In ovo injection of CHIR-99021 promotes feather follicles development via activating Wnt/ß-catenin signaling pathway during chick embryonic period.

The Wingless-types/beta-catenin (Wnt/ß-catenin) signaling pathway plays an important role in embryonic development and affects the physiological development processes of feather follicles. To investigate the role of Wnt/ß-catenin pathway in regulating feather follicles morphogenesis, in ovo injection of CHIR-99021, an activator of the Wnt/ß-catenin signaling pathway, was conducted in chick embryo model. Initially, a total of 40 embryos were used to assess feather follicles morphogenesis and the expression of ß-catenin (E9-E17). The histological results showed that feather follicle morphogenesis was mainly completed from E9 to E17. ß-catenin was involved in the processing of the appearance of dermal cell condensation (E9) and the completion of the feather follicles morphogenesis (E17). Next, a total of 160 fertilized eggs were randomly divided into 8 groups for in ovo injection at E9, including a Normal Saline injected group (CON) and the 500, 1,000, 2,000, 5,000, 10,000, 50,000, and 100,000 ng CHIR-99021 groups. Dorsal skin tissue samples were collected at E17 for investigating feather follicles morphology and expressions of ß-catenin and lymphoid enhancerbinding factor-1 (LEF1) at gene and protein levels. The results showed that feather follicle diameter in the injected groups were significantly (P < 0.05) increased with limit dose-independence compared to the CON group. CHIR-99021 significantly (P < 0.05) influenced the mRNA expressions of catenin beta-1 (CTNNB1) and downstream target LEF1. In ovo injection of CHIR-99021 caused that ß-catenin and LEF1 were significantly (P < 0.05) increased followed the increased doses as determined by western blotting. The immunochemical results showed that ß-catenin was detected in the dermal papilla of feather follicles. Given these results, this study suggests to developmental biology that in ovo injection of CHIR-99021 promoted feather follicles morphogenesis and development via activating Wnt/ß-catenin signaling pathway and upregulating downstream target LEF1 during embryonic period in chick embryo model. Moreover, CHIR-99021 may be a strong candidate to promote the animal feather/hair industry, especially as a reference for bird feather production.

Exosomal miRNA-328-3p targets ZO-3 and inhibits porcine epidemic diarrhea virus proliferation.

As essential transfer carriers for cell-to-cell communication and genetic material, exosomes carry microRNAs that participate in the regulation of various biological processes. MicroRNAs are a type of single-stranded noncoding RNA that bind to specific target gene mRNAs to degrade or inhibit their translation, thereby regulating target gene expression. Although it is known that a variety of microRNAs are involved in the viral infection process, there are few reports on specific microRNAs involved in porcine epidemic diarrhea virus (PEDV) infection. In this study, we isolated and identified exosomes in PEDV-infected Vero E6 cells. Using transcriptomics technology, we found that miRNA-328-3p was significantly downregulated in exosomes following PEDV infection. Moreover, exosomal miRNA-328-3p inhibited infection by PEDV by targeting and inhibiting tight junction protein 3 (TJP-3/ZO-3) in recipient cells. Our findings provide evidence that, after infecting cells, PEDV downregulates expression of miRNA-328-3p, and the resulting reduced inhibition of the target protein ZO-3 helps to enhance PEDV infection. These results provide new insight for understanding the regulatory mechanism of PEDV infection.

Development and application of a TaqMan-based real-time PCR assay for specifically detecting feline astrovirus.

Feline astrovirus (FeAstV), an enteric RNA virus of recent concern that is associated with diarrheal illness in cats, has been described in several countries throughout the world. However, no scientific and sensitive diagnostic method against FeAstV was reported up to now. Here, we developed a specific, sensitive and repeatable TaqMan fluorescence quantitative PCR (qPCR) assay to investigate the prevalence of FeAstV in domestic cats from China, especially low copy numbers in clinical sample. Specific assay showed that no cross-reactivity was observed with other non-FeAstV cat-derivied pathogens, suggesting this method was highly specific for FeAstV. The lowest detection limit of this assay was 3.52 copies/µl, and 1000-times more sensitive than conventional PCR. Intra- and inter-assay variability was less than 1.72%, means a high degree of repeatability. A total of 578 clinical fecal samples were collected from northeast China, and were tested for FeAstV using our developed qPCR assay. 105 samples were positive for FeAstV with an overall prevalence of 18.17%. Moreover, a higher positive rate was found in cats with diarrhea (32.26%, 80/248) than that in asymptomatic cats (7.58%, 25/330), further demonstrating that FeAstV infection was associated with diarrhea in cats. In brief, our developed assay showed high specificity, sensitivity, reproducibility for detecting FeAstV, and can be used for clinical diagnosis and epidemiological investigation of FeAstV.

Development of a cross-priming isothermal amplification assay based on the glycoprotein B gene for instant and rapid detection of feline herpesvirus type 1.

A cross-priming isothermal amplification (CPA) assay was developed for detection of feline herpesvirus type 1 (FHV-1). In this assay, the target fragment of the FHV-1 glycoprotein B gene is amplified rapidly by Bst DNA polymerase at a constant temperature (63 °C, 45 min), using a simple thermostat. The assay had no cross-reactions with four types of feline viruses, and the detection limit was 100 copies/µl. The positive rate of clinical samples from CPA was 100% consistent with qPCR but higher than ordinary PCR, indicating its superiority to ordinary PCR. Visualization was achieved using SYBR Green I dye.

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.

Development and application of a multiplex PCR method for the simultaneous detection and differentiation of feline panleukopenia virus, feline bocavirus, and feline astrovirus.

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per µl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.

Equine immunoglobulin F(ab')2 fragments protect cats against feline calicivirus infection.

Feline calicivirus (FCV) causes upper respiratory tract infections in felines and threatens the health of wild and domestic felines. Clinically, specific drugs to treat FCV have not yet been developed. Here, IgG was extracted from inactivated FCV-immunized horse sera. Equine F(ab')2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. In our study, equine immunoglobulin F(ab')2 fragments showed efficient neutralizing activity in vitro against FCV and had therapeutic and prophylactic effects in FCV-infected cats. The anti-FCV-specific F(ab')2 fragment can significantly alleviate the clinical symptoms of FCV-infected cats and reduce the viral loads of the trachea, lung and spleen. These results indicate that the F(ab')2 fragment prepared from inactivated FCV-immunized horses may be used as a prophylactic and therapeutic agent for diseases caused by FCV.

Construction of a Recombinant Lactococcus lactis Strain Expressing a Variant Porcine Epidemic Diarrhea Virus S1 Gene and Its Immunogenicity Analysis in Mice.

Porcine epidemic diarrhea caused by porcine epidemic diarrhea virus (PEDV) is a highly contagious disease in newborn piglets. The spike (S) protein is the surface glycoprotein of PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In our study, the S1 region of PEDV strain CH/JLDH/2016 spike gene was inserted into the Lactococcus lactis expression vector, pNZ8149, resulting in recombinant plasmid pNZ8149-S1, and the immunogenicity of recombinant L. lactis pNZ8149-S1/NZ3900 was evaluated in mice. After immunization, significantly higher levels of anti-PEDV serum IgG antibodies and mucosal sIgA antibodies were detected in mice orally administered with pNZ8149-S1/NZ3900, compared with control groups pNZ8149/NZ3900, NZ3900, and phosphate buffered saline (p < 0.01). Lymphocyte proliferation assay results showed that the recombinant L. lactis pNZ8149-S1/NZ3900 significantly stimulated the proliferation of splenic lymphocytes (p < 0.01). In addition, the recombinant L. lactis vaccine could induce high levels of IL-4 and IFN-γ in immunized mice (p < 0.01). The results of our study suggest that the recombinant L. lactis pNZ8149-S1/NZ3900 can provide a promising vaccine strategy against PEDV infection.

Complete genome sequence analysis of canine bocavirus 1 identified for the first time in domestic cats.

In this study, we investigated the presence of canine bocaviruses (CBoVs) in fecal samples from 105 cats with diarrhea and 92 asymptomatic cats in northeast China. One fecal sample, 17CC0312, collected from an asymptomatic cat, was found to be positive for canine bocavirus 1 (CBoV1). The nearly complete genome of this virus was cloned and sequenced. The viral genome was 5,069 nucleotides (nt) in length and combined four open reading frames (ORFs) in the order 5'-NS1-ORF4-NP1-VP1/VP2-3'. The 17CC0312 virus shared more than 90.3% nucleotide sequence identity with CBoV1 reference sequences and was placed within the CBoV1 group in a phylogenetic tree based on complete genome sequences. Further phylogenetic analysis based on the deduced amino acid sequence of the VP2 gene showed that this feline CBoV1 strain belongs to CBoV1 lineage 3. These data provide the first molecular evidence of the presence of CBoV1 in a domestic cat and suggest that cats might be carriers of CBoV1.

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.

Molecular characterization of feline astrovirus in domestic cats from Northeast China.

Feline astrovirus (FeAstV) which belonged to the genus Mamastrovirus was first identified in the feces of kittens with diarrhea in the USA in 1981 by electron microscopy, and had been reported in many countries. Presently, there are no any reports of the circulation of FeAstV in mainland China. We performed this study to investigate the apparent prevalence and genetic variability of FeAstV infected in cats in mainland China for the first time. We tested fecal samples of 105 cats with diarrhea and 92 asymptomatic cats in five cities in northeast China by RT-PCR targeting RNA-dependent RNA polymerase (RdRp) gene of FeAstV, and analyzed sequences variability and phylogenetic evolution based on the complete capsid gene of FeAstV strains obtained from positive samples. The overall prevalence of FeAstV was 23.4% (46/197) of which 38 were tested in cats with diarrhea (36.2%, 38/105) and 8 were in asymptomatic cats (8.7%, 8/92). Mixed infection with other enteroviruses including feline parvovirus (FPV), feline bocavirus (FBoV) and feline kobuvirus (FeKoV) was found in 38 FeAstV-positive samples. Phylogenetic analysis based on the complete capsid gene revealed all FeAstV strains were divided into two different groups with a 0.454±0.016 of mean amino acid genetic distance between two groups, suggesting that FeAstVs should be classified into two different genotype species. This study provided the first molecular evidence that FeAstV with considerable genetic diversity was circulating in northeast China, and analyzed genetic variability and classification of FeAstVs for the first time.

Detection and genetic characterization of feline bocavirus in Northeast China.

BACKGROUND: Bocaviruses have been reported to cause respiratory tract infection and gastroenteritis in most animal species. In cats, different genotype bocaviruses have been identified in USA, Japan, Hong Kong and Portugal. However, the clear relationship between the clinical symptoms and FBoV infection is unknown, and the prevalence of FBoV and the distribution of FBoV genotypes in China are still unclear. RESULTS: In this study, 197 fecal samples from cats with diarrhea (n = 105) and normal cats (n = 92) were collected in different regions between January 2016 and November 2017 and investigated using PCR targeting different FBoV genotypes. Screening results showed that 51 of 197 samples (25.9%) were positive for FBoV, and a higher positive rate was observed in cats with diarrhea (33.3%, 35/105) than in normal cats (17.4%, 16/92). Of these FBoV-positive samples, 35 were identified as FBoV-1, 12 as FBoV-2 and 4 as coinfection of FBoV-1 and FBoV-2. A phylogenetic analysis based on partial NS1 gene indicated that 24 sequences from randomly selected FBoV-positive samples were divided into 2 different FBoV groups: FBoV-1 and FBoV-2. Furthermore, 6 strains were randomly selected, and the complete genome was sequenced and analyzed. These strains exhibited the typical genome organization of bocavirus and were closely related to FBoV. Two FBoV-2 identified strains shared high homologies with FBoV-2 reference strains based on the complete genome and entire encoding gene, but lower identities were exhibited in the NP1 and VP1 regions for the other 4 FBoV-1 identified strains compared with FBoV-1 reference strains. CONCLUSION: These findings demonstrate that genetically diverse FBoV-1 and FBoV-2 widely circulate in cats in Northeast China and that FBoV-1 is more prevalent. The high prevalence of FBoV in cats with diarrhea symptoms suggests that FBoV infection may be associated with diarrhea in cats.