Genus Bocavirus Research Articles

Development of a novel TaqMan-based real-time PCR assay for the detection of porcine boca-like virus (Pbo-likeV)

The recently discovered porcine boca-like virus (Pbo-likeV) is a member of the Parvoviridae family, genus Bocavirus, and it is potentially associated with swine disease. Several studies have associated Pbo-likeV with postweaning multisystemic wasting syndrome in pigs, but the full spectrum of clinical disease and the epidemiology of Pbo-likeV infection remain unclear. The availability of rapid and reliable molecular diagnostics would aid future studies of this novel virus. Thus, we developed a sensitive and specific TaqMan-based real-time PCR assay to target the Pbo-likeV NP1 gene. The assay reproducibly detected 20 copies of a recombinant DNA plasmid containing the NP1 gene, with a dynamic range of six orders of magnitude (102-107 copies). The assay did not cross-react with other animal viruses. Clinical evaluation found that Pbo-likeV was present in Chinese swine herds at a frequency of 44.2% (114/258). Higher infection rates were found in diseased pigs (56.1%, 101/180) compared with healthy pigs (16.7%, 13/78) (P < 0.05). Our assay for the diagnosis and quantification of Pbo-likeV was highly sensitive and specific, and should provide a reliable real-time tool for epidemiological and pathogenetic study of Pbo-likeV infection.

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5' splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

Complete coding sequences and phylogenetic analysis of porcine bocavirus

Here we report, for the first time, the nearly full-length genome sequence of porcine bocavirus (PBoV), a recently discovered parvovirus from pigs. Phylogenetic trees based on this genome sequence showed that PBoV belongs to the branch containing the genus Bocavirus, which comprises canine minute virus (CnMV), bovine parvovirus, gorilla bocavirus and human bocavirus (HBoV), and was most closely related to the group containing CnMV. PBoV was predicted to contain three potential ORFs encoding the non-structural protein NS1, the characteristic NP1 protein and the capsid protein VP1/VP2, with an organization similar to that of known bocaviruses. Interestingly, the NS1 gene of PBoV was more similar in length to the homogeneous gene found in HBoV than to those of other known bocaviruses. In addition, highly conserved unique splice-donor and -acceptor sites were identified in the NS1 gene of HBoV and PBoV.

Detection of a novel porcine parvovirus, PPV4, in chinese swine herds

To determine whether the novel porcine parvovirus type 4 (PPV4) recently reported in America is prevalent in China, a set of specific primers was designed and used for molecular survey of PPV4 among the clinical samples collected from various provinces of China between 2006 and 2010. The results showed that PPV4 is present in Chinese swine herds at a rate of 2.09% (12/573) among the clinical samples examined and 0.76% (1/132) among the samples taken from healthy animals. We also noted that PPV4 was not detected in samples taken prior to 2009. Analysis of the coding sequences showed that the Chinese and American PPV4 genome sequences are closely related with greater than 99% nucleotide sequence identity. Similar to a previous study, viral genomes in head-to-tail configuration of various lengths of the non-coding region were detected. Our findings confirmed that PPV4 is a unique recently discovered virus in pigs. Phylogenetically, PPV4 is most closely related to bovine parvovirus 2 (BPV2, which is not a Bocavirus and is not assigned to any Parvovirinae genus) and shares limited ORF1 (33.6%) and ORF2 (24.5%) amino acid identity. With respect to genome structure and organization, PPV4 encodes an ORF3 in the middle of the viral genome that resembles the Bocavirus genus. However, the PPV4 ORF3 encoded protein shares minimal amino acid identity with the ORF3 encoded proteins of the Bocavirus genus.

Identification and Nearly Full-Length Genome Characterization of Novel Porcine Bocaviruses

The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7–56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A2 (sPLA2) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca2+-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.

Bovine parvovirus uses clathrin-mediated endocytosis for cell entry

Entry events of bovine parvovirus (BPV) were studied. Transmission electron micrographs of infected cells showed virus particles in cytoplasmic vesicles. Chemical inhibitors that block certain aspects of the cellular machinery were employed to assess viral dependency upon those cellular processes. Chlorpromazine, ammonium chloride, chloroquine and bafilamicin A1 were used to inhibit acidification of endosomes and clathrin-associated endocytosis. Nystatin was used as an inhibitor of the caveolae pathway. Cytochalasin D and ML-7 were used to inhibit actin and myosin functions, respectively. Nocodazole and colchicine were employed to inhibit microtubule activity. Virus entry was assessed by measuring viral transcription using real-time PCR, synthesis of capsid protein and assembly of infectious progeny virus in the presence of inhibitor blockage. The results indicated that BPV entry into embryonic bovine trachael cells utilizes endocytosis in clathrin-coated vesicles, is dependent upon acidification, and appears to be associated with actin and microtubule dependency. Evidence for viral entry through caveolae was not obtained. These findings provide a fuller understanding of the early cell-entry events of the replication cycle for members of the genus Bocavirus.

Studies of porcine circovirus type 2, porcine boca-like virus and torque teno virus indicate the presence of multiple viral infections in postweaning multisystemic wasting syndrome pigs

In a previous study, using random amplification and large-scale sequencing technology, we identified a novel porcine parvovirus belonging to the genus Bocavirus in the background of porcine circovirus type 2 (PCV-2) in Swedish pigs with postweaning multisystemic wasting syndrome (PMWS). In addition to bocavirus we demonstrated the presence of torque teno virus (TTV) genogroups 1 and 2 in these cases of PMWS, indicating the simultaneous presence of several viruses in this disease complex. In the present study, 34 PMWS-affected animals and 24 pigs without PMWS were screened by PCR for the presence of PCV-2, TTV-1, TTV-2 and porcine boca-like virus (Pbo-likeV). The studies revealed the following infection rates in the PMWS-affected pigs: PCV-2 100%, TTV-1 77%, TTV-2 94% and Pbo-likeV 88%. In comparison, the pigs without PMWS had the following rates: PCV-2 80%, TTV-1 79%, TTV-2 83% and Pbo-likeV 46%. The sequence identity between the different Swedish Pbo-likeV sequences ranged between 98% and 100%. By checking co-infection, it was found that 71% of the PMWS-affected pigs harbor simultaneously all these viruses. As a contrast, in the group without PMWS only 33% of the animals were positive simultaneously for these viruses. These observations indicate a multiple viral infection in PMWS-affected pigs. It has to be studied further if the clinical manifestation of PMWS might be due to synergistic effects of different viruses acting together.

Identification and molecular cloning of a novel porcine parvovirus

A novel porcine parvovirus, PPV4, was identified in the lung lavage of a diseased pig coinfected with porcine circovirus type 2. This virus exhibits limited similarity to its closest relative, bovine parvovirus 2, but resembles viruses of the genus Bocavirus (bovine parvovirus, canine minute virus and human bocavirus) that encode an additional ORF3. The ORF3 of PPV4 is predicted to encode a protein of 204 amino acid residues, which is similar in size to the ORF3-encoded proteins of the bocaviruses. Whereas the ORF3-encoded proteins of bocaviruses share significant similarity with each other, the PPV4 ORF3 encoded protein does not exhibit homology with any protein in the GenBank non-redundant database.

A Novel Bocavirus Associated with Acute Gastroenteritis in Australian Children

Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide, causing an estimated three million deaths annually. Viruses such as rotavirus, adenovirus, and caliciviruses are a major cause of AGE, but in many patients a causal agent cannot be found despite extensive diagnostic testing. Proposing that novel viruses are the reason for this diagnostic gap, we used molecular screening to investigate a cluster of undiagnosed cases that were part of a larger case control study into the etiology of pediatric AGE. Degenerate oligonucleotide primed (DOP) PCR was used to non-specifically amplify viral DNA from fecal specimens. The amplified DNA was then cloned and sequenced for analysis. A novel virus was detected. Elucidation and analysis of the genome indicates it is a member of the Bocavirus genus of the Parvovirinae, 23% variant at the nucleotide level from its closest formally recognized relative, the Human Bocavirus (HBoV), and similar to the very recently proposed second species of Bocavirus (HBoV2). Fecal samples collected from case control pairs during 2001 for the AGE study were tested with a bocavirus-specific PCR, and HBoV2 (sequence confirmed) was detected in 32 of 186 cases with AGE (prevalence 17.2%) compared with only 15 controls (8.1%). In this same group of children, HBoV2 prevalence was exceeded only by rotavirus (39.2%) and astrovirus (21.5%) and was more prevalent than norovirus genogroup 2 (13.4%) and adenovirus (4.8%). In a univariate analysis of the matched pairs (McNemar's Test), the odds ratio for the association of AGE with HBoV2 infection was 2.6 (95% confidence interval 1.2–5.7); P = 0.007. During the course of this screening, a second novel bocavirus was detected which we have designated HBoV species 3 (HBoV3). The prevalence of HBoV3 was low (2.7%), and it was not associated with AGE. HBoV2 and HBoV3 are newly discovered bocaviruses, of which HBoV2 is the thirdmost-prevalent virus, after rotavirus and astrovirus, associated with pediatric AGE in this study.

The incidence of human bocavirus infection among children admitted to hospital in Singapore

Human bocavirus (HBoV) is a parvovirus, belonging to the genus Bocavirus. The virus was identified recently in Sweden, and has now been detected in several different countries. Although it is associated with lower respiratory tract infections in pediatric patients, the incidence of HBoV infection in a developed country in South East Asia, has not been examined. The objective of this study was to determine the importance of HBoV as a cause of lower respiratory tract infections among children admitted to hospital in Singapore. Five hundred nasopharyngeal swabs were collected from anonymized pediatric patients admitted to the Kandang Kerbau Women's and Children's Hospital for acute respiratory infections. The specimens were tested for the presence of HBoV using polymerase chain reactions. HBoV was detected in 8.0% of the patients tested, and a majority of these HBoV patients exhibited lower respiratory tract infections. A significant level of coinfection with respiratory syncytial viruses and rhinoviruses was also observed in these HBoV patients. The data suggest that HBoV is an important cause of lower respiratory tract infections among children admitted to hospital in Singapore, and is the first study examining the incidence of HBoV infection in a developed country in South East Asia. J. Med. Virol. 81:82–89, 2009. © 2008 Wiley‐Liss, Inc.

Human bocavirus, a real respiratory tract pathogen

A new virus was discovered by molecular techniques in respiratory samples collected from young children with respiratory diseases in Sweden in 2005. The virus, named human bocavirus, is genetically related to the bovine parvovirus and the canine minute virus, both of which belong to the bocavirus genus of the parvoviridae family. Recent studies conducted in different countries have shown that HBoV is found in 1.5 - 19% of children with respiratory diseases. HBoV has been observed to be associated with a broad spectrum of both upper and lower respiratory tract diseases, more frequently related to lower respiratory diseases, one third of which is pneumonia. HBoV infection is of worldwide distribution, and a seasonal distribution with a peak in winter and spring is suspected. There is increasing evidence that HBoV is pathogenic for the human respiratory tract, especially in infants and young children, and HBoV has been detected from patients with gastroenteritis. However, given the frequent co-infection with bacterial or viral pathogens, the exact role played by this virus in human diseases still remains disputable. Further investigations, including population-based studies with controlled subjects, are needed to prove its pathogenic potential and epidemiologic patterns. Key words: Respiratory virus, human bocavirus, parvovirus; HBoV, respiratory infection; children.

Bocavirus: a new human parvovirus

A new virus was recently discovered by molecular techniques in respiratory samples collected from young children with respiratory disease. This virus, which represents a new member of the Parvoviridae family is genetically related to the bovine parvovirus and the canine minute virus that belong to the Bocavirus genus. It has been classified in the Bocavirus genus and named human bocavirus (HBoV). Recent studies conducted in different countries have shown that HBoV is found in 3 to 18 %of children with respiratory disease worldwide. Genetic analysis indicate that this virus shows low genetic variability. The clinical signs observed in patients infected with HBoV are not different from those caused by other respiratory viruses.A frequent association of HBoV with other respiratory pathogens may be observed. Therefore, the exact role played by this virus in human diseases still remains unclear. Further studies including control populations are needed to ascertain the pathogenic potential of this virus.

Human Bocavirus: A New Respiratory Pathogen?

A new virus was recently discovered by molecular techniques in respiratory samples collected from young children with respiratory disease. This virus is a new member of the Parvoviridae family and has been classified in the Bocavirus genus. This virus, named human bocavirus, is genetically related to the bovine parvovirus and the canine minute virus, which both belong to the Bocavirus genus. Recent studies conducted in different countries have shown that this virus is found in 3–18% of children with respiratory disease worldwide, and a seasonal distribution with a peak in winter and spring is suspected. Genetic analysis has indicated that this virus has low genetic variability. The clinical signs observed in patients infected with human bocavirus are not different from those caused by other respiratory viruses. Given the frequent association of human bocavirus with other respiratory pathogens, the exact role played by this virus in human diseases still remains unclear, and further studies including control subjects are needed to ascertain the pathogenic potential of this virus.

Human bocavirus DNA detected by quantitative real-time PCR in two children hospitalized for lower respiratory tract infection

Viral pathogens such as respiratory syncytial virus, human metapneumovirus, rhinovirus, adenovirus, parainfluenza virus and influenza virus are the most frequent causes of acute respiratory tract infection and hospitalization in infants and young children. However, no pathogen can be identified in about 30% of suspected respiratory infections [1]. Recently, a new human virus belonging to the Bocavirus genus of the subfamily Parvovirinae was cloned from pooled human respiratory samples and its pathogenic potential was proposed according to its association with respiratory illness [2]. Later studies suggested human bocavirus (HBoV) may be causative for lower respiratory tract disease in young children [3–6]. In this report, we describe the detection by quantitative real-time PCR of HBoV DNA in the nasopharyngeal aspirates of two German children hospitalized for pneumonia. Sequencing of an NP-1 gene fragment and phylogenetic analysis revealed high sequence identity for this HBoV strain compared with the prototype strain HBoV st1 [2] and other strains worldwide [2–6]. Our finding of a high viral load combined with symptoms of acute respiratory tract infection may support the assertion of others that HBoV is an important emerging pathogen [2–6]. In March 2004, a 32-month-old boy presented with a 3day history of high fever and cough. He was treated with corticosteroids and bronchodilators to control acute bronchoconstriction. No antibiotics were administered. Due to increasing tachypnea and dyspnea, the child was hospitalized. Clinical examination upon admission revealed a body temperature of 39°C, tachypnea and dyspnea, as well as subcostal retractions. Wheezing and dry bilateral fine rales were present upon lung auscultation. Transcutaneously measured oxygen saturation was decreased to 87% and the patient required oxygen supplementation (4 l/min) for 6 days and intravenous treatment with corticosteroids for persistent bronchoconstriction. Laboratory tests showed a leukocyte count of 7.7×10/l, a blood pH level of 7.4, base excess of −4.7 mmol/l, and a C-reactive protein value of 5.6 mg/l. Chest radiograph revealed beginning right paracardial pulmonary infiltration without pleural effusion, indicating pneumonia. Under antiobstructive therapy with sultanol and intravenous corticosteroids, the boy’s respiratory condition improved. On day 4 after admission, he developed rotavirus gastroenteritis, probably of nosocomial origin, which rapidly improved under symptomatic therapy. By discharge on day 9, his general condition was good, although a mild fever (38°C) was still present. In the same period, an 18-month-old boy presented with a 2-day history of increasing dyspnea, fever (39.5°C), and cough. Clinical examination revealed a weakened general condition, tachypnea, a body temperature of 38.8°C, and modest wheezing as well as bilateral basal rales upon auscultation. The patient required oxygen supplementation (2 l/min) for 4 days. Initial laboratory tests showed an elevated leukocyte count of 12.5×10/l, a blood pH level of Eur J Clin Microbiol Infect Dis (2007) 26:147–149 DOI 10.1007/s10096-006-0244-6

Human Bocavirus in Febrile Children, the Netherlands

Human Bocavirus in Febrile Children, the Netherlands

Le bocavirus humain (HboV) un nouveau pathogene respiratoire?

RésuméUn nouveau virus a été très récemment identifié par biologie moléculaire dans des prélèvements respiratoires chez de jeunes enfants. Ce virus est un nouveau représentant de la famille des Parvoviridae et a été classé dans le genre Bocavirus aux côtés du parvovirus bovin (BPV) et du virus minute canin (MCV). Quelques études ont depuis été conduites sur ce virus qui est désormais appelé le bocavirus humain (HboV). Les premières conclusions sur l'épidémiologie de ce nouveau virus révèlent une prévalence assez importante, comprise entre 3 à 10 % des infections respiratoires de l'enfant, une association fréquente avec d'autres virus respiratoires, une répartition mondiale et l'absence de réelle distribution saisonnière. Un seul type viral semble circuler dans la population cible qui apparaît être essentiellement les jeunes enfants. Les données cliniques disponibles ne permettent pas la mise en évidence de spécificité par rapport aux autres viroses respiratoires. A ce jour, les liens de causalité entre la présence de ce nouveau virus découvert par génie moléculaire et une pathologie respiratoire sont loin d'être établis. Le rôle exact du HBoV en pathologie humaine reste donc à confirmer.

Human Bocavirus: Developing Evidence for Pathogenicity

number of individuals without respiratory symptoms were included as controls, and HBoV either was not found or was found very infrequently in this group of individuals. As described in both of these articles, HBoV is a member of the genus Bocavirus and is closely related to parvovirus B19, which also is in thesubfamilyParvovirinae in the family Parvoviridae. Thereareother bocaviruses found in cattle and in dogs; the name“bocavirus”itselfisderivedfrom the combination of “bo” (from “bovine”) and “ca” (from “canine”). One of these bocaviruses, bovine parvovirus, primarily causes diarrhea, and the other, minute virus of canines, causes neonatal respiratory disease and embryopathy. The human virus appears to be very common. In the 9 published studies of HBoV, the virus was detected in 1.5%–11.3% of individuals with acute respiratory illness who had respiratory samples screenedforHBoV,with a frequency of detection of 5.0%–5.5% noted for most of the studies [1–9]. All of the studies but one [5] have found that the virus is most frequently detected in infants !3 years of age. This frequency of detection makes HBoV less common than respiratory syncytial virus and probably also rhinoviruses in infants with respiratory illnesses; approximately as common as influenz viruses, human metapneumovirus, parainfluenz virus type 3, and adenoviruses; and probably more common than coronaviruses and the other parainfluenz viruses. What else do we know about HBoV? The answer is not much yet, even though the genome has been fully sequenced. The firs question we should ask aboutanymicroorganism that is found in the respiratorytractduringillnessiswhetheritcauses any disease. Could it simply be carried asymptomatically in the respiratory tract like, for example, the adenovirus-associated viruses, which are also members of the Parvoviridae family? Finding, in 2 studies, a zero or very low incidence among control infants of the same age, sampled over the same time period in the same hospitals, is a huge advance in this regard, because it provides a statistical association of the virus with disease. It does not, however, prove causality, although it adds important corroborative support.

Human Bocavirus in Hospitalized Children, South Africa

Human Bocavirus in Hospitalized Children, South Africa

Human Bocavirus in French Children

Human bocavirus (HBoV), a new member of the genus Bocavirus in the family Parvoviridae, has been recently associated with respiratory tract infections. We report the epidemiologic and clinical features observed from a 1-year retrospective study of HBoV infection in young children hospitalized with a respiratory tract infection.

Human Bocavirus in French Children

Human bocavirus (HBoV), a new member of the genus Bocavirus in the family Parvoviridae, has been recently associated with respiratory tract infections. We report the epidemiologic and clinical features observed from a 1-year retrospective study of HBoV infection in young children hospitalized with a respiratory tract infection.