Epidemiological investigation reveals genetic diversity and high co-infection rate of canine bocavirus strains circulating in Heilongjiang province, Northeast China.

To trace evolution of CBoV in Northeast China, 201 fecal samples from rectal swabs of diarrheic dogs collected from May 2014 to April 2015 were investigated using PCR targeting partial NS1 gene (440bp). Furthermore, phylogenetic analysis of the identified CBoV strains was conducted using nucleotide sequences of the partial NS1 gene. The results indicated that 15 of 201 fecal samples (7.5%) were positive for CBoV; the partial NS1 genes of the 15 CBoV strains exhibited 83.1%-100% nucleotide identity, and 75.8%-100% amino acid identity; the entire VP2 gene of five selected CBoV strains exhibited 82.9%-96.8% nucleotide identity, and 90.4%-99.1% amino acid identity. The 15 CBoV strains exhibited high co-infection rates with CPV-2 (40%), CCoV (20%), and CaKV (26.67%). Phylogenetic analysis of the partial NS1 gene revealed that the 15 CBoV strains were divided into different subgroups of CBoV-2 when compared with CBoV-2 strains from South Korea, USA, Germany, and Hong Kong in China. Moreover, phylogenetic analysis of the VP2 gene indicated that five selected CBoV strains were divided into three different genetic groups of CBoV-2, involving in CBoV-2HK group, CBoV-2C group, and CBoV-2B group. The recombination analysis using the entire VP2 gene revealed three potential recombination events that occurred among five selected strains in our study. These data demonstrated that the CBoV strains circulating in Heilongjiang province, Northeast China showed genetic diversities, potential recombination events, and high co-infection rate. Further studies will be required to address the potential pathogenic role of these diverse CBoV strains.

Co-Circulation of Canine Coronavirus I and IIa/b with High Prevalence and Genetic Diversity in Heilongjiang Province, Northeast China.

To trace the evolution of canine coronavirus (CCoV), 201 stool samples from diarrheic dogs in northeast China were subjected to reverse transcription-polymerase chain reactions (RT-PCRs) targeting the partial M and S genes of CCoV, followed by an epidemiological analysis. M gene RT-PCRs showed that 28.36% (57/201) of the samples were positive for CCoV; of the 57 positive samples, CCoV-I and CCoV-II accounted for 15.79% (9/57) and 84.21% (48/57), respectively. A sequence comparison of the partial M gene revealed nucleotide homologies of 88.4%-100% among the 57 CCoV strains, and 88.7%-96.2% identity between the 57 CCoV strains and the Chinese reference strain HF3. The CCoV-I and CCoV-II strains exhibited genetic diversity when compared with reference strains from China and other countries. The 57 CCoV strains exhibited high co-infection rates with canine kobuvirus (CaKV) (33.33%) and canine parvovirus-2 (CPV-2) (31.58%). The CCoV prevalence in diarrheic dogs differed significantly with immunization status, regions, seasons, and ages. Moreover, 28 S genes were amplified from the 57 CCoV-positive samples, including 26 CCoV-IIa strains, one CCoV-IIb strain, and one CCoV-I strain. A sequence comparison of the partial S gene revealed 86.3%-100% nucleotide identity among the 26 CCoV-IIa strains, and 89.6%-92.2% identity between the 26 CCoV-IIa strains and the Chinese reference strain V1. The 26 CCoV-IIa strains showed genetic diversity when compared with reference strains from China and other countries. Our data provide evidence that CCoV-I, CCoV-IIa, and CCoV-IIb strains co-circulate in the diarrhoetic dogs in northeast China, high co-infection rates with CaKV and CPV-2 were observed, and the CCoV-II strains exhibited high prevalence and genetic diversity.

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China.

Prevalence and phylogenetic analysis of canine kobuviruses in diarrhoetic dogs in northeast China.

Canine kobuviruses (CaKVs) are newly recognized picornaviruses that have been recently detected in dogs in the U.S.A., Italy, U.K., the Republic of Korea and Tanzania. To trace the evolution of CaKV strains, a total of 201 fecal samples from rectal swabs of diarrheic dogs, which were obtained from May 2014 to April 2015 in northeast China, were detected by reverse transcription-PCR targeting a partial (504 bp) fragment of the 3D gene. Furthermore, a phylogenetic analysis of the CaKV strains identified in northeast China was conducted based on the partial 3D gene sequence. The results indicated that 36 fecal samples (17.91%, 36/201) were positive for CaKV, in which the co-infection rates of canine coronavirus, canine parvovirus-2 and canine bocavirus were 58.33%, 41.67%, and 11.11%, respectively. Sequence comparison of the partial 3D gene revealed nucleotide homologies of 94.4-100%, 95.6-98.6%, 94.3-97.6%, 94.4-96.3% and 93.3-95.1% within the 36 Chinese CaKV strains, and between the 36 Chinese CaKV strains and four CaKV reference strains from South Korea, Italy, U.S.A. and Tanzania, respectively. A phylogenetic tree revealed that the 36 Chinese CaKV strains formed one specific CaKV lineage with CaKVs that have recently been identified in other countries. The 36 Chinese CaKV strains were closely related to CaKV reference strains from Asia and Europe, but differed genetically from CaKV reference strains from North America and Africa. This study provides evidence that CaKVs circulate in diarrhoetic dogs in China and that they exhibit substantial genetic diversity and high co-infection rates with other enteric viruses.

Co-Circulation of the Rare CPV-2c with Unique Gln370Arg Substitution, New CPV-2b with Unique Thr440Ala Substitution, and New CPV-2a with High Prevalence and Variation in Heilongjiang Province, Northeast China.

To trace evolution of canine parvovirus-2 (CPV-2), a total of 201 stool samples were collected from dogs with diarrhea in Heilongjiang province of northeast China from May 2014 to April 2015. The presence of CPV-2 in the samples was determined by PCR amplification of the VP2 gene (568 bp) of CPV-2. The results revealed that 95 samples (47.26%) were positive for CPV-2, and they showed 98.8%-100% nucleotide identity and 97.6%-100% amino acid identity. Of 95 CPV-2-positive samples, types new2a (Ser297Ala), new2b (Ser297Ala), and 2c accounted for 64.21%, 21.05%, and 14.74%, respectively. The positive rate of CPV-2 and the distribution of the new2a, new2b and 2c types exhibited differences among regions, seasons, and ages. Immunized dogs accounted for 48.42% of 95 CPV-2-positive samples. Coinfections with canine coronavirus, canine kobuvirus, and canine bocavirus were identified. Phylogenetic analysis revealed that the identified new2a, new2b, and CPV-2c strains in our study exhibited a close relationship with most of the CPV-2 strains from China; type new2a strains exhibited high variability, forming three subgroups; type new2b and CPV-2c strains formed one group with reference strains from China. Of 95 CPV-2 strains, Tyr324Ile and Thr440Ala substitutions accounted for 100% and 64.21%, respectively; all type new2b strains exhibited the Thr440Ala substitution, while the unique Gln370Arg substitution was found in all type 2c strains. Recombination analysis using entire VP2 gene indicated possible recombination events between the identified CPV-2 strains and reference strains from China. Our data revealed the co-circulation of new CPV-2a, new CPV-2b, and rare CPV-2c, as well as potential recombination events among Chinese CPV-2 strains.

Preparation of monoclonal antibodies against bovine haptoglobin.

Female, 8-week-old BALB/c mice were immunized with purified recombinant proteins of the predicted immunodominant region of bovine haptoglobin (pirBoHp). Two monoclonal antibodies (MAbs), named 1B3 and 6D6, were prepared by conventional B lymphocyte hybridoma technique. Titers of ascitic fluid and cell culture supernatant of MAb 1B3 were 1:9.6 × 10(8) and 1:8.2 × 10(4), respectively, and that of MAb 6D6 were 1:4.4 × 10(5) and 1:1.0 × 10(4), respectively. The subtype of MAbs 1B3 and 6D6 was IgG1κ. In Western blot analysis, MAbs 1B3 and 6D6 could recognize the α-chain of native BoHp from plasma of dairy cows. These data indicated that MAbs 1B3 and 6D6 have a potential use for developing diagnostic reagents of BoHp.