Complete nucleotide sequence and genome organization of bovine parvovirus.

We determined the complete nucleotide sequence of bovine parvovirus (BPV), an autonomous parvovirus. The sequence is 5,491 nucleotides long. The terminal regions contain nonidentical imperfect palindromic sequences of 150 and 121 nucleotides. In the plus strand, there are three large open reading frames (left ORF, mid ORF, and right ORF) with coding capacities of 729, 255, and 685 amino acids, respectively. As with all parvoviruses studied to date, the left ORF of BPV codes for the nonstructural protein NS-1 and the right ORF codes for the major parts of the three capsid proteins. The mid ORF probably encodes the major part of the nonstructural protein NP-1. There are promoterlike sequences at map units 4.5, 12.8, and 38.7 and polyadenylation signals at map units 61.6, 64.6, and 98.5. BPV has little DNA homology with the defective parvovirus AAV, with the human autonomous parvovirus B19, or with the other autonomous parvoviruses sequenced (canine parvovirus, feline panleukopenia virus, H-1, and minute virus of mice). Even though the overall DNA homology of BPV with other parvoviruses is low, several small regions of high homology are observed when the amino acid sequences encoded by the left and right ORFs are compared. From these comparisons, it can be shown that the evolutionary relationship among the parvoviruses is B19 in equilibrium with AAV in equilibrium with BPV in equilibrium with MVM. The highly conserved amino acid sequences observed among all parvoviruses may be useful in the identification and detection of parvoviruses and in the design of a general parvovirus vaccine.     

Phylogenetic evidence for the rapid evolution of human B19 erythrovirus

Human B19 erythrovirus is a ubiquitous viral pathogen, commonly infecting individuals before adulthood. As with all autonomous parvoviruses, its small single-stranded DNA genome is replicated with host cell machinery. While the mechanism of parvovirus genome replication has been studied in detail, the rate at which B19 virus evolves is unknown. By inferring the phylogenetic history and evolutionary dynamics of temporally sampled B19 sequences, we observed a surprisingly high rate of evolutionary change, at approximately 10(-4) nucleotide substitutions per site per year. This rate is more typical of RNA viruses and suggests that high mutation rates are characteristic of the Parvoviridae.     

Structure comparisons of Aedes albopictus densovirus with other parvoviruses

Parvoviridae is a family of the smallest viruses known with a wide variety of hosts. The capsid structure of the Aedes albopictus C6/36 cell densovirus (C6/36 DNV) at 1.2-nm resolution was obtained by electron cryomicroscopy (cryoEM) and three-dimensional (3D) image reconstruction. Structure comparisons between the C6/36 DNV and other parvoviruses reveal that the degree of structural similarity between C6/36 DNV and the human parvovirus B19 is higher than that between C6/36 DNV and other insect parvoviruses. The amino acid sequence comparisons of structural and non-structural proteins also reveal higher levels of similarity between C6/36 DNV and parvovirus B19 than those between C6/36 DNV and other parvoviruses. These findings indicate that C6/36 DNV is closely related to the human virus B19, and the former might evolve from the human species other than from other insect viruses.     

Discovery and characterization of mammalian endogenous parvoviruses

Public databases of nucleotide sequences contain exponentially increasing amounts of sequence data from mammalian genomes. Through the use of large-scale bioinformatic screening for sequences homologous to exogenous mammalian viruses, we found several sequences related to human and animal parvoviruses (PVs) in the Parvovirus and Dependovirus genera within genomes of several mammals, including rats, wallabies, opossums, guinea pigs, hedgehogs, African elephants, and European rabbits. However, phylogenetic analysis of these endogenous parvovirus (EnPV) sequences demonstrated substantial genetic divergence from exogenous mammalian PVs characterized to date. Entire nonstructural and capsid gene sequences of a novel EnPV were amplified and genetically characterized from rat (Rattus norvegicus) genomic DNA. Rat EnPV sequences were most closely related to members of the genus Parvovirus, with >70% and 65% amino acid identities to nonstructural and capsid proteins of canine parvovirus, respectively. Integration of EnPV into chromosome 5 of rats was confirmed by PCR cloning and sequence analysis of the viral and chromosomal junctions. Using inverse PCR, we determined that the rat genome contains a single copy of rat EnPV. Considering mammalian phylogeny, we estimate that EnPV integrated into the rat genome less than 30 million years ago. Comparative phylogenetic analysis done using all known representative exogenous parvovirus (ExPV) and EnPV sequences showed two major genetic groups of EnPVs, one genetically more similar to genus Parvovirus and the other genetically more similar to the genus Dependovirus. The full extent of the genetic diversity of parvoviruses that have undergone endogenization during evolution of mammals and other vertebrates will be recognized only once complete genomic sequences from a wider range of classes, orders, and species of animals become available.     

Nucleotide sequence and genome organization of canine parvovirus.

The genome of a canine parvovirus isolate strain (CPV-N) was cloned, and the DNA sequence was determined. The entire genome, including ends, was 5,323 nucleotides in length. The terminal repeat at the 3' end of the genome shared similar structural characteristics but limited homology with the rodent parvoviruses. The 5' terminal repeat was not detected in any of the clones. Instead, a region of DNA starting near the capsid gene stop codon and extending 248 base pairs into the coding region had been duplicated and inserted 75 base pairs downstream from the poly(A) addition site. Consensus sequences for the 5' donor and 3' acceptor sites as well as promotors and poly(A) addition sites were identified and compared with the available information on related parvoviruses. The genomic organization of CPV-N is similar to that of feline parvovirus (FPV) in that there are two major open reading frames (668 and 722 amino acids) in the plus strand (mRNA polarity). Both coding domains are in the same frame, and no significant open reading frames were apparent in any of the other frames of both minus and plus DNA strands. The nucleotide and amino acid homologies of the capsid genes between CPV-N and FPV were 98 and 99%, respectively. In contrast, the nucleotide and amino acid homologies of the capsid genes for CPV-N and CPV-b (S. Rhode III, J. Virol. 54:630-633, 1985) were 95 and 98%, respectively. These results indicate that very few nucleotide or amino acid changes differentiate the antigenic and host range specificity of FPV and CPV.     

Structure comparisons of Aedes albopictus densovirus with other parvoviruses

Parvoviridae is a family of the smallest viruses known with a wide variety of hosts. The capsid structure of the Aedes albopictus C6/36 cell densovirus (C6/36 DNV) at 1.2-nm resolution was obtained by elec-tron cryomicroscopy (cryoEM) and three-dimensional (3D) image reconstruction. Structure compari-sons between the C6/36 DNV and other parvoviruses reveal that the degree of structural similarity be-tween C6/36 DNV and the human parvovirus B19 is higher than that between C6/36 DNV and other in-sect parvoviruses. The amino acid sequence comparisons of structural and non-structural proteins also reveal higher levels of similarity between C6/36 DNV and parvovirus B19 than those between C6/36 DNV and other parvoviruses. These findings indicate that C6/36 DNV is closely related to the human virus B19, and the former might evolve from the human species other than from other insect viruses.     

The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage

The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.     

Analysis of the termini of the DNA of bovine parvovirus: demonstration of sequence inversion at the left terminus and its implication for the replication model.

The distribution of terminal-sequence orientations in the viral DNA of bovine parvovirus (BPV), an autonomous parvovirus, was studied by end labeling and restriction enzyme digestion and also by cloning. The left (3') end of the minus strand of BPV was found in two alternative sequence orientations (designated as flip and flop, which are reverse complements of each other), with a 10-fold excess of flip. This is in contrast to the autonomous rodent parvoviruses which encapsidate minus-strand DNA with only the flip orientation at this end. The right (5') end of the minus strand of BPV contained both sequence orientations with equal frequencies, as in the rodent parvoviruses. Sequence inversions were also detected at both ends of the plus strand, which makes up about 10% of the encapsidated BPV DNA. Each terminus of BPV DNA had a characteristic ratio of flip to flop forms, and this ratio was restored in the progeny DNA resulting from transfection with genomic clones of different defined terminal conformations. Replicative-form DNA showed the same distribution of terminal-sequence orientations as the reannealed plus and minus virion DNAs, suggesting that the distribution of flip and flop forms observed in virion DNA is not due to selective encapsidation, but rather to the specific distribution of replicative forms. The current replication model for autonomous parvoviruses, which was based on the available data for the rodent parvoviruses, cannot account for the observed distribution of BPV DNA. An alternative model is suggested.     

人博卡病毒1型感染性克隆的构建及鉴定

人博卡病毒1 (human bocaparvovirus 1, HBoV1)为感染人并引起疾病的两种细小病毒之一。其感染2−5岁婴幼儿,能引起轻度或重度急性呼吸道疾病,严重时可危及生命。HBoV1基因组末端含末端反向重复序列(repeat the sequence in reverse, ITR),为病毒基因组复制所必需,但是难以进行PCR扩增合成。本研究通过分步合成末端ITR及分子克隆方法成功构建HBoV1的全长感染性克隆pSKHBoV1。经转染HEK293细胞后,分别从重要非结构蛋白的表达、病毒RNA转录后修饰与加工、病毒基因组复制水平以及子代病毒粒子基因组鉴定等方面,证实构建的感染性克隆在转染HEK293细胞后能够进入正常的复制周期并具有拯救出病毒粒子的潜力,这为后续研究HBoV1的复制增殖、病毒与宿主互作关系以及病毒疫苗的研发奠定了基础。     

Parvovirus infection-induced cell death and cell cycle arrest

The cytopathic effects induced during parvovirus infection have been widely documented. Parvovirus infection-induced cell death is often directly associated with disease outcomes (e.g., anemia resulting from loss of erythroid progenitors during parvovirus B19 infection). Apoptosis is the major form of cell death induced by parvovirus infection. However, nonapoptotic cell death, namely necrosis, has also been reported during infection of the minute virus of mice, parvovirus H-1 and bovine parvovirus. Recent studies have revealed multiple mechanisms underlying the cell death during parvovirus infection. These mechanisms vary in different parvoviruses, although the large nonstructural protein (NS)1 and the small NS proteins (e.g., the 11 kDa of parvovirus B19), as well as replication of the viral genome, are responsible for causing infection-induced cell death. Cell cycle arrest is also common, and contributes to the cytopathic effects induced during parvovirus infection. While viral NS proteins have been indicated to induce cell cycle arrest, increasing evidence suggests that a cellular DNA damage response triggered by an invading single-stranded parvoviral genome is the major inducer of cell cycle arrest in parvovirus-infected cells. Apparently, in response to infection, cell death and cell cycle arrest of parvovirus-infected cells are beneficial to the viral cell lifecycle (e.g., viral DNA replication and virus egress). In this article, we will discuss recent advances in the understanding of the mechanisms underlying parvovirus infection-induced cell death and cell cycle arrest.     

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.     

Widespread endogenization of densoviruses and parvoviruses in animal and human genomes

Parvoviruses infect humans and a broad range of animals, from mammals to crustaceans, and generally are associated with a variety of acute and chronic diseases. However, many others cause persistent infections and are not known to be associated with any disease. Viral persistence is likely related to the ability to integrate into the chromosomal DNA and to establish a latent infection. However, there is little evidence for genome integration of parvoviral DNA except for Adeno-associated virus (AAV). Here we performed a systematic search for homologs of parvoviral proteins in publicly available eukaryotic genome databases followed by experimental verification and phylogenetic analysis. We conclude that parvoviruses have frequently invaded the germ lines of diverse animal species, including mammals, fishes, birds, tunicates, arthropods, and flatworms. The identification of orthologous endogenous parvovirus sequences in the genomes of humans and other mammals suggests that parvoviruses have coexisted with mammals for at least 98 million years. Furthermore, some of the endogenized parvoviral genes were expressed in eukaryotic organisms, suggesting that these viral genes are also functional in the host genomes. Our findings may provide novel insights into parvovirus biology, host interactions, and evolution.     

Structure of the 3' hairpin termini of four rodent parvovirus genomes: nucleotide sequence homology at origins of DNA replication.

The nucleotide sequences of the 3' termini of the DNA from four autonomous rodent parvoviruses have been determined. The terminus of each genome exists as a Y-shaped hairpin structure involving 115 or 116 nucleotides. The sequence of this region of DNA is highly conserved and shows no evidence of internal sequence heterogeneity, a characteristic which is observed in the terminal nucleotide sequence of the helper-dependent, adeno-associated viruses (Berns et al., 1978a). The implications of these results with respect to the models of parvovirus DNA replication are discussed.     

Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins.

Empty parvovirus B19 capsids were isolated from insect cells infected with a recombinant baculovirus expressing parvovirus B19 VP2 alone and also with a double-recombinant baculovirus expressing both VP1 and VP2. That VP2 alone can assemble to form capsids is a phenomenon not previously observed in parvoviruses. The stoichiometry of the capsids containing both VP1 and VP2 was similar to that previously observed in parvovirus B19-infected cells. The capsids were similar to native capsids in size and appearance, and their antigenicity was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assay with B19-specific antibodies.     

Molecular characterization of a newly recognized mouse parvovirus.

Mouse parvovirus (MPV), formerly known as orphan parvovirus, is a newly recognized rodent parvovirus distinct from both serotypes of minute virus of mice (MVM). Restriction analysis of the MPV genome indicated that many restriction sites in the capsid region were different from those of MVM, but most sites in the nonstructural (NS) region of the genome were conserved. MPV resembled MVM in genome size, replication intermediates, and NS proteins. Replication intermediates in infected cells were the same for MPV and MVM, including packaging of the 5-kb minus (V) strand. Furthermore, the MPV NS proteins were the same size as and present at the same ratio as the MVM(i) proteins in infected cells. Cloning and sequencing of the MPV genome revealed a genome organization closely resembling that of MVM, with conservation of open reading frames, promoter sequences, and splice sites. The left terminal hairpin was identical to that of MVM(i), but the right terminus was not conserved. Also, the MPV genome was unique in that it contained 1.8 copies of the terminal repeat sequence rather than the 1 or 2 copies found in other parvoviruses. The predicted amino acid sequence of the NS proteins of MPV and MVM(i) were nearly identical. In contrast, the predicted amino acid sequence of the capsid proteins of MPV was different from sequences of other parvoviruses. These results confirm that MPV is a distinct murine parvovirus and account for the antigenic differences between MPV and MVM.