TaqMan-based real-time polymerase chain reaction assay for specific detection of bocavirus-1 in domestic cats.

Feline bocavirus-1 (FBoV-1) was first discovered in Hong Kong in 2012, and studies have indicated that the virus may cause feline hemorrhagic enteritis. Currently, there is a lack of an effective and quantitative method for FBoV-1 detection. In this study, a TaqMan-based quantitative real-time PCR (qPCR) for FBoV-1 detection was established. Primers and probes were designed to target the conserved region of the FBoV-1 NS1 gene. The sensitivity analysis indicated that the minimum detection limit was 4.57 × 101 copies/µL. The specificity test revealed no cross-reaction with seven other common feline viruses, including the same species-FBoV-2 and FBoV-3. The sensitivity of this method was 100 times higher than that of conventional PCR (cPCR). The established method showed good repeatability, with the intra-assay and inter-assay coefficients of variation of 0.18%-1.00% and 0.27%-0.45%, respectively. Furthermore, the analysis of feline feces revealed that the detection rate by qPCR was 7.0% (9/128), whereas that by cPCR was 4.7% (6/128). In conclusion, the established qPCR assay can quantitatively detect FBoV-1 with a high sensitivity, high specificity, and good reproducibility, making it a promising technique for the clinical detection of and basic and epidemiological research on FBoV-1.

Identification of a novel bocaparvovirus in a wild squirrel in Kunming, Yunnan Province, China.

In December 2017, a squirrel (Callosciurus phayrei) died 2 days after capture in Kunming, and its intestinal tract, heart, liver, spleen, lung, and kidney were subjected to metagenomics analysis. Reassembly and verification by reverse transcription PCR of contigs generated by next-generation sequencing yielded a 5176-nt sequence, which was designated "squirrel bocaparvovirus" (SQBOV). Phylogenetic trees based on the aa sequences of NS1, NP1, and VP1 showed that SQBOV formed an independent branch in the bocaparvovirus phylogenetic tree. The amino acid sequence identity of the NS1 of SQBOV to those of other bocaparvoviruses was below the threshold of 85% that is used to demarcate species within the genus, indicating that it should be considered a member of a new bocaparvovirus species. To our knowledge, this is the first report of a bocaparvovirus in squirrels. Our findings will enable further studies of viral diversity in rodents and of the genetic diversity and host range of bocaparvoviruses.

Detection, genetic, and codon usage bias analyses of the VP2 gene of mink bocavirus.

Mink bocavirus 1 (MiBoV1), a novel virus detected from the feces of domestic minks in China in 2016, may be related to gastrointestinal diseases. However, its prevalence and genetic characteristics are poorly described. In this study, we examined 192 samples collected from minks in the major mink industry province from northern China. PCR results showed that 10 samples (5.2%) were positive for MiBoV1, and 60% of MiBoV1-positive samples were co-infected with Aleutian mink disease virus or mink circovirus. MiBoV1 was detected in six serum samples. Sequence analysis demonstrated that the VP2 gene of MiBoV1 was highly conserved and had low viral diversity over the VP2 region and unique nucleotide mutations. Phylogenetic analysis of the VP2 sequence demonstrated that MiBoV1 strains formed two clades and were grouped with California sea lion bocavirus, Canine bocavirus, and Feline bocavirus. Codon usage analysis revealed that most of the preferentially used codons in MiBoV1 were A- or U-ended codons, and no evident codon usage bias was found. This study provides evidence that MiBoV1 has a low prevalence in Jilin and Hebei provinces in China. Moreover, it contributes information regarding the expansion of the limited mink bocavirus sequence and determines the codon usage bias of the VP2 gene for the first time. Epidemiological surveillance is necessary to understand the importance and evolution of MiBoV1.

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.

Seroprevalence of porcine bocavirus in pigs in north-central China using a recombinant-NP1-protein-based indirect ELISA.

Porcine bocavirus (PBoV), which belongs the genus Bocaparvovirus, has been identified throughout the world. However, serological methods for detecting anti-PBoV antibodies are presently limited. In the present study, an indirect enzyme-linked immunosorbent assay (PBoV-rNP1 ELISA) based on a recombinant form of nucleoprotein 1 (NP1) of PBoV was established for investigating the seroprevalence of PBoV in 2025 serum specimens collected in north-central China from 2016 to 2018, and 42.3% of the samples tested positive for anti-PBoV IgG antibodies, indicating that the seroprevalence of PBoV is high in pig populations in China.

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.

Presence of rat bocavirus in oropharyngeal and fecal samples from murine rodents in China.

In this study, we detected and genetically characterized rat bocavirus (RBoV) carried in 496 murine rodents that were captured in four provinces in China between May 2015 and May 2017. RBoV-positive samples were found in all four provinces, with a total positive rate of 24.8% (123/496) in throat swabs and 58.1% (286/492) in fecal samples. Twelve nearly full-length genome sequences of RBoV were determined, and the average sequence identity was 96.2%. Phylogenetic analysis showed that RBoVs formed a distinct clade that was distinguishable from the bocaviruses discovered in humans and other animals.

Genetic analysis of three porcine bocaparvoviruses and identification of a natural recombinant breakpoint in NS1.

In this study, we obtained the whole genomes of three porcine bocaparvovirus (PBoV) strains (GD6, GD10, and GD23) by polymerase chain reaction. Sequence analysis showed that all three field strains belonged to PBoV group 3 (G3). The phylogenetic trees based on NS1, NP1, and VP1 differed to the extent that these PBoVs were potentially more closely related to bocaparvoviruses known to infect other animals than to other PBoVs. GD6, GD10, and GD23 all included the conserved sequences YLGPF and HDXXY, with known phospholipase A2 activity. Using recombination-detection software we identified a natural recombinant breakpoint in the NS1 region of PBoV G3. The results of this study will further the epidemiological characterization of PBoVs.

Canine Bocavirus Type 2 Infection Associated With Intestinal Lesions.

Bocaviruses are small nonenveloped DNA viruses belonging to the Bocaparvovirus genus of the Parvoviridae family and have been linked to both respiratory and enteric disease in humans and animals. To date, 3 bocaviruses, canine bocaviruses 1 to 3 (CBoV-1-3), have been shown to affect dogs with different disease manifestations reported for infected animals. We used next-generation sequencing to identify a novel strain of canine CBoV-2 (CBoV TH-2016) in a litter of puppies that died in Thailand from acute dyspnea and hemoptysis, for which no causal pathogen could be identified in routine assays. Analysis of the complete coding sequences of CBoV TH-2016 showed that this virus was most closely related to a strain previously identified in South Korea (isolate 14D193), with evidence of genetic recombination in the VP2 gene with related strains from South Korea and Hong Kong. Use of quantitative polymerase chain reaction showed the presence of CBoV TH-2016 in several tissues, suggesting hematogenous virus spread, while only intestinal tissue was found to be positive by in situ hybridization and electron microscopy. Histologic small intestinal lesions associated with CBoV TH-2016 infection were eosinophilic intranuclear inclusion bodies within villous enterocytes without villous atrophy or fusion, similar to those previously considered pathognomonic for CBoV-1 infection. Therefore, this study provides novel insights in the pathogenicity of canine bocavirus infections and suggests that a novel recombinant CBoV-2 may result in atypical findings of CBoV infection. Although the specific cause of death of these puppies remained undetermined, a contributory role of enteric CBoV TH-2016 infection is possible.

Two novel dromedary camel bocaparvoviruses from dromedaries in the Middle East with unique genomic features.

The recent emergence of Middle East respiratory syndrome (MERS) coronavirus and its discovery from dromedary camels has boosted interest in the search for novel viruses in dromedaries. While bocaparvoviruses are known to infect various animals, it was not known that they exist in dromedaries. In this study, we describe the discovery of two novel dromedary camel bocaparvoviruses (DBoVs), DBoV1 and DBoV2, from dromedary faecal samples in Dubai. Among 667 adult dromedaries and 72 dromedary calves, 13.9 % of adult dromedaries and 33.3 % of dromedary calves were positive for DBoV1, while 7.0 % of adult dromedaries and 25.0 % of dromedary calves were positive for DBoV2, as determined by PCR. Sequencing of 21 DBoV1 and 18 DBoV2 genomes and phylogenetic analysis showed that DBoV1 and DBoV2 formed two distinct clusters, with only 32.6-36.3 % amino acid identities between the DBoV1 and DBoV2 strains. Quasispecies were detected in both DBoVs. The amino acid sequences of the NS1 proteins of all the DBoV1 and DBoV2 strains showed <85 % identity to those of all the other bocaparvoviruses, indicating that DBoV1 and DBoV2 are two bocaparvovirus species according to the ICTV criteria. Although the typical genome structure of NS1-NP1-VP1/VP2 was observed in DBoV1 and DBoV2, no phospholipase A2 motif and associated calcium binding site were observed in the predicted VP1 sequences for any of the 18 sequenced DBoV2, and no start codons were found for their VP1. For all 18 DBoV2 genomes, an AT-rich region of variable length and composition was present downstream to NP1. Further studies will be crucial to understand the pathogenic potential of DBoVs in this unique group of animals.

Virome of US bovine calf serum.

Using viral metagenomics we analyzed four bovine serum pools assembled from 715 calves in the United States. Two parvoviruses, bovine parvovirus 2 (BPV2) and a previously uncharacterized parvovirus designated as bosavirus (BosaV), were detected in 3 and 4 pools respectively and their complete coding sequences generated. Based on NS1 protein identity, bosavirus qualifies as a member of a new species in the copiparvovirus genus. Also detected were low number of reads matching ungulate tetraparvovirus 2, bovine hepacivirus, and several papillomaviruses. This study further characterizes the diversity of viruses in calf serum with the potential to infect fetuses and through fetal bovine serum contaminate cell cultures.

Identification and interspecies transmission of a novel bocaparvovirus among different bat species in China.

We report the discovery of a novel bocaparvovirus, bat bocaparvovirus (BtBoV), in one spleen, four respiratory and 61 alimentary samples from bats of six different species belonging to three families, Hipposideridae, Rhinolophidae and Vespertilionidae. BtBoV showed a higher detection rate in alimentary samples of Rhinolophus sinicus (5.7 %) than those of other bat species (0.43-1.59 %), supporting R. sinicus as the primary reservoir and virus spillover to accidental bat species. BtBoV peaked during the lactating season of R. sinicus, and it was more frequently detected among female than male adult bats (P<0.05), and among lactating than non-lactating female bats (P<0.0001). Positive BtBoV detection was associated with lower body weight in lactating bats (P<0.05). Ten nearly complete BtBoV genomes from three bat species revealed a unique large ORF1 spanning NS1 and NP1 in eight genomes and conserved splicing signals leading to multiple proteins, as well as a unique substitution in the conserved replication initiator motif within NS1. BtBoV was phylogenetically distantly related to known bocaparvoviruses with ≤57.3 % genome identities, supporting BtBoV as a novel species. Ms-BtBoV from Miniopterus schreibersii and Hp-BtBoV from Hipposideros pomona demonstrated 97.2-99.9 % genome identities with Rs-BtBoVs from R. sinicus, supporting infection of different bat species by a single BtBoV species. Rs-BtBoV_str15 represents the first bat parvovirus genome with non-coding regions sequenced, which suggested the presence of head-to-tail genomic concatamers or episomal forms of the genome. This study represents the first to describe interspecies transmission in BoVs. The high detection rates in lactating female and juvenile bats suggest possible vertical transmission of BtBoV.

Molecular characteristics of a novel strain of canine minute virus associated with hepatitis in a dog.

A 5-year-old female Yorkshire terrier dog died a few days following hernia and ovariohysterectomy surgeries. Necropsy performed on the dog revealed that the surgeries were not the cause of death; however, degenerative viral hepatitis, showing intranuclear inclusion bodies in hepatic cells, was observed in histopathologic examination. Several diagnostic methods were used to screen for the cause of disease, and minute virus of canines (MVC) was detected in all parenchymal organs, including the liver. Other pathogens that may cause degenerative viral hepatitis were not found. Infection with MVC was confirmed by in situ hybridization, which revealed the presence of MVC nucleic acid in the liver tissue of the dog. Through sequencing and phylogenetic analysis of the nearly complete genome sequence, the strain was found to be distinct from other previously reported MVC strains. These results indicate that this novel MVC strain might be related to degenerative viral hepatitis in dogs.

Genetic characterization of feline bocavirus detected in cats in Japan.

Feline bocavirus (FBoV) has been classified into three genotypes (FBoV1-FBoV3). FBoVs are mainly detected in feces. In the present study, we collected rectal swabs from cats in Japan and examined the samples for the presence of FBoV. The FBoV infection rate was 9.9 % in 101 cats. No significant association was observed between FBoV infection and clinical symptoms. Based on the full-length NS1 protein, the three strains of FBoVs detected in the present study shared high homologies with the genotype 2 FBoV POR1 strain. This is the first study to report FBoV in Japan.

A novel bocavirus from domestic mink, China.

Bocaviruses have been found in the feces of humans and a variety of animals, including pigs, cattle, dogs, gorillas, cats, and sea lions. Here, we have characterized the almost complete genome (5224 nt) of a novel bocavirus from feces of domestic minks, which has been provisionally named mink bocavirus. The NS1 protein of mink bocavirus shared 36.9-52 % amino acid sequence identities with those of other known bocaviruses and phylogenetically clustered with bocaviruses from other carnivores. According to the genetic distance-based criteria, mink bocavirus qualifies as a novel species of bocavirus. PCR of feces from a group of domestic minks, which included both healthy animals and animals suffering from diarrhea, revealed that 30 % (9/30) shed virus. However, no association between viral shedding and the presence of diarrhea could be determined.

Ungulate copiparvovirus 1 (bovine parvovirus 2): characterization of a new genotype and associated viremia in different bovine age groups.

A novel bovine parvovirus 2 (BPV2) genotype comprising 5394 nt was identified by next generation sequencing from sera of healthy cattle at different age groups farmed in the state of Rio Grande do Sul, Brazil. The genome organization of new BPV2 genotype retains the two ORFs typical of members of the Parvovirinae with 86.4 % of overall nucleotide sequence identities in comparison to other members of the subfamily. Phylogenetic analysis revealed similar clustering with two previously described bovine BPV2 within the genus Copiparvovirus. No significant differences (P ≥ 0.05) were detected in the distribution of BPV2 infection in cattle at different age groups. This is the third complete or near complete genome sequence of BPV2 reported to date and may contribute to a better understanding of the biology of copiparvoviruses and its interactions with the host.

[Detection and analysis of bocavirus in hospitalized children with respiratory infection].

OBJECTIVE: To detect human bocavirus (HBoV) and investigate its genetic and evolutionary characteristics in children with acute respiratory infection in Tianjin, China. METHODS: A total of 1,259 samples of nasopharyngeal aspirates were collected from children with a confirmed diagnosis of acute respiratory infection between January and December, 2012. Viral nucleic acid was extracted, HBoV was detected by real-time quantitative PCR, and the gene segments of nucleocapsid protein of HBoV in positive samples were amplified by PCR. Several products were randomly selected and sequenced.The sequence obtained was compared with the known sequence of HBoV, and a phylogenetic analysis was performed. All the samples were examined to detect for other common respiratory tract viruses. RESULTS: Among the 1,259 samples, the positive rate of HBoV was 4.53% (57/1,259), and among the 57 samples with positive HBoV, 75% (43/57) were positive in children with an age of 6-36 months. The positive rate of HBoV in children peaked in summer (from June to August), and there was a mixed infection with other viruses. Sequence analysis was performed for the PCR products from 36 positive samples, and the presence of HBoV was confirmed, with a higher homology to the known sequence of HBoV. CONCLUSIONS: In Tianjin, acute respiratory infection in some children may be associated with HBoV infection, which is commonly seen in infants with an age of 6-36 months. The peak of HBoV infection occurs in summer. The phylogenetic analysis shows a high homology to the known sequence of HBoV, with few gene sequence variations.

High prevalence and genetic diversity of porcine bocaviruses in pigs in the USA, and identification of multiple novel porcine bocaviruses.

Viruses in the genus Bocavirus are associated with respiratory and enteric disease in dogs and cattle. In addition, novel porcine bocaviruses (PBoVs) have been identified in domestic and wild pigs in recent years, but are of unknown relevance to date. The objectives of this study were to determine the prevalence ra tes and genetic diversity of PBoVs in pigs in the USA. Using newly established multiplex real-time PCR assays, 385 lung, lymph node, serum and faecal samples from pigs with various disease conditions were investigated. A high PBoV prevalence rate ranging from 21.3 to 50.8 % was identified in the investigated samples and often two or more PBoV species were detected in the same sample. Cloning and sequencing analysis of the partial non-structural protein NS1 and the capsid proteins VP1 and VP2 of DNA samples positive for PBoV groups 1 (n = 6), 2 (n = 16) and 3 (n = 42), including subgroups 3A, 3B or 3C, revealed a high genetic diversity especially for the PBoV G3 VP2 gene, whereas the PBoV group 1 VP1 gene displayed a low nucleotide polymorphism. Using primer walking, 18 partial or nearly complete genomes of PBoVs were obtained and six of the 18 nearly complete genomes represented novel PBoV species. Recombination analysis using partial NS1, VP1 and VP2 genes and the nearly complete genomes indicated possible recombination events within and between PBoVs. Further studies will be required to reveal the possible pathogenic role of these diverse PBoVs.

Molecular detection and genetic analysis of porcine bocavirus in Korean domestic swine herds.

Several porcine bocaviruses have been detected worldwide, and this report is the first to describe this virus in a Korean domestic swine herd. We identified porcine bocavirus in various samples, including serum, tissue, feces and saliva, which revealed that porcine bocavirus predominates in Korean domestic swine populations. The results of this study also suggested that porcine bocaviruses primarily infect weaned piglets. Phylogenetic analysis of the ORF3 gene was performed to determine the genetic relationship of the Korean strains to reference strains from other countries.

Complete sequence and phylogenetic analysis of a porcine bocavirus strain swBoV CH437.

Porcine bocavirus (PBoV), a member of genus Bocavirus, family Parvoviridae, was first identified in 2009 in Swedish swine herds suffering from postweaning multisystemic wasting syndrome. Up to date, the different species of PBoVs have been reported in different countries. Especially, the virus isolated in China was complicated. In this study, we detected a novel PBoV strain swBoV CH437 from clinical samples collected in Gansu Province, Northwest China. The complete genome of swBoV CH437 was 5,275 nucleotides (nt) in length and contains three ORFs: ORF1 encodes NS1 (2,004 nt, 667 aa), ORF3 encodes NP1 (681 nt, 226 aa), and ORF2 encodes VP1 (2,049 nt, 682 aa) and VP2 (1,641 nt, 546 aa). Sequence analysis demonstrated that the NS1 gene shared 24.2-88.6 % nucleotide sequence identity, the NP1 shared 21.3-89.9 %, less than 95 % nucleotide sequence identity with other PBoV strains. Therefore, we propose that swBoV CH437 should be classified as a novel PBoV species.