猪博卡病毒环状附加体的发现与鉴定

为了证实博卡病毒可以环状附加体形式存在于宿主体内,本研究利用半巢式PCR方法在健康猪粪便标本中筛查出2株猪博卡病毒环状附加体PBoV_G2-episome和PBoV_G3-episome。通过反复测序和序列拼接得到其末端非编码区序列（405nt和511nt）。经过对其进行序列分析及二级结构的预测,发现PBoV_G2-episome与人博卡病毒3附加体（HBoV3-episome）结构相似,而PBoV_G3-episome与博卡病毒属其他成员的末端二级结构存在较大差别。猪博卡病毒环状附加体的发现证实了有些博卡病毒与其他细小病毒的复制方式存在一定差异,也为今后博卡病毒感染性克隆的构建提供了一条研究思路。     

Genomic characterization and high prevalence of bocaviruses in swine

Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ～2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45-75% and 55-70% respectively, with 30-47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses.     

Porcine Bocavirus: A 10-Year History since Its Discovery

Virologica Sinica - Porcine bocavirus (PBoV) is a single-stranded DNA virus, belongs to the genus Bocaparvovirus of family Parvoviridae. It was discovered along with porcine circovirus 2 (PCV 2)...     

Acute diarrhea in west african children: diverse enteric viruses and a novel parvovirus genus

Parvoviruses cause a variety of mild to severe symptoms or asymptomatic infections in humans and animals. During a viral metagenomic analysis of feces from children with acute diarrhea in Burkina Faso, we identified in decreasing prevalence nucleic acids from anelloviruses, dependoviruses, sapoviruses, enteroviruses, bocaviruses, noroviruses, adenoviruses, parechoviruses, rotaviruses, cosavirus, astroviruses, and hepatitis B virus. Sequences from a highly divergent parvovirus, provisionally called bufavirus, were also detected whose NS1 and VP1 proteins showed <39% and <31% identities to those of previously known parvoviruses. Four percent of the fecal samples were PCR positive for this new parvovirus, including a related bufavirus species showing only 72% identity in VP1. The high degree of genetic divergence of these related genomes from those of other parvoviruses indicates the presence of a proposed new Parvoviridae genus containing at least two species. Studies of the tropism and pathogenicity of these novel parvoviruses will be facilitated by the availability of their genome sequences.     

Complete genome sequence of a novel species of Porcine Bocavirus, PBoV5

Porcine bocavirus 5 is a novel porcine bocavirus species found in a pig with clinical diarrhea from a farm in China. Here, we report the complete genome sequence of strain PBoV5/JS677, which will help toward understanding the molecular and evolutionary characteristics of the porcine bocavirus.     

Identification and Characterization of a New Bocavirus Species in Gorillas

A novel parvovirus, provisionally named Gorilla Bocavirus species 1 (GBoV1), was identified in four stool samples from Western gorillas (Gorilla gorilla) with acute enteritis. The complete genomic sequence of the new parvovirus revealed three open reading frames (ORFs) with an organization similar to that of known bocaviruses. Phylogenetic analysis using complete capsid and non structural (NS) gene sequence suggested that the new parvovirus is most closely related to human bocaviruses (HBoV). However, the NS ORF is more similar in length to the NS ORF found in canine minute virus and bovine parvovirus than in HBoV. Comparative genetic analysis using GBoV and HBoV genomes enabled characterization of unique splice donor and acceptor sites that appear to be highly conserved among all four HBoV species, and provided evidence for expression of two different NS proteins in all primate bocaviruses. GBoV is the first non-human primate bocavirus identified and provides new insights into the genetic diversity and evolution of this highly prevalent and recently discovered group of parvoviruses.     

Ungulate bocaparvovirus 4 and rodent bocavirus are different genotypes of the same species of virus

Bocaviruses are associated with many human infectious diseases, such as respiratory tract infections, gastroenteritis, and hepatitis. Rats are known to be reservoirs of bocaviruses, including rodent bocavirus and rat bocavirus. Recently, ungulate bocaparvovirus 4, a known porcine bocavirus, has also been found in rats. Thus, investigating bocaviruses in rats is important for determining the origin of the viruses and preventing and controlling their transmission. To the best of our knowledge, no study to date has investigated bocaviruses in the livers of rats. In this report, a total of 624 rats were trapped in southern China between 2014 and 2017. Liver and serum samples from rats were tested for the prevalence of bocaviruses using PCR. Sequences related to ungulate bocaparvovirus 4 and rodent bocavirus were detected in both liver and serum samples. Interestingly, the prevalence of ungulate bocaparvovirus 4 (reference strain:KJ622366.1) was higher than that of rodent bocavirus (reference strain:KY927868.1) in both liver (2.24% and 0.64%, respectively) and serum samples (2.19% and 0.44%, respectively). The NS1 regions of ungulate bocaparvovirus 4 and rodent bocavirus related sequences displayed over 84% and 88% identity at the nucleic acid and amino acid levels, respectively. Furthermore, these sequences had similar genomic structure, genomic features, and codon usage bias, and shared a common ancestor. These viruses also displayed greater adaptability to rats than pigs. Our results suggested that ungulate bocaparvovirus 4 and rodent bocavirus may originate from rats and may be different genotypes of the same bocavirus species.     

Prevalence and genomic characterization of porcine parvoviruses detected in Chiangmai area of Thailand in 2011

Porcine parvovirus (PPV) causes reproductive failure in sows and has spread worldwide. Several new types of porcine parvoviruses have recently been identified in pig herds. The prevalence of five porcine parvoviruses in the Chiangmai area of Thailand was studied. The prevalence in 80 pigs was 53% for PPV (PPV‐Kr or ‐NADL2 being the new abbreviations), 83% for PPV2 (CnP‐PARV4), 73% for PPV3 (P‐PARV4), 44% for PPV4 (PPV4), and 18% for PBo‐likeV (PBoV7). Over 60% of the pigs carried more than three of the five porcine parvoviruses and occurrence together of the two pairs of viral genes, PPV1/PPV3 and PPV2/PBo‐likeV were observed. Phylogenetic analyses for PPV2 and PPV3 indicated the existence of only two major clades of PPV2 and one major clade of PPV3.     

Bocavirus episome in infected human tissue contains non-identical termini

Human bocaviruses (HBoV) are highly prevalent human infections whose pathogenic potential remains unknown. Recent identification of the first non-human primate bocavirus [1] in captive gorillas raised the possibility of the persistent nature of bocavirus infection. To characterize bocavirus infection in humans, we tested intestinal biopsies from 22 children with gastrointestinal disease for the presence of HBoV DNA. Four HBoV-positive tissue samples were analyzed to determine whether viral DNA was present in the linear genomic, the episomal closed circular or the host genome-integrated form. Whereas one tissue sample positive for HBoV3 contained the episomal form (HBoV3-E1), none had the genome-integrated form. The complete genome sequence of HBoV3-E1 contains 5319 nucleotides of which 513 represent the non-coding terminal sequence. The secondary structure of HBoV3-E1 termini suggests several conserved and variable features among human and animal bocaviruses. Our observation that HBoV genome exists as head-to-tail monomer in infected tissue either reflects the likely evolution of alternative replication mechanism in primate bocaviruses or a mechanism of viral persistence in their host. Moreover, we identified the HBoV genomic terminal sequences that will be helpful in developing reverse genetic systems for these widely prevalent parvoviruses. Significance: HBoV have been found in healthy human controls as well as individuals with respiratory or gastrointestinal disease. Our findings suggest that HBoV DNA can exist as episomes in infected human tissues and therefore can likely establish persistent infection in the host. Previous efforts to grow HBoV in cell culture and to develop reverse genetic systems have been unsuccessful. Complete genomic sequence of the HBoV3 episome and its genomic termini will improve our understanding of HBoV replication mechanism and its pathogenesis.     

猪博卡病毒的研究进展

猪博卡病毒是细小病毒科细小病毒亚科博卡病毒属的新成员,2009年发现于患仔猪断奶后多系统衰竭综合征的瑞典猪群中。目前,猪博卡病毒作为新发现的病原,已是世界各国科研人员的研究热点。本文结合已发表的文献,对猪博卡病毒的发现、分类、基因组结构与复制、流行病学、与疾病的相关性、培养与检测等方面进行了阐述,为广大科研人员对猪博卡病毒的研究提供一定的理论依据。     

Evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo.

A related group of parvoviruses infects members of many different carnivore families. Some of those viruses differ in host range or antigenic properties, but the true relationships are poorly understood. We examined 24 VP1/VP2 and 8 NS1 gene sequences from various parvovirus isolates to determine the phylogenetic relationships between viruses isolated from cats, dogs, Asiatic raccoon dogs, mink, raccoons, and foxes. There were about 1.3% pairwise sequence differences between the VP1/VP2 genes of viruses collected up to four decades apart. Viruses from cats, mink, foxes, and raccoons were not distinguished from each other phylogenetically, but the canine or Asiatic raccoon dog isolates formed a distinct clade. Characteristic antigenic, tissue culture host range, and other properties of the canine isolates have previously been shown to be determined by differences in the VP1/VP2 gene, and we show here that there are at least 10 nucleotide sequence differences which distinguish all canine isolates from any other virus. The VP1/VP2 gene sequences grouped roughly according to the time of virus isolation, and there were similar rates of sequence divergence among the canine isolates and those from the other species. A smaller number of differences were present in the NS1 gene sequences, but a similar phylogeny was revealed. Inoculation of mutants of a feline virus isolate into dogs showed that three or four CPV-specific differences in the VP1/VP2 gene controlled the in vivo canine host range.     

WLL-1株博卡病毒(Bocavirus)全基因组序列分析

儿童下呼吸道感染已成为当前儿科发病率最高的一种疾病,而病毒是小儿下呼吸道感染的重要原因。临床上有相当比例的小儿下呼吸道感染病因未能作出明确的实验室诊断,给临床诊断与治疗带来了较大的困难。小儿下呼吸道感染可以由不同病毒引起,其中包括呼吸道合胞病毒、流感病毒、腺     

Structure of Bombyx mori densovirus 1, a silkworm pathogen

Bombyx mori densovirus 1 (BmDNV-1), a major pathogen of silkworms, causes significant losses to the silk industry. The structure of the recombinant BmDNV-1 virus-like particle has been determined at 3.1-? resolution using X-ray crystallography. It is the first near-atomic-resolution structure of a virus-like particle within the genus Iteravirus. The particles consist of 60 copies of the 55-kDa VP3 coat protein. The capsid protein has a β-barrel "jelly roll" fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. Most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. In contrast to vertebrate parvoviruses, the N-terminal β-strand of BmDNV-1 VP3 is positioned relative to the neighboring 2-fold related subunit in a "domain-swapped" conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the Densovirinae.     

The recognition of parvovirus capsids by antibodies

The parvoviruses are small, non-enveloped icosahedral viruses which infect many animals, including vertebrates and arthropods. Vertebrate parvoviruses can be classified into the autonomous and the adeno-associated viruses — the autonomous parvoviruses have been examined in detail for antigenic structure. The protective immunity against parvoviruses in animals appears to be primarily antibody-mediated. The capsid of the autonomous parvoviruses is assembled from two proteins, VP1 and VP2, which overlap in sequence, with VP1 having additional N-terminal residues. Empty capsids can be assembled from VP2 alone.The structures of canine parvovirus (CPV) and feline panleukopenia virus (FPV) have been solved to better than 3·5 Å resolution, and the structure of human parvovirus, B19, has been solved to 8 Å resolution. In each case the T = 1 icosahedron is made up to 60 copies of a structural motif common to VP1 and VP2, consisting of an eight-stranded anti-parallel β-barrel. The surface of the capsid is made up primarily of large elaborate loops which connect the β-strands that make up the barrel. Antigenic epitopes have been mapped utilizing escape mutants, natural variants, peptide analysis and by expression of viral proteins. In CPV two major antigenic determinants were defined by escape mutant analysis, while peptide analysis revealed antigenic determinants in many different regions of the capsid protein, including the amino terminus of VP2. Neutralizing epitopes of B19 were found by peptide analysis in the VP1-unique region and in sequences common to VP1 and VP2. Other antigenic, but non-neutralizing, epitopes were found in the VP1–VP2 junction, as well as various other parts of the VP2 protein.The binding of a Fab derived from one neutralizing anti-CPV Mab has been examined by cryo-electron microscopy image reconstruction, which showed that 60 copies of the Fab were bound per virion. The Fab footprint covered approximately 796 Ųof the capsid surface, in a region where escape mutations to that Mab had been previously shown to cluster. The mechanism of neutralization was not clear, but could involve interference with cell attachment, cell entry or uncoating during the process of cell infection.     

Sequence analysis of an isolate of minute virus of canines in China reveals the closed association with bocavirus

In the present study, we have cloned and sequenced the nearly-full-length genome of minute virus of canines (MVC), SH26, in China. The genome of MVC, 5,132 nucleotides (nts) in length, contains three open reading frames (ORFs), which are 2,325-bp of NS1, 561-bp of NP1 and 2,112-bp of VP1/VP2 encoding three proteins of 774, 186 and 703 residues, respectively. Predicted amino acids sequence of NS1 of MVC has 44% identity with human bocavirus (HBoV) and human boacvirus 2 (HBoV2), NP1 has 48 and 45% identity with HBoV and HBoV2, VP1/VP2 has 45 and 46% identity with HBoV and HBoV2, respectively. Phylogenetic analysis showed that the present Chinese MVC strain was also closely clustered with the previous American and Japanese MVC isolates, and MVCs formed a different branch together with bovine parvovirus and HBoVs from other parvoviruses classified into Parvovirinae.     

The small nonstructural protein NP1 of human bocavirus 1 directly interacts with Ku70 and RPA70 and facilitates viral DNA replication

Human bocavirus 1 (HBoV1), a member of the genus Bocaparvovirus of the family Parvoviridae, causes acute respiratory tract infections in young children. Well-differentiated pseudostratified human airway epithelium cultured at an air-liquid interface (HAE-ALI) is an ideal in vitro culture model to study HBoV1 infection. Unique to other parvoviruses, bocaparvoviruses express a small nonstructured protein NP1 of ~25 kDa from an open reading frame (ORF) in the center of the viral genome. NP1 plays an important role in viral DNA replication and pre-mRNA processing. In this study, we performed an affinity purification assay to identify HBoV1 NP1-inteacting proteins. We identified that Ku70 and RPA70 directly interact with the NP1 at a high binding affinity, characterized with an equilibrium dissociation constant (K_D) of 95 nM and 122 nM, respectively. Furthermore, we mapped the key NP1-interacting domains of Ku70 at aa266-439 and of RPA70 at aa181-422. Following a dominant negative strategy, we revealed that the interactions of Ku70 and RPA70 with NP1 play a significant role in HBoV1 DNA replication not only in an in vitro viral DNA replication assay but also in HBoV1-infected HAE-ALI cultures. Collectively, our study revealed a novel mechanism by which HBoV1 NP1 enhances viral DNA replication through its direct interactions with Ku70 and RPA70.     

SAT: a late NS protein of porcine parvovirus

The genomes of all members of the Parvovirus genus were found to contain a small open reading frame (ORF), designated SAT, with a start codon four or seven nucleotides downstream of the VP2 initiation codon. Green fluorescent protein or FLAG fusion constructs of SAT demonstrated that these ORFs were expressed. Although the SAT proteins of the different parvoviruses are not particularly conserved, they were all predicted to contain a membrane-spanning helix, and mutations in this hydrophobic stretch affected the localization of the SAT protein. SAT colocalized with calreticulin in the membranes of the endoplasmic reticulum and the nucleus. A knockout mutant (SAT(-)), with an unmodified VP sequence, showed a "slow-spreading" phenotype. These knockout mutants could be complemented with VP2(-) SAT(+) mutant. The SAT protein is a late nonstructural (NS) protein, in contrast to previously identified NS proteins, since it is expressed from the same mRNA as VP2.     

Interfacial enzymology of parvovirus phospholipases A2

The capsid of parvoviruses proteins were recently shown to contain secreted phospholipase A(2) (sPLA(2))-like activity that is required during host cell entry. Parvoviral PLA(2) domains have little sequence identity with sPLA(2)s and lack disulfide bonds. In the present study, after bacterial expression and purification, the biochemical characterizations of these first PLA(2)s identified in viruses have been investigated, and a comparison has been made with other known PLA(2)s. The specific activities of three viral PLA(2)s differed by 3 orders of magnitude, with porcine parvovirus PLA(2) displaying a specific activity similar to that of the most active sPLA(2)s (e.g. human group IIA) and the human AAV2 and B19 parvoviral enzymes displaying approximately 10(3) lower specific activities (similar to human sPLA(2) groups IIE and XIIA). These differences were not caused by weaker Ca(2+) or interfacial binding. The specific activities of the viral PLA(2)s on zwitterionic or anionic phospholipid vesicles were comparable. The viral PLA(2)s did not display a preference for unsaturated versus saturated sn-2 fatty acyl chains and hydrolyzed all major classes of glycero-phospholipids except phosphatidylinositol. Incubation of mammalian cells with porcine parvovirus PLA(2) led to the release of arachidonic acid into the culture medium. Interestingly, among nine previously known sPLA(2) inhibitors, only a subset showed inhibition of the viral PLA(2)s and with weak potency, indicating that the active sites of these new enzymes are structurally distinct from those of sPLA(2)s. Based on these distinct enzymatic and structural properties, we propose to classify the parvovirus PLA(2)s within the PLA(2) superfamily as group XIII enzymes.