Molecular characterization of HLJ-073, a recombinant canine coronavirus strain from China with an ORF3abc deletion.

Canine enteric coronaviruses (CCoVs) are important enteric pathogens of dogs. CCoVs with different variations are typically pantropic and pathogenic in dogs. In this study, we isolated a CCoV, designated HLJ-073, from a dead 6-week-old male Pekingese with gross lesions and diarrhea. Interestingly, sequence analysis suggested that HLJ-073 contained a 350-nt deletion in ORF3abc compared with reference CCoV isolates, resulting in the loss of portions of ORF3a and ORF3c and the complete loss of ORF3b. Phylogenetic analysis based on the S gene showed that HLJ-073 was more closely related to members of the FCoV II cluster than to members of the CCoV I or CCoV II cluster. Furthermore, recombination analysis suggested that HLJ-073 originated from the recombination of FCoV 79-1683 and CCoV A76, which were both isolated in the United States. Cell tropism experiments suggested that HLJ-073 could effectively replicate in canine macrophages/monocytes and human THP-1 cells. This is the first report of the isolation of strain HLJ-073 in China, and this virus has biological characteristics that are different from those of other reported CCoVs.

One-step triplex PCR/RT-PCR to detect canine distemper virus, canine parvovirus and canine kobuvirus.

To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 102.1 TCID50 for CDV, 101.9 TCID50 for CPV and 103 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for the rapid clinical detection and epidemiological surveillance of CDV, CPV and CaKoV in dogs.

First report of feline bocavirus associated with severe enteritis of cat in Northeast China, 2015.

Feline bocavirus (FBoV) is a newly identified bocavirus of cats in the family Parvoviridae. A novel FBoV HRB2015-LDF was first identified from the cat with severe enteritis in Northeast China, with an overall positive rate of 2.78% (1/36). Phylogenetic and homologous analysis of the complete genome showed that FBoV HRB2015-LDF was clustered into the FBoV branch and closely related to other FBoVs, with 68.7-97.5% identities. And the genes of VP1, NPA and NS1 shared 70.7-97.6, 72.4-98.6 and 67.2-98.0% nucleotide identities with other FBoVs, respectively. The results suggested that the FBoVs could be divided into two distinct lineages, and the difference of nucleotide identities was >20-30% between the lineages.

Transmissible Gastroenteritis Virus Papain-Like Protease 1 Antagonizes Production of Interferon-ß through Its Deubiquitinase Activity.

Coronaviruses (CoVs), such as human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome CoV (SARS-CoV), murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), encode papain-like (PL) proteases that inhibit Sendai virus- (SeV-) induced interferon (IFN-ß) production. Recently, the crystal structure of transmissible gastroenteritis virus (TGEV) PL1 has been solved, which was similar to that of SARS-CoV PL2pro, which may antagonize host innate immunity. However, very little is known about whether TGEV PL1 can antagonize host innate immune response. Here, we presented evidence that TGEV PL1 encoded by the replicase gene could suppress the IFN-ß expression and inhibit the nuclear translocation of interferon regulatory factor 3 (IRF3). The ability to antagonize IFN-ß production was dependent on the intact catalytic activity of PL1. Furthermore, TGEV PL1 exerted deubiquitinase (DUB) activity which strongly inhibited the retinoic acid-induced gene I- (RIG-1-) and stimulator of interferon gene- (STING-) dependent IFN expression. Our data collectively suggest that TGEV PL1 can inhibit the IFN-ß expression and interfere with RIG-1- and STING-mediated signaling through a viral DUB activity. Our study has yielded strong evidence for the TGEV PL1 mechanisms that counteract the host innate immunity.

Feline Panleucopenia Virus NS2 Suppresses the Host IFN-ß Induction by Disrupting the Interaction between TBK1 and STING.

Feline panleucopenia virus (FPV) is a highly infectious pathogen that causes severe diseases in pets, economically important animals and wildlife in China. Although FPV was identified several years ago, little is known about how it overcomes the host innate immunity. In the present study, we demonstrated that infection with the FPV strain Philips-Roxane failed to activate the interferon ß (IFN-ß) pathway but could antagonize the induction of IFN stimulated by Sendai virus (SeV) in F81 cells. Subsequently, by screening FPV nonstructural and structural proteins, we found that only nonstructural protein 2 (NS2) significantly suppressed IFN expression. We demonstrated that the inhibition of SeV-induced IFN-ß production by FPV NS2 depended on the obstruction of the IFN regulatory factor 3 (IRF3) signaling pathway. Further, we verified that NS2 was able to target the serine/threonine-protein kinase TBK1 and prevent it from being recruited by stimulator of interferon genes (STING) protein, which disrupted the phosphorylation of the downstream protein IRF3. Finally, we identified that the C-terminus plus the coiled coil domain are the key domains of NS2 that are required for inhibiting the IFN pathway. Our study has yielded strong evidence for the FPV mechanisms that counteract the host innate immunity.

Identification of Inonotus obliquus polysaccharide with broad-spectrum antiviral activity against multi-feline viruses.

Inonotus obliquus polysaccharides (IOPs) are a potential drug for the prevention and treatment of cancer, cardiopathy, diabetes, AIDs, pancreatitis and other diseases. In this study, we found that IOP can act as a broad-spectrum antiviral drug against feline viruses in the in vitro experiment. Using cell models of feline calicivirus (FCV), we demonstrated that IOP treatment was capable of exhibiting anti-FCV strain F9 activity in cell-based assays and also showed low cytotoxicity. Investigation of the mechanism of action of the compound revealed that IOP treatment induces its inhibitory actions directly on virus particles through blocking viral binding/absorpting. The inhibitory activity against other FCV isolates from China was also identified. More importantly, we found that IOP exhibited broad-spectrum antiviral activity against the feline herpesvirus 1, feline influenza virus H3N2 and H5N6, feline panleukopenia virus and feline infectious peritonitis virus that can contribute to respiratory and gastrointestinal diseases in cats. These findings suggest that IOP may be a potential broad-spectrum antiviral drug against feline viruses.

Molecular characterization of a feline calicivirus isolated from tiger and its pathogenesis in cats.

Feline calicivirus (FCV) is a virus that causes respiratory disease in cats. In this study, the FCV TIG-1 was isolated from Siberian tiger feces collected in 2014 in Heilongjiang Province, China. Phylogenetic analysis among TIG-1 and other FCVs showed that TIG-1 does not share the same lineage with other FCV isolates from Heilongjiang or other regions in China but is located in the same cluster with the FCV strain Urbana, which was isolated from the United States. The growth kinetics in vitro and the pathogenicity in cats between TIG-1 and the domestic cat-origin FCV strain F9 (vaccine strain) and strain 2280 were compared. We found that the growth kinetics of strains TIG-1 and 2280 were faster than that of strain F9 from 12h to 36h post-infection, indicating that strains TIG-1 and 2280 produce infectious virions and reach peak yields earlier. Challenge experiments in cats showed that TIG-1 grew faster than the other two strains in the lungs of cats and that TIG-1 is a virulent FCV with 100% morbidity and lethality. In addition, the histopathological results showed that the virulent TIG-1 strain directly led to severe lung tissue damage and indirectly led to intestinal damage. The results presented here show that a tiger-origin FCV exhibits high virulence in cats.

Characterization and pathogenicity of a novel mammalian orthoreovirus from wild short-nosed fruit bats.

Mammalian orthoreoviruses (MRVs) have a wide range of geographic distribution and have been isolated from humans and various animals. This study describes the isolation, molecular characterization and analysis of pathogenicity of MRV variant B/03 from wild short-nosed fruit bats. Negative stain electron microscopy illustrated that the B/03 strain is a non-enveloped icosahedral virus with a diameter of 70nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) migration patterns showed that the B/03 viral genome contains 10 segments in a 3:3:4 arrangement. The isolate belongs to MRV serotype 1 based on S1 gene nucleotide sequence data. BALB/c mice experimentally infected with B/03 virus by intranasal inoculation developed severe respiratory distress with tissue damage and inflammation. Lastly, B/03 virus has an increased transmission risk between bats and humans or animals.

A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

BACKGROUND: Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. METHODS AND RESULTS: To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. CONCLUSIONS AND SIGNIFICANCE: We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

In vitro antiviral effect of germacrone on feline calicivirus.

Feline calicivirus (FCV) often causes respiratory tract and oral disease in cats and is a highly contagious virus. Widespread vaccination does not prevent the spread of FCV. Furthermore, the low fidelity of the RNA-dependent RNA polymerase of FCV leads to the emergence of new variants, some of which show increased virulence. Currently, few effective anti-FCV drugs are available. Here, we found that germacrone, one of the main constituents of volatile oil from rhizoma curcuma, was able to effectively reduce the growth of FCV strain F9 in vitro. This compound exhibited a strong anti-FCV effect mainly in the early phase of the viral life cycle. The antiviral effect depended on the concentration of the drug. In addition, germacrone treatment had a significant inhibitory effect against two other reference strains, 2280 and Bolin, and resulted in a significant reduction in the replication of strains WZ-1 and HRB-SS, which were recently isolated in China. This is the first report of antiviral effects of germacrone against a calicivirus, and extensive in vivo research is needed to evaluate this drug as an antiviral therapeutic agent for FCV.

Molecular cloning and functional characterization of feline MAVS.

The mitochondrial anti-viral signaling protein (MAVS) plays an important role in the type I IFN response. In this study, two feline MAVS transcripts were cloned. Both transcripts have the same open reading frame encoding 523 amino acids. The putative protein shares 76.6 % similarity with canine and exhibits similarity to human, mouse, rat, bovine and porcine, ranging from 46.1 to 65.8 %. Deletion mutant analysis indicated that the transmembrane (TM) domain is necessary for localization in the mitochondrial membrane, and both the caspase activation and recruitment domain and TM domain are indispensible for activating the IFN-ß response. Additionally, Sendai virus-induced IFN-ß promoter activation was significantly inhibited by siRNA targeting MAVS. Finally, miniMAVS, a second protein encoded by MAVS mRNA, was identified, which interfered with the IFN-ß response via the inhibition of NF-κB activation. The identification of MAVS will promote the understanding and control of feline infectious diseases.

Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus.

For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV.

Isolation and pathogenicity of the mammalian orthoreovirus MPC/04 from masked civet cats.

Mammalian reoviruses (MRVs) are associated with pulmonary infections and have been isolated from humans and various animals experiencing respiratory illness. We report here the first case of an MRV detected in the masked palm civet, which showed the highest similarity to the serotype 3 MRV. Reovirus particles were identified by electron microscopic examination of both negative-stain and thin-section. Genomic pattern analysis on SDS-PAGE showed that MPC/04 had 10-segmented double-strand RNA genome. Intranasal infection of four-week-old female BALB/c mice resulted in fatal respiratory distress but not other routes. Infections caused tissue damage and inflammation. MPC/04 grew to higher titers in the lungs than in other tissues. This research strongly suggests a need for additional experimentation to understand the pathogenic mechanisms of mammalian orthoreoviruses in infected animals and humans.

Antiviral effect of lithium chloride on feline calicivirus in vitro.

Feline calicivirus (FCV) is a highly contagious pathogen that causes oral and upper respiratory tract disease in cats. Despite widespread vaccination, the prevalence of FCV remains high. Furthermore, a high gene mutation rate has led to the emergence of variants, and some infections are lethal. To date, there is no effective antiviral drug available for treating FCV infection. Here, we show that lithium chloride (LiCl) effectively suppresses the replication of FCV strain F9 in Crandell-Reese feline kidney (CRFK) cells. The antiviral activity of LiCl occurred primarily during the early stage of infection and in a dose-dependent manner. LiCl treatment also inhibited the cytopathic effect. LiCl treatment exhibited a strong inhibitory effect against a panel of other two reference strains and two recent FCV isolates from China. These results demonstrate that LiCl might be an effective anti-FCV drug for controlling FCV disease. Further studies are required to explore the antiviral activity of LiCl against FCV replication in vivo.

Complete Genome Sequence of Feline Panleukopenia Virus Strain HRB-CS1, Isolated from a Domestic Cat in Northeastern China.

Here, we report the complete genome sequence of feline panleukopenia virus (FPLV) strain HRB-CS1, isolated from a dead domestic cat showing enteric symptoms in China in 2014. The genome of HRB-CS1 was sequenced and analyzed, which will help to understand the genetic characteristics and evolution of FPLV in China.

Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals.

Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers-two internal and two external-were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65°C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10(-1)TCID50ml(-1). The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.

Complete Genome Sequence of Feline Calicivirus Strain HRB-SS from a Cat in Heilongjiang Province, Northeastern China.

Here, we report the complete genome sequence of feline calicivirus (FCV) strain HRB-SS isolated in 2014 from a cat in Heilongjiang Province of northeastern China. The virus genome is 7,705 bases, excluding the poly(A) tail. This analysis will help to understand the genetic characteristics and evolution of FCV in China.

Identification and genetic characterization of avian-origin H3N2 canine influenza viruses isolated from the Liaoning province of China in 2012.

A total of 158 serum samples and 510 nasal swab specimens were collected between September 2010 and May 2012, from dogs exhibiting respiratory symptoms, in order to investigate the epidemiology of H3N2 canine influenza viruses (CIVs) in the Liaoning province of China. Serological surveillance demonstrated that 10.8 % (17/158) of serum samples were positive for H3N2 canine influenza. Two H3N2 influenza viruses, A/canine/Liaoning/27/2012 and A/canine/Liaoning/H6/2012, were isolated from pet dogs in 2012. Phylogenetic analysis indicated that the genes from these two viruses were closely related to those of avian-origin, H3N2 subtype CIVs from China and Thailand. Genetic analysis of eight genes revealed that these two H3N2 canine influenza isolates were highly similar (99.2-99.8 %) to the current common strains in Asia. Analysis of the genotype demonstrated that each gene of the two strains in this study had the same genotype (K, G, E, 3B, F, 2D, F, 1E) as those prevalent in H3N2 CIVs. Our findings further confirm that avian-origin H3N2 canine influenza has become established in China. Conducting extensive serological and epidemiological surveillance is necessary to develop an effective vaccine against this disease.

Emerging multiple reassortant H5N5 avian influenza viruses in ducks, China, 2008.

Three highly pathogenic H5N5 avian influenza viruses (HPAI), A/duck/Guangdong/wy11/2008 (WY11), A/duck/Guangdong/wy19/2008 (WY19), and A/duck/Guangdong/wy24/2008 (WY24) were isolated from ducks in southern China in April 2008. Here, we characterized these viruses by performing sequencing and phylogenetic analyses of their viral genes, assessing their virulence in ducks and mice, and performing cross-protection experiments in chickens. Sequence analysis revealed that the HA genes of these H5N5 viruses showed 97.1-97.8% homology to A/wild duck/Hunan/211/2005 (H5N1) influenza virus and that their NA genes showed 96.4-96.8% nucleotide identity to the NA gene of A/duck/Hunan/5613/2003 (H6N5) influenza virus, which belongs to the Eurasian lineage. Genotypic analysis indicated that these H5N5 viruses were multiple reassortants among H5N1, H5N2, H6N2, and H6N5 viruses. The analysis of HA clade showed that these H5N5 viruses are clustering into clade 2.3.4. In animal experiments, these H5N5 viruses caused 50% mortality in ducks and 100% mortality in chickens. In cross-protection experiments, the clade 2.3.2 avian influenza vaccine could provide only 75% protection with chickens against H5N5 virus challenge. Moreover, the H5N5 virus replicated efficiently in the lungs of mice, which suggested that the H5N5 viruses have the potential to infect mammalian hosts. Since ducks have served as reassortant vessels, playing pivotal roles in the generation of new subtypes of influenza viruses, it is important to monitor the emergence of this novel subtype of influenza viruses in waterfowl to understand their ecology and evolution and to control the spread of new viruses.

Comparison of the protective efficacy of recombinant adenoviruses against classical swine fever.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF.