Bovine Parvovirus Research Articles

Structural Insights into Human Bocaparvoviruses.

Bocaparvoviruses are emerging pathogens of the Parvoviridae family. Human bocavirus 1 (HBoV1) causes severe respiratory infections and HBoV2 to HBoV4 cause gastrointestinal infections in young children. Recent reports of life-threatening cases, lack of direct treatment or vaccination, and a limited understanding of their disease mechanisms highlight the need to study these pathogens on a molecular and structural level for the development of therapeutics. Toward this end, the capsid structures of HBoV1, HBoV3, and HBoV4 were determined to a resolution of 2.8 to 3.0 Å by cryo-electron microscopy and three-dimensional image reconstruction. The bocaparvovirus capsids, which display different tissue tropisms, have features in common with other parvoviruses, such as depressions at the icosahedral 2-fold symmetry axis and surrounding the 5-fold symmetry axis, protrusions surrounding the 3-fold symmetry axis, and a channel at the 5-fold symmetry axis. However, unlike other parvoviruses, densities extending the 5-fold channel into the capsid interior are conserved among the bocaparvoviruses and are suggestive of a genus-specific function. Additionally, their major viral protein 3 contains loops with variable regions at their apexes conferring capsid surface topologies different from those of other parvoviruses. Structural comparisons at the strain (HBoV) and genus (bovine parvovirus and HBoV) levels identified differences in surface loops that are functionally important in host/tissue tropism, pathogenicity, and antigenicity in other parvoviruses and likely play similar roles in these viruses. This study thus provides a structural framework to characterize determinants of host/tissue tropism, pathogenicity, and antigenicity for the development of antiviral strategies to control human bocavirus infections.IMPORTANCE Human bocaviruses are one of only a few members of the Parvoviridae family pathogenic to humans, especially young children and immunocompromised adults. There are currently no treatments or vaccines for these viruses or the related enteric bocaviruses. This study obtained the first high-resolution structures of three human bocaparvoviruses determined by cryo-reconstruction. HBoV1 infects the respiratory tract, and HBoV3 and HBoV4 infect the gastrointestinal tract, tissues that are likely targeted by the capsid. Comparison of these viruses provides information on conserved bocaparvovirus-specific features and variable regions resulting in unique surface topologies that can serve as guides to characterize HBoV determinants of tissue tropism and antigenicity in future experiments. Based on the comparison to other existing parvovirus capsid structures, this study suggests capsid regions that likely control successful infection, including determinants of receptor attachment, host cell trafficking, and antigenic reactivity. Overall, these observations could impact efforts to design antiviral strategies and vaccines for HBoVs.

Alternative Polyadenylation of Human Bocavirus at Its 3' End Is Regulated by Multiple Elements and Affects Capsid Expression.

ABSTRACTAlternative processing of human bocavirus (HBoV) P5 promoter-transcribed RNA is critical for generating the structural and nonstructural protein-encoding mRNA transcripts. The regulatory mechanism by which HBoV RNA transcripts are polyadenylated at proximal [(pA)p] or distal [(pA)d] polyadenylation sites is still unclear. We constructed a recombinant HBoV infectious clone to study the alternative polyadenylation regulation of HBoV. Surprisingly, in addition to the reported distal polyadenylation site, (pA)d, a novel distal polyadenylation site, (pA)d2, which is located in the right-end hairpin (REH), was identified during infectious clone transfection or recombinant virus infection. (pA)d2 does not contain typical hexanucleotide polyadenylation signal, upstream elements (USE), or downstream elements (DSE) according to sequence analysis. Further study showed that HBoV nonstructural protein NS1, REH, and cis elements of (pA)d were necessary and sufficient for efficient polyadenylation at (pA)d2. The distance and sequences between (pA)d and (pA)d2 also played a key role in the regulation of polyadenylation at (pA)d2. Finally, we demonstrated that efficient polyadenylation at (pA)d2 resulted in increased HBoV capsid mRNA transcripts and protein translation. Thus, our study revealed that all the bocaviruses have distal poly(A) signals on the right-end palindromic terminus, and alternative polyadenylation at the HBoV 3′ end regulates its capsid expression.IMPORTANCE The distal polyadenylation site, (pA)d, of HBoV is located about 400 nucleotides (nt) from the right-end palindromic terminus, which is different from those of bovine parvovirus (BPV) and canine minute virus (MVC) in the same genus whose distal polyadenylation is located in the right-end stem-loop structure. A novel polyadenylation site, (pA)d2, was identified in the right-end hairpin of HBoV during infectious clone transfection or recombinant virus infection. Sequence analysis showed that (pA)d2 does not contain typical polyadenylation signals, and the last 42 nt form a stem-loop structure which is almost identical to that of MVC. Further study showed that NS1, REH, and cis elements of (pA)d are required for efficient polyadenylation at (pA)d2. Polyadenylation at (pA)d2 enhances capsid expression. Our study demonstrates alternative polyadenylation at the 3′ end of HBoV and suggests an additional mechanism by which capsid expression is regulated.

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.

Inactivation and Disinfection of Porcine Parvovirus on a Nonporous Surface

Animal parvoviruses have historically been accorded status as “highly resistant to inactivation”. This status has been based largely on the well-known heat and chemical inactivation resistance of the animal parvoviruses (especially porcine, canine, bovine, and murine parvoviruses) in liquid inactivation settings. On the other hand, less is known about the relative resistance of parvoviruses to disinfection after being dried on surfaces. In the present article, we evaluate the ability of sodium hypochlorite and two proprietary aldehyde-based disinfectants to inactivate porcine parvovirus (PPV) dried on glass carriers in the presence and absence of varying organic load. Sodium hypochlorite and Microbide-G (a glutaraldehyde-based agent) caused rapid and complete (≥ 3 to 4 log10) inactivation of PPV deposited on glass carriers in a low organic load (5% serum) matrix. Microbide-G displayed the greatest inactivation efficacy for PPV deposited onto a glass surface in a blood matrix. In that case, a contact time of 10 min resulted in 3.5 log10 inactivation at ambient temperature.

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.

A metagenomics and case-control study to identify viruses associated with bovine respiratory disease.

Bovine respiratory disease (BRD) is a common health problem for both dairy and beef cattle, resulting in significant economic loses. In order to identify viruses associated with BRD, we used a metagenomics approach to enrich and sequence viral nucleic acids in the nasal swabs of 50 young dairy cattle with symptoms of BRD. Following deep sequencing, de novo assembly, and translated protein sequence similarity searches, numerous known and previously uncharacterized viruses were identified. Bovine adenovirus 3, bovine adeno-associated virus, bovine influenza D virus, bovine parvovirus 2, bovine herpesvirus 6, bovine rhinitis A virus, and multiple genotypes of bovine rhinitis B virus were identified. The genomes of a previously uncharacterized astrovirus and picobirnaviruses were also partially or fully sequenced. Using real-time PCR, the rates of detection of the eight viruses that generated the most reads were compared for the nasal secretions of 50 animals with BRD versus 50 location-matched healthy control animals. Viruses were detected in 68% of BRD-affected animals versus 16% of healthy control animals. Thirty-eight percent of sick animals versus 8% of controls were infected with multiple respiratory viruses. Significantly associated with BRD were bovine adenovirus 3 (P < 0.0001), bovine rhinitis A virus (P = 0.005), and the recently described bovine influenza D virus (P = 0.006), which were detected either alone or in combination in 62% of animals with BRD. A metagenomics and real-time PCR detection approach in carefully matched cases and controls can provide a rapid means to identify viruses associated with a complex disease, paving the way for further confirmatory tests and ultimately to effective intervention strategies. Bovine respiratory disease is the most economically important disease affecting the cattle industry, whose complex root causes include environmental, genetics, and infectious factors. Using an unbiased metagenomics approach, we characterized the viruses in respiratory secretions from BRD cases and identified known and previously uncharacterized viruses belonging to seven viral families. Using a case-control format with location-matched animals, we compared the rates of viral detection and identified 3 viruses associated with severe BRD signs. Combining a metagenomics and case-control format can provide candidate pathogens associated with complex infectious diseases and inform further studies aimed at reducing their impact.

Sequence Comparison and Phylogenetic Analysis of Several Emerging Porcine Bocaviruses and a Proposal Regarding Nomenclature

The first Bocavirus (bovine parvovirus 1, BPV1) was discovered in calves with diarrhea in 1961. Since then, a series of bocaviruses have emerged in many animals including dogs, humans, swine, gorillas, California sea lions, and cats. These viruses are associated with digestive tract and respiratory tract diseases. Several emerging porcine bocaviruses (PBoVs) have been reported on pig farms in the past four years. However, the nomenclature used to describe these viruses has been confused and irregular. The goal of this study was to compare the sequences of these emerging PBoVs, perform phylogenetic analysis, and present a proposal regarding their nomenclature. Genome sequences of some PBoVs and other bocaviruses were downloaded from the GenBank database. Sequence similarity, sequence mutation, deletion, or insertion, and sequence recombination among reference bocaviruses were obtained using DNAStar software, and its genetic evolution was determined using MEGA 5.1 software. According to the differences in their sequences and genome structure, the nomenclature of PBoVs was proposed and they were divided into four different species (from PBoV1 to PBoV4) and many different genotypes. Among them, both PBoV1 and PBoV2 had two genotypes, PBoV1-a and PBoV1-b and PBoV2-a and PBoV2-a and PBoV2-b, respectively. PBoV3 included three genotypes, PBoV3-a, PBoV3-b and PBoV3-c, respectively. In addition, for PBoV4, there were at least eight genotypes (PBoV4-a to PBoV4-h) in intraspecies. In conclusion, PBoVs were highly genetically diversified. The proposed nomenclature of PBoVs was used in the present studies, it could help us to better understand and standardize the nomenclature of PBoVs.

Use of tangential flow filtration for improving detection of viral adventitious agents in cell substrates

In this study, we assessed the feasibility of tangential flow filtration (TFF) for primary concentration of viral adventitious agents (AAs) from large volumes of cell substrate-derived samples, such as cell-free Chinese hamster ovary (CHO) culture supernatants (500 mL) and CHO cell lysates (50 mL), prior to virus detection in them by nucleic acid-based methods (i.e., qPCR and massively parallel sequencing (MPS). The study was conducted using the samples spiked with four model DNA viruses (bovine herpesvirus type 4, human adenovirus type 5, simian polyomavirus SV-40, and bovine parvovirus). The results showed that the combined TFF/MPS approach enables reliable detection of as low as 1000 genome equivalents (GE) of each of the four viruses spiked into the cell substrate samples. The final achieved sensitivities of 2 GE/mL for cell culture supernatant and 20 GE/mL for cell lysate make this approach more sensitive than virus-specific PCR and qPCR assays. The study results allowed us to propose that TFF might be useful and valuable method for simple and rapid concentration of potential AAs in cell substrate samples prior to AAs detection by conventional in vivo, in vitro, or molecular methods.

Viral infection risks for patients using the finished product Hirudo verbana (medicinal leech).

The virological safety of medicinal leeches has to be ensured prior to their use on patients. While leeches can be kept and bred under standardized conditions, feeding them horse blood adds a non-standardized component, which poses some risk of infection of the treated patients. Here, we investigated the speed at which blood-borne viruses are degraded by the microbial flora in the leech intestine, in order to define the safety of the product and the length of the necessary quarantine period prior to its administration to patients. Feeding blood was spiked with bovine viral diarrhea virus (BVDV), reovirus, and murine parvovirus (10(7) ID₅₀ ml(-1)). The virus titer in the intestinal contents of the leeches was determined using permissive cell cultures and compared to that of the original virus titer at the following time points: immediately after feeding; after 3, 14, and 30 days; and monthly thereafter until the 7th month. The BVDV titer was below the detection limit of 10(1) TCID₅₀ ml(-1) after 3 months, while reovirus and murine parvovirus titers were undetectable after 4 months. No positive virus findings were obtained at later time points. Thus, when fed the blood of vertebrates, the finished product "Medicinal leech, Hirudo verbana" can be considered virologically safe if the animals are maintained at 20 °C, which corresponds to their natural habitat conditions and ensures a high metabolic rate. Therefore, after the last feeding, a quarantine period of 4-6 months and appropriate care at room temperature, which supports microbial degradation and digestive processes, are recommended.

Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture

Fetal bovine serum (FBS) and trypsin are reagents used in cell culture and have been the source of viral contamination of pharmaceutical products. We performed high throughput sequencing (HTS) of two pools of commercial batches of FBS and three commercial batches of trypsin. Taxonomies were assigned by comparing sequences of contigs and singletons to the entire NCBI nucleic acid and protein databases. The same major viral species were evidenced between batches of a given reagent but the proportion of viral reads among total reads varied markedly between samples (from 0.002% to 22.7%). In FBS, the sequences found were mainly from bovine viral diarrhea virus (BVDV) 1 to 3 and bovine parvovirus 3 (BPV3). The BVDV sequences derived from FBS showed only minor discrepancies with primers generally used for the screening of BVDV. Viral sequences in trypsin were mainly from porcine circovirus type 2. Other known viral sequences at lower read counts and potential new viral species (bovine parvovirus and bovine pegivirus) were evidenced. The load of some known and new viruses detected by HTS could be quantified by qPCR. Results of HTS provide a framework for evaluating the pertinence of control measures including the design of PCRs, bioassays and inactivation procedures.

БИОЛОГИЧЕСКИЕ СВОЙСТВА ПАРВОВИРУСА И ЕГО РОЛЬ В ПАТОЛОГИИ КРУПНОГО РОГАТОГО СКОТА (обзор)

Basing on analysis of extensive investigations in many countries, the properties and prevalence of bovine parvovirus are described. The isolates of bovine parvovirus I from the United States, Europe, North America, Africa, and Australia are identical on antigenic properties, but differ from other animal and human parvoviruses. Japanese isolates which differ from bovine parvovirus I on antigenic patterns are referred to the bovine parvovirus II. Bovine parvovirus can agglutinate the human and Guinea pig red cells. This is a DNA-containing virus with the virion size of 23 nm. Virus replication and the assembling occur in the cell nucleus. The highest titеrs (10 6.75-10 8.00 TCID 50/ml) are produced by parvovirus I cultivation in primary cell cultures of lung, kidney, spleen, testis and adrenals from cattle embryos. To isolate parvovirus I, the cattle fetal serum free of specific antibodies must be used. Oral or intravenous infection of the newborn calves or the calves who were not fed with сolostrum resultes in the diarrhea syndrome within 24-48 hours. The virus is commonly found in the intestines, blood leukocytes, lymph nodes, spleen, thymus and in the adrenal cells. In cows it persists for a long time and penetrates through the placental barrier causing abortions and embryo mortality. The virus was found in the thymus, lungs and spleen of mummified fetuses. The rate of seropositive animals in different herds in the UK, Australia, Germany, Austria, Italy, Algeria, etc. varies from 14 to 100 %. According to our data, 26.6 to 58.4 % of pregnant cows and calves from nine regions of the Middle Volga are positive on antibodies to parvovirus in ELISA. This percentage was higher in the herds with imported cattle. Parvovirus is involved in mixed respiratory and intestinal infections in calves and the reproduction abnormalities in adult cattle. To control the infections caused by bovine parvovirus in the national cattle husbandry, the enzyme immunoassay (EIA) diagnostic kit and the inactivated associated vaccine with a wide range of specific activity have been developed. The patented EIA test meets the international standards, and the vaccine is also patentable.

Characterization of a Novel Porcine Parvovirus Tentatively Designated PPV5

A new porcine parvovirus (PPV), provisionally designated as PPV5, was identified in U.S. pigs. Cloning and sequencing from a circular or head-to-tail concatemeric array revealed that the PPV5 possesses the typical genomic organization of parvoviruses with two major predicted open reading frames (ORF1 and ORF2), and is most closely related to PPV4 with overall genomic identities of 64.1–67.3%. The amino acid identities between PPV5 and PPV4 were 84.6%–85.1% for ORF1 and 54.0%–54.3% for ORF2. Unlike PPV4, but similar to bovine parvovirus 2 (BPV2), PPV5 lacks the additional ORF3 and has a much longer ORF2. Moreover, the amino acid sequences of ORF1 and ORF2 of BPV2 showed higher homologies to PPV5 than to PPV4. The conserved motifs of the Ca2+ binding loop (YXGXG) and the catalytic center (HDXXY) of phospholipase A2 (PLA2) were identified in VP1 (ORF2) of PPV5, as well as in BPV2, but were not present in PPV4. Phylogenetic analyses revealed that PPV5, PPV4 and BPV2 form a separate clade different from the genera Parvovirus and Bocavirus. Further epidemiologic investigations of PPV4 and PPV5 in U.S. pigs of different ages indicated a slightly higher prevalence for PPV5 (6.6%; 32/483) compared to PPV4 (4.1%; 20/483), with detection of concurrent PPV4 and PPV5 in 15.6% (7/45) of lungs of infected pigs. Evidence for potential vertical transmission or association with reproductive failure was minimal for both PPV4 and PPV5. The high similarity to PPV4 and the lack of ORF3 may suggest PPV5 is an intermediate of PPV4 during the evolution of parvoviruses in pigs.

Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1

Human bocavirus (HBoV), closely related to canine minute virus (MVC) and bovine parvovirus (BPV), is a new member of the Bocavirus genus within the Parvoviridae family. The non-structural protein NP1 of HBoV is a nuclear localized protein and plays an important role in DNA replication as well as in the evasion of host innate immunity. In the current study, we provide the first evidence that NP1 possesses a non-classical nuclear localization signal (ncNLS) (amino acids 7-50). Embedded within this ncNLS is a classical bipartite nuclear localization signal (cNLS) (amino acids 14-28), capable of transporting a heterologous cytoplasmic protein β-galactosidase fusion protein (β-gal-EGFP) to the nucleus via the classical importin α/β1-mediated pathway. Amino acids 7-50 containing the cNLS and the ncNLS of NP1 or full-length NP1 interact with importin α1, importin β1 and importin β1Δ, which lacks the importin α binding domain, indicating that the nuclear import of NP1 is through both conventional importin α/β1 heterodimer- and non-classical importinß1-mediated pathways. Given that the arrangement of a cNLS embedded within an ncNLS is unusual in viral proteins, our data together reveal a novel molecular mechanism underlying the nuclear import of HBoV NP1, providing a basis for further understanding its biological function.

소 가죽 유래 Type I Collagen 생산 공정에서 바이러스 불활화

세포치료제 또는 조직공학제제에 사용되는 동물 유래 콜라겐은 원료물질 유래 바이러스가 오염될 가능성이 있기 때문에 생산과정 중 바이러스가 오염되지 않도록 하여야 한다. 이를 위해 콜라겐 생산공정은 오염될 가능성이 있는 바이러스들을 불활화 하거나 제거하는 과정을 포함하여야 하며, 바이러스 불활화/제거 능력은 제품의 안전성을 보증하는 중요한 지표로 사용된다. 본 연구의 목적은 소 가죽을 원료로 하여 type I 콜라겐을 생산하는 공정에서 소 유래 바이러스들의 불활화/제거 효능을 평가하는 데 있다. 이를 위해 70% 에탄올 처리 공정과 펩신 처리 공정(pH 2)에서 바이러스 불활화 효과를 평가하였다. 바이러스 불활화 효과 평가를 위해 bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza 3 virus (BPIV-3), bovine parvovirus (BPV)를 모델 바이러스로 선정하였다. 바이러스 불활화를 위해 24시간 동안 70% 에탄올을 처리하는 공정에서 BHV, BVDV, BPIV-3, BPV 모두 처리 1시간 안에 검출 한계 이하로 불활화되었으며, 바이러스 로그 감소 값은 각각 <TEX>${\geq}5.58$</TEX>, <TEX>${\geq}5.32$</TEX>, <TEX>${\geq}5.11$</TEX>, <TEX>${\geq}3.42$</TEX>이었다. 또한 소 조직으로부터 콜라겐을 추출하기 위한 14일간의 펩신 처리 공정에서 BHV, BVDV, BPIV-3, BPV 모두 처리 5일 안에 검출한계 이하로 불활화되었으며, 바이러스 로그 감소 값은 각각 <TEX>${\geq}7.08$</TEX>, <TEX>${\geq}6.60$</TEX>, <TEX>${\geq}5.60$</TEX>, <TEX>${\geq}3.59$</TEX>이었다. 두 공정에서 BHV, BVDV, BPIV-3, BPV의 누적 바이러스 로그 감소 값은 각각 <TEX>${\geq}12.66$</TEX>, <TEX>${\geq}11.92$</TEX>, <TEX>${\geq}10.71$</TEX>, <TEX>${\geq}7.01$</TEX>이었다. 이상의 결과에 의하면, 소 가죽 유래 type I 콜라겐제조공정은 바이러스 안전성 보증을 위한 충분한 바이러스 불활화 능력을 가지고 있는 것으로 판단된다. Most types of collagen used for biomedical applications, such as cell therapy and tissue engineering, are derived from animal tissues. Therefore, special precautions must be taken during the production of these proteins in order to assure against the possibility of the products transmitting infectious diseases to the recipients. The ability to remove and/or inactivate known and potential viral contaminants during the manufacturing process is an ever-increasingly important parameter in assessing the safety of biomedical products. The purpose of this study was to evaluate the efficacies of the 70% ethanol treatment and pepsin treatment at pH 2.0 for the inactivation of bovine viruses during the manufacture of collagen type I from bovine hides. A variety of experimental model viruses for bovine viruses including bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), bovine parainfluenza 3 virus (BPIV-3), and bovine parvovirus (BPV), were chosen for the evaluation of viral inactivation efficacy. BHV, BVDV, BPIV-3, and BPV were effectively inactivated to undetectable levels within 1 h of 70% ethanol treatment for 24 h, with log reduction factors of <TEX>${\geq}5.58$</TEX>, <TEX>${\geq}5.32$</TEX>, <TEX>${\geq}5.11$</TEX>, and <TEX>${\geq}3.42$</TEX>, respectively. BHV, BVDV, BPIV-3, and BPV were also effectively inactivated to undetectable levels within 5 days of pepsin treatment for 14 days, with the log reduction factors of <TEX>${\geq}7.08$</TEX>, <TEX>${\geq}6.60$</TEX>, <TEX>${\geq}5.60$</TEX>, and <TEX>${\geq}3.59$</TEX>, respectively. The cumulative virus reduction factors of BHV, BVDV, BPIV-3, and BPV were <TEX>${\geq}12.66$</TEX>, <TEX>${\geq}11.92$</TEX>, <TEX>${\geq}10.71$</TEX>, and <TEX>${\geq}7.01$</TEX>. These results indicate that the production process for collagen type I from bovine hides has a sufficient virus-reducing capacity to achieve a high margin of virus safety.

Inactivation of enveloped and non‐enveloped viruses in the process of chemical treatment and gamma irradiation of bovine‐derived grafting materials

Xenografts, unlike other grafting products, cannot be commercialized unless they conform to stringent safety regulations. Particularly with bovine-derived materials, it is essential to remove viruses and inactivate infectious factors because of the possibility that raw materials are imbrued with infectious viruses. The removal of the characteristics of infectious viruses from the bovine bone grafting materials need to be proved and inactivation process should satisfy the management provision of the Food and Drug Administration (FDA). To date, while most virus inactivation studies were performed in human allograft tissues, there have been almost no studies on bovine bone. To evaluate the efficacy of virus inactivation after treatment of bovine bone with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, we selected a variety of experimental model viruses that are known to be associated with bone pathogenesis, including bovine parvovirus (BPV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza-3 virus (BPIV-3). The cumulative virus log clearance factor or cumulative virus log reduction factor for the manufacturing process was obtained by calculating the sum of the individual virus log clearance factors or log reduction factors determined for individual process steps with different physicochemical methods. The cumulative log clearance factors achieved by three different virus inactivation processes were as follows: BPV ≥ 17.73, BHV ≥ 20.53, BVDV ≥ 19.00, and BPIV-3 ≥ 16.27. On the other hand, the cumulative log reduction factors achieved were as follows: BPV ≥ 16.95, BHV ≥ 20.22, BVDV ≥ 19.27, and BPIV-3 ≥ 15.58. Treatment with 70% ethanol, 4% sodium hydroxide, or gamma irradiation was found to be very effective in virus inactivation, since all viruses were at undetectable levels during each process. We have no doubt that application of this established process to bovine bone graft manufacture will be effective and essential.

Host range of Canine minute virus in cell culture

Canine minute virus (CnMV) is a member of the Bocavirus genus, together with Bovine parvovirus (BPV), which emerged as a new pathogen of dogs in 1967. The genus Bocavirus gained more recognition after the recent identification of Human bocavirus in pooled specimens from the respiratory tract of children. The cell culture host range of CnMV appears to be restricted to the Walter Reed canine cell (WRCC) line, although there is a report that indicates susceptibility of the Madin-Darby canine kidney (MDCK) cell line. In order to study the susceptibility of different cell lines to CnMV, the replication in cell cultures of canine, bovine, and human origin and the interaction of the virus with freshly isolated canine peripheral blood mononuclear cells were evaluated. The in vitro host range was unexpectedly wide. As shown by indirect fluorescent antibody and polymerase chain reaction assays, CnMV replicates efficiently in the A72 and MDCK canine cell lines. Bovine and human cell lines support the replication of CnMV less efficiently. In contrast, canine mononuclear blood cells are permissive to replication of CnMV in vitro. The present study revealed the ability of CnMV to replicate in continuous cell lines of different origin and, partially, in canine mononuclear cells.

Development of a loop-mediated isothermal amplification assay for rapid detection of bovine parvovirus

A loop-mediated isothermal amplification (LAMP) assay was developed for detection of bovine parvovirus (BPV) DNA. Four primers were designed to recognize six distinct regions on the target DNA based on a highly conserved sequence in the VP2 region of the BPV genome. The optimized LAMP reaction conditions were 8mM Mg2+, 1.2mM betaine, and an incubation at 63°C for 45min. After amplification the products were detected either by observing a ladder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. The detection limit of the LAMP assay was 9 copies of BPV-DNA and was 100 times more sensitive than conventional PCR. A ladder pattern of bands after gel electrophoresis was observed for only BPV isolates and showed that the BPV LAMP assay was highly specific without any cross-reactivity with other related viruses. The LAMP assay was evaluated further using 59 field samples and the results were comparable to conventional PCR. The LAMP assay is a simple, rapid and economic detection method; it can provide a useful technique suitable for detection of BPV infection in both field conditions and laboratory settings.

ELISA test system for serological diagnosis of bovine parvovirus infection and determination of postvaccinal antibody level

An ELISA test system was developed on the basis of using bovine parvovirus type I intended for revealing antibodies in bovine blood serum in sick, convalescent, and vaccinated animals.

Genomic Features of the Human Bocaviruses

The human bocavirus (HBoV) was initially discovered in 2005 as the second pathogenic member of the parvovirus family, next to the human parvovirus B19. HBoV has since been shown to be extremely common worldwide and to cause a systemic infection in small children often resulting in respiratory disease. Three more, presumably enteric, human bocaviruses (HBoV2-4) have been identified in stool samples. Parvoviruses are assumed to replicate via their genomic terminal hairpin-like structures in a so-called 'rolling-hairpin model'. These terminal sequences have recently been partially identified in head-to-tail HBoV-PCR amplicons from clinical samples, and are most likely hybrid relics of HBoV's predecessors, namely bovine parvovirus 1 on the left-hand side and minute virus of canines on the right, shown for the first time in this article. Thereby, the replication model postulated for HBoV remains questionable as the occurrence of head-to-tail sequences is not a typical feature of the rolling-hairpin replication model. However, such episomes can also be persistent storage forms of the genome.

Seroepidemiology of Human Bocaviruses 1–4

Recently, 3 new members of the genus Bocavirus, human bocavirus 2 (HBoV2), human bocavirus 3 (HBoV3), and human bocavirus 4 (HBoV4), were discovered. HBoV2-4 occur mainly in the gastrointestinal tract but rarely in the respiratory tract, contrary to human bocavirus 1 (HBoV1). To investigate HBoV1-4 seroepidemiology among 195 adults and 252 wheezing children, we conducted immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme immunoassays with recombinant viruslike particles (VLPs). The children's sera were also tested for HBoV1-4 DNA by quantitative polymerase chain reaction (qPCR). Both rabbit and human antibodies to HBoV1-4 VP2 VLPs were found to be cross-reactive. After depletion of HBoV1-reactive antibodies, the HBoV2-4 approximate seroprevalences in adults were 34%, 15%, and 2% and in children aged 1-2 years 25%, 10%, and 5%, respectively. After depletion of HBoV2-4-reactive antibodies, the HBoV1 seroprevalence among adults decreased from 96% to 59%. No cross-reactivity of human anti-HBoV IgG was observed with bovine parvovirus1, parvovirus B19 or PARV4. No child was HBoV2-4 viremic. HBoV2-4 infect humans less commonly and elicit weaker B-cell responses than HBoV1. In our study HBoV2-4 did not seem to have a major etiological role in wheezing. Cross-reactivity with HBoV2-4 IgG partially accounts for the high HBoV1 seroprevalences previously reported. Correction for cross-reactivity is a prerequisite for VLP-based HBoV seroepidemiology.