Establishment and preliminary application of duplex fluorescence quantitative PCR for porcine circoviruses type 2 and type 3.

Porcine circovirus types 2 (PCV2) and 3 (PCV3) are the two most prevalent porcine circoviruses in China, all of which can infect swine herds and cause serious diseases. To detect coinfection with PCV2 and PCV3, primers and probes for duplex PCV2 and PCV3 real-time PCR were designed to target their cap genes based on the constructed plasmids pUC57-PCV2 and pUC57-PCV3. The established duplex PCV2 and PCV3 real-time PCRs were specific to PCV2 and PCV3 and showed no cross-reactions with other porcine viral pathogens. The limit of detection was 5 and 50 copies for the PCV2 and PCV3 plasmids, respectively. The intra- and interassay repeatability had coefficients of variation below 3 %. The established methods were used to analyze clinical samples from Liaoning and Jilin provinces of China. The coinfection rates of PCV2 and PCV3 in pigs extensively fed in Liaoning and Jilin, large-scale farmed pigs in Liaoning and large-scale farmed pigs in Jilin were 15.0 % (6/40), 36.7 % (11/30) and 35.4 % (62/175), respectively. This study established a useful duplex PCV2 and PCV3 real-time PCR method that can be used for the detection of PCV2 and PCV3 in local clinical samples.

An updated review of feline coronavirus: mind the two biotypes.

Feline coronavirus (FCoV) includes two biotypes: feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). Although both biotypes can infect cats, their pathogenicities differ. The FIPV biotype is more virulent than the FECV biotype and can cause peritonitis or even death in cats, while most FECV biotypes do not cause lesions. Even pathogenic strains of the FECV biotype can cause only mild enteritis because of their very low virulence. This article reviews recent progress in FCoV research with regard to FCoV etiological characteristics; epidemiology; clinical symptoms and pathological changes; pathogenesis; and current diagnosis, prevention and treatment methods. It is hoped that this review will provide a reference for further research on FCoV and other coronaviruses.

Mind the feline coronavirus: Comparison with SARS-CoV-2.

Both feline coronavirus (FCoV) and SARS-CoV-2 are coronaviruses that infect cats and humans, respectively. However, cats have been shown to be susceptible to SARS-CoV-2, and FCoV also had been shown to infect human. To elucidate the relationship between FCoV and SARS-CoV-2, we highlight the main characteristics of the genome, the receptor usage, and the correlation of the receptor-binding domain (RBD) of spike proteins in FCoV and SARS-CoV-2. It is demonstrated that FCoV and SARS-CoV-2 are closely related to the main characteristics of the genome, receptor usage, and RBD of spike proteins with similar furin cleavage sites. In particular, the affinity of the conserved feline angiotensin-converting enzyme 2 (fACE2) receptor to the RBD of SARS-CoV-2 suggests that cats are susceptible to SARS-CoV-2. In addition, cross-species of coronaviruses between cats and humans or other domesticated animals are also discussed. This review sheds light on cats as potential intermediate hosts for SARS-CoV-2 transmission, and cross-species transmission or zoonotic infection of FCoV and SARS-CoV-2 between cats and humans was identified.

Goose parvovirus and the protein NS1 induce apoptosis through the AIF-mitochondrial pathway in goose embryo fibroblasts.

In this study, the effects of Goose parvovirus (GPV) infection as well as the possible role of NS1 protein on apoptosis induction in goose embryo fibroblast (GEF) cells were examined. Flow cytometry analysis and TUNEL assays revealed that GPV infection and NS1 transfection induced significant apoptosis in GEF cells compared to what was observed in mock-infected cells. Interestingly, the increase in the rate of apoptosis detected in GPV-infected GEFs was accompanied by an increased viral load in the cells. In addition, the apoptotic pathway was mediated by apoptosis-inducing factors (AIFs) and internal factors that influence the release of AIFs. The results indicated that the mitochondrial membrane potential was decreased, and AIF expression was increased in the nucleus (P < 0.01). Reactive oxygen species (ROS) increased gradually within 48 h (P < 0.001). Cathepsin D activities were also increased (P < 0.05). The results demonstrated that the AIF-mediated pathway is a new mitochondrial apoptotic pathway and that mitochondrial depolarization, ROS content, and cathepsin D activities are the key factors influencing apoptosis in GEF cells.

Genomic Characterization of Diverse Gyroviruses Identified in the Feces of Domestic Cats.

Gyroviruses (GyVs) are small, single-stranded, circular DNA viruses in the genus Gyrovirus, which consists of the chicken anemia virus (CAV) prototype and nine other viral species. These different GyV species have been reported in chickens, humans, mice, and companion animals. To date, CAV has been identified in the feces of domestic cats, while the circulation of other GyV species in cats is currently unknown. In the present study, 197 fecal samples were collected from pet cats in northeast China, and samples were screened for different GyV species by PCR. Twelve GyV strains were identified from the feces of pet cats. These included 4 positive for CAV, 3 for HGyV/AGV2, 3 for GyV3 and 2 positive for GyV6. The complete genome sequences of the 12 cat-sourced GyV strains showed 93.9-99.7% nucleotide identities to the homologous reference GyV strains. Phylogenetic analyses based on the complete genomes, VP1, VP2 and VP3 genes showed the identical classification of GyV species with previous reports. Moreover, one and four unique amino acid substitutions were identified in the VP1 protein of the cat-sourced HGyV/AGV2 and GyV6 strains, respectively, and one substitution was also observed in the VP2 protein of one GyV6 strain identified in this study. In conclusion, our investigation demonstrates that the diverse GyV species were circulating in domestic cats, and provides the first molecular evidence for the circulation of HGyV/AGV2, GyV3 and GyV6 in domestic cats. These cat-origin GyVs possessed considerable genetic diversity. This study also raises the possibility that domestic cats, as reservoirs for gyroviruses, may inadvertently disseminate viruses to other species, e.g., humans and chickens.

Serological evidence of hepatitis E virus and influenza A virus infection in farmed wild boars in China.

Hepatitis E virus (HEV) and influenza A virus (IAV) are two important pathogens which can infect humans and various animals causing public health problems. In this study, the seroprevalence and risk factors associated with HEV and IAV infection in farmed wild boars were investigated in China. A total of 758 serum samples were collected from farmed wild boars between 2015 and 2016, and antibodies against HEV and IAV were examined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits. The overall prevalence of anti-HEV antibodies was 24.54% (186/758, 95% CI 21.48-27.60) in farmed wild boars. There were statistically significant differences in the HEV seroprevalence in farmed wild boars of different ages (<22 days: 8.33%; 22-66 days: 18.89%; >66 days: 26.36%) (P < 0.05) and different genders (50.00% in male and 23.49% in female) (P < 0.01). However, there was no statistically significant difference in the HEV seroprevalence in farmed wild boars of different regions and different years. The overall IAV seroprevalence was 5.80% (44/758, 95% CI 4.14-7.46), and there was no statistically significant difference in the IAV seroprevalence in farmed wild boars of different ages and genders, collected from different regions and different years. Our results indicate that HEV and IAV infections in farmed wild boars may pose a potential risk for human infection. To our knowledge, this is the first report of HEV and IAV seroprevalence in farmed wild boars in China, which provides baseline data for further studies and for control of HEV and IAV infection in farmed wild boars.

Molecular Characterization and Comparative Pathogenicity of Goose Parvovirus Isolated from Jilin Province, Northeast China.

Goose parvovirus (GPV) is a highly contagious disease caused by GPV in goslings and young Muscovy ducklings. In recent years, frequent GPV outbreaks have occurred in many regions of Jilin Province, China. In this study, to understand the immune-related characteristics of the currently prevailing GPV strains in some regions of Jilin Province, six GPV strains were isolated from six different regions of Jilin Province in 2016-2018. The results of phylogenetic analysis, clinical signs, and histopathologic analysis showed that four strains were virulent and two strains were attenuated. Specifically, we found that the two attenuated strains have the same amino acid mutation at the same position in the virus protein 3 (VP3) gene, and the virulent strains have more mutation sites than the attenuated strains. This finding suggests that changes in these sites may result in GPV replication or reduction in the immune response in goslings, thereby producing strong pathogenicity, and that attenuated strains are more conservative than virulent strains.

Caracterización molecular y patogenicidad comparativa de parvovirus de ganso aislados en la provincia de Jilin, noreste de China. El parvovirus del ganso (GPV, por sus siglas en inglés) es una enfermedad altamente contagiosa causada por el parvovirus de ganso en gansitos y patitos reales jóvenes. En los últimos años, se han presentado brotes frecuentes por el parvovirus del ganso en muchas regiones de la provincia de Jilin, en China. En este estudio, para comprender las características relacionadas con el sistema inmunológico de las cepas del parvovirus del ganso prevalentes actualmente en algunas regiones de la provincia de Jilin, se aislaron seis cepas de parvovirus del ganso de seis regiones diferentes de la provincia de Jilin entre los años 2016 al 2018. Los resultados del análisis filogenético, los signos clínicos y el análisis histopatológico mostraron que las cuatro cepas fueron virulentas y dos fueron atenuadas. Específicamente, se encontró que las dos cepas atenuadas tienen la misma mutación de aminoácidos en la misma posición en el gene de la proteína viral 3 (VP3) y las cepas virulentas tienen más sitios de mutación que las cepas atenuadas. Este hallazgo sugiere que los cambios en estos sitios pueden resultar en la replicación o reducción de la respuesta inmune en los gansitos, lo que induce una fuerte patogenicidad y que las cepas atenuadas son más conservadas que las virulentas.

Hepatic Metabolomics Investigation in Acute and Chronic Murine Toxoplasmosis.

Toxoplasma gondii poses a great threat to human health, with no approved vaccine available for the treatment of T. gondii infection. T. gondii infections are not limited to the brain, and may also affect other organs especially the liver. Identification of host liver molecules or pathways involved in T. gondii replication process may lead to the discovery of novel anti-T. gondii targets. Here, we analyzed the metabolic profile of the liver of mice on 11 and 30 days postinfection (dpi) with type II T. gondii Pru strain. Global metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 389 significant metabolites from acutely infected mice; and 368 from chronically infected mice, when compared with control mice. Multivariate statistical analysis revealed distinct metabolic signatures from acutely infected, chronically infected and control mice. Infection influenced several metabolic processes, in particular those for lipids and amino acids. Metabolic pathways, such as steroid hormone biosynthesis, primary bile acid biosynthesis, bile secretion, and biosynthesis of unsaturated fatty acids were perturbed during the whole infection process, particularly during the acute stage of infection. The present results provide insight into hepatic metabolic changes that occur in BALB/c mice during acute and chronic T. gondii infection.

First Report of Chlamydia Seroprevalence in Farmed Wild Boars in China.

Chlamydia is Gram-negative obligate bacterium, which can cause human diseases worldwide and has huge economic impact on animals. It is yet to know whether farmed wild boars are infected with Chlamydia in China. To assess risk factors of Chlamydia infection in farmed wild boars in China, from April 2015 to February 2016, a total of 837 serum samples of farmed wild boars were collected in Jilin province, northeastern China, and antibodies against Chlamydia were examined by the indirect hemagglutination assay. The investigation showed that antibodies to Chlamydia were detected in 332 (39.67%, 95% CI 33.36-42.98) of 837 serum samples of farmed wild boars, seroprevalence ranged from 33.71% to 44.42% among different regions and the differences were statistically significant by SPSS analysis (p = 0.0248). These results indicated that Chlamydia is highly prevalent in farmed wild boars in Jilin province, northeastern China, and may pose a potential risk for human health. To our knowledge, this is the first report of Chlamydia seroprevalence in farmed wild boars in China, which provided baseline data for preventing and controlling Chlamydia infection in wild boars in China.

First report of Mycoplasma hyopneumoniae seroprevalence in farmed wild boars in China.

Porcine enzootic pneumonia caused by Mycoplasma hyopneumoniae affects the global pig industry with significant economic losses. It is yet to know whether wild boars in China were infected with M. hyopneumoniae. The present study was conducted to examine the seroprevalence and to evaluate risk factors of M. hyopneumoniae infection in farmed wild boars in China. A total of 882 serum samples were collected from farmed wild boars in Jilin City, Siping City and Baishan City in Jilin Province, northeastern China from April 2015 to February 2016, and were examined by the double sandwich enzyme-linked immunosorbent assay (ELISA). Seventy-eight out of 882 (8.8%) serum samples were M. hyopneumoniae-seropositive. Among region groups, wild boars from Jilin city (11.7%, 33/281) had the highest seropositivity, followed by Siping city (11%, 29/263) and Baishan city (4.7%, 16/338), and the difference was statistically significant (P = 0.0031). The M. hyopneumoniae seroprevalence in the female wild boars (9.0%, 75/831) was higher than that in the male wild boars (5.9%, 3/51) (P = 0.4429). The results of this investigation showed that farmed wild boars were susceptible to M. hyopneumoniae. Logistic regression analysis showed that there is a significant correlation between the geographical area and M. hyopneumoniae infection, which may be related to the regional environment. This is the first report of M. hyopneumoniae seroprevalence in farmed wild boars in China, which provided baseline information for further studies and control of M. hyopneumoniae infection in wild boars in China.

Detection of Enterocytozoon bieneusi in White Yaks in Gansu Province, China.

Enterocytozoon bieneusi, the most common zoonotic pathogen of microsporidiosis, has been found in various animals and humans, but no information is available concerning the prevalence and genotypes of E. bieneusi in white yaks (Bos grunniens). In the present study, 353 faecal samples from white yaks in Tianzhu Tibetan Autonomous County, Gansu Province, Northwestern China, were collected and examined by PCR amplification of the internal transcribed spacer gene to estimate E. bieneusi prevalence and identify their genotypes. Of the 353 faecal samples, 4 (1.13%) were tested E. bieneusi-positive. Sequences analysis revealed that two known genotypes, namely, I (n = 1) and BEB4 (n = 2), and a novel genotype, namely, WCY1 (n = 1), were found in this study. Among them, genotype WCY1 was clustered into Group 1, and genotypes I and BEB4 belonged to Group 2. The present study firstly indicates the existence of E. bieneusi in yaks in Gansu Province, Northwestern China. This is also the first record of E. bieneusi in white yaks. Effective measures should be taken to control E. bieneusi infection in white yaks, other animals, and humans.

Profiling of the perturbed metabolomic state of mouse spleen during acute and chronic toxoplasmosis.

BACKGROUND: Toxoplasma gondii, a common opportunistic protozoan, is a leading cause of illness and mortality among immunosuppressed individuals and during congenital infections. Current therapeutic strategies for toxoplasmosis are not fully effective at curtailing disease progression in these cases. Given the parasite ability to influence host immunity and metabolism, understanding of the metabolic alterations in the host's immune organs during T. gondii infection may enhance the understanding of the molecular mechanisms that define the pathophysiology of T. gondii infection. METHODS: We investigated the global metabolic changes in the spleen of BALB/c mice at early and late stage of infection with T. gondii using LC-MS/MS-based metabolomics. Multivariate data analysis methods, principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were used to identify metabolites that are influenced by T. gondii infection. RESULTS: Multivariate analyses clearly separated the metabolites of spleen of infected and control mice. A total of 132 differential metabolites were identified, 23 metabolites from acutely infected versus control mice and 109 metabolites from chronically infected versus control mice. Lipids, hormones, lactones, acids, peptides, antibiotics, alkaloids and natural toxins were the most influenced chemical groups. There were 12 shared differential metabolites between acutely infected versus control mice and chronically infected versus control mice, of which 4,4-Dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol was significantly upregulated and ubiquinone-8 was significantly downregulated. Major perturbed metabolic pathways included primary bile acid biosynthesis, steroid hormone biosynthesis, biotin metabolism, and steroid biosynthesis, with arachidonic acid metabolism being the most significantly impacted pathway. These metabolic changes suggest a multifactorial nature of the immunometabolic responses of mouse spleen to T. gondii infection. CONCLUSIONS: This study demonstrated that T. gondii infection can cause significant metabolomic alterations in the spleen of infected mice. These findings provide new insights into the molecular mechanisms that underpin the pathogenesis of T. gondii infection.

Seroprevalence and Risk Factors of Bluetongue Virus Infection in Tibetan Sheep and Yaks in Tibetan Plateau, China.

Bluetongue (BT), caused by bluetongue virus (BTV), is an arthropod-borne viral disease in ruminants. However, information about BTV infection in yaks in China is limited. Moreover, no such data concerning BTV in Tibetan sheep is available. Therefore, 3771 serum samples were collected from 2187 Tibetan sheep and 1584 yaks between April 2013 and March 2014 from Tibetan Plateau, western China, and tested for BTV antibodies using a commercially available ELISA kit. The overall seroprevalence of BTV was 17.34% (654/3771), with 20.3% (443/2187) in Tibetan sheep and 13.3% (211/1584) in yaks. In the Tibetan sheep group, the seroprevalence of BTV in Luqu, Maqu, Tianzhu, and Nyingchi Prefecture was 20.3%, 20.8%, 20.5%, and 19.1%, respectively. The seroprevalence of BTV in different season groups varied from 16.5% to 23.4%. In the yak group, BTV seroprevalence was 12.6%, 15.5%, and 11.0% in Tianzhu, Maqu, and Luqu counties, respectively. The seroprevalence in different seasons was 12.6%, 15.5%, 15.4%, and 9.0% in spring, summer, autumn, and winter, respectively. The season was the major risk factor concerning BTV infection in yaks (P < 0.05). The date of the BTV seroprevalence in Tibetan sheep and yaks provides baseline information for controlling BT in ruminants in western China.

Seroprevalence and risk factors of bovine viral diarrhoea virus (BVDV) infection in yaks (Bos grunniens) in northwest China.

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is an important pathogen of cattle worldwide, causing reproductive disorders in adult cattle and mucosal disease in calves. However, limited information about BVDV infection in yaks (Bos grunniens) in China is available, especially in white yaks which is a unique yak breed that only lives in Tianzhu Tibetan Autonomous County (TTAC), Gansu Province, northwest China. Therefore, we conducted a cross-sectional study to estimate the seroprevalence and risk factors associated with BVDV infection in 1584 yaks in Gansu province, northwest China, between April 2013 and March 2014 using an indirect ELISA test. The overall seroprevalence of BVDV in yaks was 37.56 % (595/1584), with 45.08 % (275/610) in black yaks and 32.85 % (320/974) in white yaks. Moreover, positive yaks were found in all four regions, varied from 33.22 to 40.31 %. Male yaks had a similar seroprevalence (37.84 %) with that of the female yaks (37.11 %). Season, species and geographical origins of yaks were considered as risk factors analyzed by logistic regression model. To our knowledge, this is the first report of seroprevalence and risk factors associated with BVDV infection in white yaks in China.

First Report of Bovine Leukemia Virus Infection in Yaks (Bos mutus) in China.

Enzootic bovine leukosis (EBL) is a chronic lymphosarcoma disease of cattle caused by bovine leukemia virus (BLV). No information is available concerning the epidemiology of BLV infection in yaks (Bos mutus). One thousand five hundred and eighty-four serum samples from 610 black yaks and 974 white yaks from Gansu province, northwest China, were collected between April 2013 and March 2014 and tested for BLV antibodies using a commercially available ELISA kit. The overall BLV seroprevalence in yaks was 21.09% (334/1584), with 24.26% (148/610) black yaks and 19.10% (186/974) white yaks yielding positive results. Risk factor analysis indicated that with the exception of breed (OR = 1.36, 95% CI = 1.06-1.73, P < 0.05), the age, region, gender, farm, and the numbers of pregnancies were not considered as risk factors for the presence of BLV in yaks included in this study. This is the first report of BLV infection in yaks in China, which provides information for controlling BLV infection in yaks.

Isolation, Identification, and Sequencing of a Goose-Derived Newcastle Disease Virus and Determination of Its Pathogenicity.

In August 2010, geese in the Meihekou area of Jilin province in China were found to be infected by a pathogen that caused a disease similar to Newcastle disease. To determine the causative agent of the infections, a virus was isolated from liver tissues of infected geese, followed by a pathogenicity determination. The isolated virus was named NDV/White Goose/China/Jilin(Meihekou)/MHK-1/2010. Specific primers were designed to amplify the whole genome of the MHK-1 virus, followed by sequencing and splicing of the entire genome. Sequencing and phylogenetic analysis of MHK-1 showed that the isolate was a virulent strain of Newcastle disease virus. The MHK-1 genome is 15,192 nucleotides long, and it belongs to the class II branch of Newcastle disease viruses, as evidenced by the amino acid sequence (112R-R-Q-K-R-F117) of the F protein. The hemagglutinin titer was 1:128 to 1:512. The chicken embryo mean death time, the intracerebral pathogenicity index, and the median lethal dose of chicken embryos of MHK-1 were 43 hr, 1.63, and 10(9)/ml, respectively, which revealed that the newly isolated MHK-1 strain is strongly pathogenic to geese.

Isolation and identification of a canine coronavirus strain from giant pandas (Ailuropoda melanoleuca).

Two giant pandas (Ailuropoda melanoleuca) died of unknown causes in a Chinese zoo. The clinical disease profile suggested that the pandas may have suffered a viral infection. Therefore, a series of detection including virus isolation, electron microscopy, cytobiological assay, serum neutralization and RT-PCR were used to identify the virus. It was determined that the isolated virus was a canine coronavirus (CCV), on the basis of coronavirus, neutralization by canine anti-CCV serum, and 84.3% to 100% amino acid sequence similarity with CCV. The results suggest that the affected pandas had been infected with CCV.

[Construction of bicistronic eukaryotic expression vector containing HA and M2 genes derived from high pathogenic avian influenza virus (HPAI) H5N1 (Anhui strain)and its efficient expression in mammalian cells].

HA and M2 genes derived from human highly pathogenic avian influenza H5N1 virus (A/Anhui/ 1/2005) isolated from China, were amplified and cloned into the DNA vaccine expression vector pVRC. In order to improve the expression of hemagglutinin, the human codon usage preference was made and the whole length of HA gene of H5NI (A/Anhui/1/2005) influenza virus was synthesized,named HA/YH/K, and inserted to pVRC vector, the expression of HA/YH/K protein in eukaryotic cells was significantly improved according to internal control of actin protein. Furthermore, the M2 and HA/YH/K genes were cloned into bicistronic eukaryotic expressing vector pIRES to yield the recombinant plasmid pIRES-HA/ YH/K-M2/YS/K, which could expressed HA and M2 protein simultaneously by transfection of one plasmid. Western blot and IFA showed that the recombinant pIRES-HA/YH/K-M2/YS/K plasmid was successfully expressed in several mammalian cells (Hela, MDCK and 293FT). The above results may help to identify the function and pathogenic mechanism of HA, M2 genes derived from HPAI H5N1 (Anhui strain) and pave a way for the development of novel bivalent vaccines against human highly pathogenic avian influenza virus and for preparedness for influenza pandemic.

[Construction of recombinant canine adenovirus type 2 expressing Canine coronavirus spike glycoprotein and its immunogenicity].

In order to construct a recombinant Canine adenovirus type 2 (CAV-2) expressing the spike glycoprotein of Canine coronavirus (CCV), the S1 gene fragment of CCV strain DXMV, encoding major antigenic region A, B, C and D of S protein, was amplified by RT-PCR and cloned into pVAX1 vector. The complete S1 expression cassette was subcloned into the shuttle vector pVAXE3, then further cloned into the backbone vector pPoly2-CAV2 containing complete genome of CAV-2. To gain the recombinant Canine adenovirus, the recombinant plasmid pCAV-2-CCV-S1 was linearized by Cla I/Asc I to release recombinant genome, and then transfected into MDCK cell. The recombinant virus CAV-2-S1 was gained through 4 passages in MDCK, which showed classical CPE of CAV-2. The expressed S1 protein of CCV, which was identified by RT-PCR and Western blot, can be specifically recognized by polyclonal antibody against CCV. The immunization in dogs indicated that the recombinant CAV-2 could effectively induce the specific antibodies against CCV and CAV.