenicity of the co-infection.

A novel porcine reproductive and respiratory syndrome virus 2 (PRRSV2) was isolated from diseased piglets in Shandong, China in 2017 and denominated as SD17-38. ORF5 sequencing showed that SD17-38 contains a unique serine/asparagine deletion at position 33 and an asparagine insertion at position 60 of GP5, which has never been described. The SD17-38 complete genome was then determined, and genome-based phylogenetic analysis showed that SD17-38 is clustered with NADC30-like isolates. Sequence alignment and recombination analyses by RDP4 and SimPlot all indicated that SD17-38 is a recombinant virus from NADC30 (lineage 1), BJ-4 (lineage 5) and TJ (lineage 8) isolates. Animal challenge study in 4-week piglets showed that SD17-38 causes high fever (≥41°C), 100% morbidity and 40% mortality. In addition, significantly lower weight gain and severe histopathological lung lesions could be observed in SD17-38-infected pigs. In particular, the unique deletion and insertion in GP5 were stable during the challenge study. This study provides direct evidence for the natural occurrence of recombination events among three lineages of PRRSV2 in Chinese swine herds, resulting in the emergence of novel PRRSV variant with unique genetic property and high pathogenicity.

Rotavirus A (RVA) G9 genotype is recognized as an emerging genotype which is spreading worldwide, however, our knowledge on pathogenicity of this virus is limited. In this study, porcine RVA strain HN03 was successfully isolated on MA-104 cells, and the isolate was propagated continuously for 7 passages after a virus cloning at passage 3. The virus titers from 4 to 10 passages ranged from 107.1 to 108.1 TCID50/ml. The growth curve of HN03 strain in cell culture was determined, and the virus production dynamics was confirmed by immunoperoxidase monolayer assay (IPMA). Sequence and phylogenetic analyses based on full-length VP7 and partial VP4 genes indicated that HN03 strain belongs to genotype G9P[23]. In addition, the sixth passage of strain HN03 in cell culture was subjected to 3-day-old piglets. All infected piglets developed severe watery diarrhea within 24 hr post-inoculation (hpi), but recovered from disease after 72 hpi. RVA antigen could be detected by IHC in the cytoplasm of villous enterocytes as early as 2 hr after appearance of clinical symptoms and virus antigen load kept increasing in the next 30 hr. The dynamics of RVA HN03 strain proliferation on cells and in pigs extended our understanding of rotavirus pathogenicity.

Canine parvovirus (CPV) can cause severe disease in animals, especially in dogs, and continuously generates new variants. In this study, the complete VP2 genes of 59 CPV isolates from clinical diseased dogs between 2015 and 2016 in 4 Chinese provinces were sequenced and analyzed. The results showed that new CPV-2a was still the prominent CPV genotype, followed by CPV-2c and new CPV-2b. CPV-2c with Ala5Gly and Gln370Arg mutations was first detected in Henan, Guangxi and Jiangsu provinces in China. A phylogenetic tree based on VP2 sequences showed that all isolates in this study formed a close lineage which separated from foreign isolates. The above results indicated that point mutations in VP2 were constantly occurring along with the widespread of CPV in China. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13337-018-0427-7) contains supplementary material, which is available to authorized users.

NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) has widely spread in China and become locally dominant virus strain in some provinces. Although they are not pathogenic as highly pathogenic PRRSV (HP-RRRSV) that outbreaks since 2006, NADC30-like PRRSVs distinguished themselves by high incidence of recombination with other virus strains which lead to change of virulence. The outbreaks of NADC30-like PRRSV in the vaccinated pig herds suggested that current commercial PRRSV vaccines cannot provide complete protection to the infection. In this review, we have described in detail the current situation of NADC30 PRRSV including epidemiology, genomic characterization, pathogenicity, and efficacy of current commercial vaccines in China.

Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

Background: Canine parvovirus (CPV) emerged in 1970s as a highly infectious disease. CPV modified live vaccines have been widely used to control the disease. It is urgent to develop specific monoclonal antibodies to differentiate field virus from vaccine virus in vaccinated dogs.

A canine parainfluenza virus type 5 strain was isolated from a lung sample from a diseased dog. The genome sequene of this isolate, named HeN0718, was determined and compared tho those of other previously reported canine parainfluenza viruses. Unlike previously reported viruses, the HeN0718 strain contained several nucleotide mutations in the SH gene that led to a frame shift in the open reading frame. Phylogenetic analysis based on the complete virus genome and the P, F, and HN genes showed that HeN0718 was genetically closest to D277, a Korean strain that was isolated in 2008.

Summary Constant surveillance of live poultry markets (LPMs) is currently the best way to predict and identify emerging avian influenza viruses (AIVs) that pose a potential threat to public health. Through surveillance of LPMs from 16 provinces and municipalities in China during 2014–2016, we identified 3,174 AIV-positive samples and isolated and sequenced 1,135 AIVs covering 31 subtypes. Our analysis shows that H5N6 has replaced H5N1 as one of the dominant AIV subtypes in southern China, especially in ducks. Phylogenetic analysis reveals that H5N6 arose from reassortments of H5 and H6N6 viruses, with the hemagglutinin and neuraminidase combinations being strongly lineage specific. H5N6 viruses constitute at least 34 distinct genotypes derived from various evolutionary pathways. Notably, genotype G1.2 virus, with internal genes from the chicken H9N2/H7N9 gene pool, was responsible for at least five human H5N6 infections. Our findings highlight H5N6 AIVs as potential threats to public health and agriculture.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a huge threat to the modern pig industry, and current vaccine prevention strategies could not provide full protection against it. Therefore, exploring new anti-PRRSV strategies is urgently needed. Ginsenoside Rg1, derived from ginseng and notoginseng, is shown to exert anti-inflammatory, neuronal apoptosis-suppressing and anti-oxidant effects. Here we demonstrate Rg1-inhibited PRRSV infection both in Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose-dependent manner. Rg1 treatment affected multiple steps of the PRRSV lifecycle, including virus attachment, replication and release at concentrations of 10 or 50 &micro, M. Meanwhile, Rg1 exhibited broad inhibitory activities against Type 2 PRRSV, including highly pathogenic PRRSV (HP-PRRSV) XH-GD and JXA1, NADC-30-like strain HNLY and classical strain VR2332. Mechanistically, Rg1 reduced mRNA levels of the pro-inflammatory cytokines, including IL-1&beta, IL-8, IL-6 and TNF-&alpha, and decreased NF-&kappa, B signaling activation triggered by PRRSV infection. Furthermore, 4-week old piglets intramuscularly treated with Rg1 after being challenged with the HP-PRRSV JXA1 strain display moderate lung injury, decreased viral load in serum and tissues, and an improved survival rate. Collectively, our study provides research basis and supportive clinical data for using Ginsenoside Rg1 in PRRSV therapies in swine.

We reported a severe human pseudorabies encephalitis case and described a dynamic clinical manifestation with cerebrospinal fluid analyses and cytological and serological evaluation, which may elucidate the mechanism of PRV infection and facilitate clinical diagnosis and treatment in human.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA-PRRSV2), highly pathogenic (HP-PRRSV2) and NADC30-like (NL-PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real-time RT-PCR assays using TaqMan-MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real-time RT-PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real-time RT-PCR assays were further validated by comparing with a universal PRRSV conventional RT-PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real-time RT-PCR assays and the conventional RT-PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV-positive, including 2 samples that were determined to be co-infected with NL-PRRSV2 and HP-PRRSV2 isolates by the quadruplex real-time RT-PCR assay. ORF5 sequencing confirmed the real-time RT-PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA-PRRSV2, NL-PRRSV2 and HP-PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.

Background: Newcastle disease virus (NDV) genotype VII has become the dominant genotype in China. However, NDV genotype II was used to make current commercial NDV vaccines. The mismatch of genotypes between circulating and vaccine strains of viruses may compromise the efficacy of vaccines.

Epidemiological outbreak investigations were conducted on NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the prevalence of the disease in south-east China in 2015. Two more provinces were found to have NADC30-like PRRSV circulating besides previously reported six provinces. Phylogenetic analysis showed that these virus isolates were clustered in an independent branch and shared high nucleotide similarity to NADC30, a type 2 PRRSV that has been isolated in Unite States in 2008. One NADC30-like PRRSV strain from Henan province was successfully isolated on porcine alveolar macrophages and was tested on 6-week-old specific pathogen-free pigs for pathogenic study. The virus-inoculated pigs showed typical PRRSV clinical symptoms, but all pigs survived throughout the study with a period of 14 days. At necropsy, the lungs of infected pigs developed PRRSV-specific interstitial pneumonia, and virus antigen was detected in lung samples. Therefore, our results indicated NADC30-like PRRSV has widely spread in China and could cause clinical disease on pigs.

We report the strategies leading to the large-production of soluble non-tag full-length porcine circovirus type 2 (PCV2) Cap protein in Escherichia coli. Under neutral pH condition, the purified recombinant Cap protein derived from E. coli expression self-assembles into homogenous round virus-like particle at the similar size of that of the intact PCV2 virus, which is further characterized by Cryo-EM single particle structure determined at 4.5Å. The engineered PCV2 rCap VLP was tested as a subunit vaccine for the protective efficacy against PCV2 challenge on 3-week old piglets. Similar to commercial available PCV2 vaccine, the Cap VLP-immunized piglets developed specific antibody-mediated response and were protected from the virulent SH PCV2 strain challenge. Hence, the production of E. coli based PCV2Cap-VLP could be applied as a cost-friendly and effective subunit vaccine to control PCV2 spreading in developing countries.

The truncated spike protein (S1, 390-789aa) of porcine epidemic diarrhea virus was expressed as a recombinant protein in Baculorvirus expression system. Female BALB/c mice were immunized with the purified recombinant S1 (rS1) protein and two monoclonal antibodies, designated 8A3A10 and 12C4G12, were generated by hybridoma technique. The two monoclonal antibodies specifically reacted with PEDV rS1 protein, as proven by Western blot and indirect immunofluorescence assay. 8A3A10 monoclonal antibody showed a neutralizing activity to PEDV, as demonstrated in the results of fluorescent focus neutralization assay. These two newly established monoclonal antibodies may work as a useful tool to study the pathogenic mechanisms of PED virus and to develop diagnostic reagents.

Feline panleukopenia virus (FPV) and canine parvovirus (CPV) infections are highly contagious and cause serious enteric diseases, with high fatality rates of cats and dogs. Given the importance of cats as a potential source of genetic diversity for parvoviruses, parvovirus strains detected in cats with gastroenteritis signs were analysed, and molecular characterisation, sequence analysis and phylogeny were evaluated on the VP2 gene. The results showed that FPV, new CPV-2a, and CPV-2 are co-circulating in the cat population in Henan province of China. Moreover, CPV-2 strains (F2016020, and F2016021) with Ser297Ala substitution in VP2 protein was for the first time detected in cats with clinical gastroenteritis. This study provided new important findings about the evolutionary of parvovirus infection in domestic cats.

Porcine circoviruses (PCV) include PCV1, PCV2, and the new-emerging PCV3. PCV2 is pathogenic to pigs, but the pathogenicity of PCV3 in pigs is debatable. Recently, there have been frequent reports of PCV2 and PCV3 co-infections in clinical samples. Thus, it would be practical to develop a duplex PCR method to detect PCV2 and PCV3 simultaneously. In this study, specific primers and probes were designed to target PCV2 cap and PCV3 rep genes. A duplex real-time PCR method was then developed to detect the two viruses. The assay was found to be highly specific, sensitive, and reproducible for PCV2/3 without cross-reactions with other swine pathogens. The sensitivity of this assay was 2.9 copies for the PCV2 plasmid and 22.5 copies for the PCV3 plasmid. The established assay was then used to detect PCV2/3 infection in 340 clinical samples collected in the first half of 2017. The results showed that the co-infection rate of PCV2/3 in the samples was 27.6%. Our study provides an important tool that can be used to perform urgently needed surveys for the two porcine circoviruses to evaluate their impact on the swine industry. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-018-0025-2) contains supplementary material, which is available to authorized users.

Porcine cirvovirus type 3 (PCV3) is a newly emerging swine virus with worldwide distribution. The pathogenesis of PCV3 remains unknown due to failures in propagating and isolating the virus on successive cell lines. The virus cap protein is the only structural protein of PCV3 and no monoclonal antibodies against it are available. Although antisera are available from PCV3-infected pigs, they are not suitable for immunoassays such as immunohistochemical staining of infected tissues due to high non-specific background. Developing monoclonal antibodies against the cap protein is indispensable for elucidating PCV3 biological properties. In this study, a monoclonal antibody (mAb 1H1) with a titer of 1/25,600 that recognized the PCV3 cap protein was developed. Western-blot results showed that mAb 1H1 reacted strongly with the recombinant PCV3 cap protein preparation but not with the PCV2 cap protein. MAb 1H1 was also applied successfully for the detection of the cap protein in PCV3-infected pig tissues using immunochemistry staining. In conclusion, mAb 1H1 has significant potential uses in PCV3 diagnosis as well as the study of PCV3 biology.

New-emerging variants of Pseudorabies virus (PRV) compromise the protection provided by current vaccines and cause the death of all ages of vaccinated pigs since 2011. New vaccines based on current circulating PRV strain are needed to control the spread of disease since the variants are antigenically different from classical strains of virus. In this study, a TK/gE/gI triple gene-deleted PRV derived from current circulating field isolate was generated by using bacterial artificial chromosome techniques, and the rescued virus showed similar growth properties in vitro to its parent strain but reduced plaque size. To evaluate it as vaccine candidate, 9 day-old pigs were vaccinated and challenged with a virulent PRV variant. The results showed that vaccination can generate high level of protective gB-specific antibodies after vaccination and provide complete protection to the viral challenge. By contrast, the unvaccinated piglets all died within 6 days after viral challenge. Therefore, the TK/gE/gI triple gene-deleted PRV could be a promising vaccine candidate to control the wide spreading of PR variants in China.

Background: The spreading of H9N2 avian influenza viruses in poultry in Eurasia and Africa accompanied with the great economic losses to poultry industry in past decades has attracted the great attention of whole world. Domestic ducks play a critical role in the ecology of avian influenza viruses.

Porcine circovirus type 3 (PCV3) was initially reported in 2016 in the United States of America. Since then, the virus has been detected on swine farms in Poland, South Korea, and China using PCR. However, a serological survey of PCV3 in pig populations has never been conducted. In this study, for the first time, we established an indirect enzyme-linked immunosorbent (ELISA) assay and performed a national retrospective serological survey for PCV3. Our results showed that the PCV3-postive rate increased from 22.35% to 51.88% between 2015 and 2017. The above results suggest PCV3 has spread widely in China with increased positive rates since 2015.

An early and yet indispensable step in the alphaherpesvirus infection is the engagement of host receptors by the viral envelope glycoprotein D (gD). Of the thus-far identified gD receptors, nectin-1 is likely the most effective in terms of its wide usage by multiple alphaherpesviruses for cell entry. The molecular basis of nectin-1 recognition by the gD protein is therefore an interesting scientific question in the alphaherpesvirus field. Previous studies focused on the herpes simplex virus (HSV) of the Simplexvirus genus, for which both the free gD structure and the gD/nectin-1 complex structure were reported at high resolutions. The structural and functional features of other alphaherpesviral gDs, however, remain poorly characterized. In the current study, we systematically studied the characteristics of nectin-1 binding by the gD of a Varicellovirus genus member, the pseudorabies virus (PRV). We first showed that PRV infects host cells via both human and swine nectin-1, and that its gD exhibits similar binding affinities for nectin-1 of the two species. Furthermore, we demonstrated that removal of the PRV gD membrane-proximal residues could significantly increase its affinity for the receptor binding. The structures of PRV gD in the free and the nectin-1-bound states were then solved, revealing a similar overall 3D fold as well as a homologous nectin-1 binding mode to its HSV counterpart. However, several unique features were observed at the binding interface of PRV gD, enabling the viral ligand to utilize different gD residues (from those of HSV) for nectin-1 engagement. These observed binding characteristics were further verified by the mutagenesis study using the key-residue mutants of nectin-1. The structural and functional data obtained in this study, therefore, provide the basis of receptor recognition by PRV gD., Author summary Both herpes simplex virus (HSV) and pseudorabies virus (PRV) recognize nectin-1 as the cellular receptor. They utilize the envelope glycoprotein D (gD) on the virion surface to interact with nectin-1, initiating the virus infection. Although the molecular basis of nectin-1 binding by HSV gD has been successfully elucidated with high resolution structures, the atomic features of PRV gD interacting with the same receptor remains uncharacterized. Here, we show that PRV gD exhibits nano-molar affinities for both human and swine nectin-1, and deletion of the membrane-proximal loop in the gD ectodomain dramatically increases its receptor-binding avidity. We further solved the free and the nectin-1-bound PRV gD structures. The free gD structure reveals a canonical fold of an IgV-like core wrapped by the N- and C-terminal extensions as observed in the HSV homologs. The N-terminus of PRV gD, however, is shorter than that of HSV gDs. The solved complex structure demonstrates that PRV gD exhibits a homologous receptor-binding mode to the HSV counterpart. Nevertheless, several unique features at the PRV gD binding interface suffice the viral ligand to engage nectin-1 with a series of residues differing from the HSV amino acids. These observations not only delineated the molecular basis of PRV engaging nectin-1 but also enriched our knowledge on the receptor-binding mechanism of the Alphaherpesvirinae subfamily.

Pseudorabies (PR) is an economically important viral disease of pigs which can infect numerous species of mammals including rodents. Commercial PR vaccines have been widely used worldwide to control and eradicate this disease. However, some PRV vaccines such as Bartha-K61 were occasionally reported to be lethal to mice. Since mice are commonly found in pig farms, the safety issue of PRV live vaccine across different species was never addressed. In this study, PRV vaccine strain Bartha-K61 was in vivo propagated in mice for five passages. The mortality of mice ranged from 80%-100% at each passage of PRV infection. The fifth passage of PRV was used to infect piglets to test its virulence on this species. The infected piglets clinically behaved normally and survived by the end of study (terminated at 10days post-infection). Histopathologically, there was infiltration of eosinophile granulocyte in tonsil and lung and no other changes were observed in other organs of infected pigs. Immunohistochemistry staining results showed that PRV antigen was only found in lung sample of one piglet. Therefore, the above results suggested there was no safety concern of Bartha-K61 PRV vaccine on pigs after the vaccine virus was passaged in mice for 5 times. The result of this study may suggest that mice may play a minimal role in the derivation of PRV vaccine-like field viruses that are believed to cause disease in young pigs.