Identification of human parvovirus 4 genotypes 1 and 2 in Chinese source plasma pools.

Human parvovirus B19 (B19V) and human parvovirus 4 (PARV4) are known to infect humans and transmit through contaminated blood and blood products. Globally, three genotypes of B19V, as well as PARV4, have been identified, respectively. The existence of different B19V genotypes in Chinese plasma donors has been investigated, however, the data regarding PARV4 were not available. The main objective of this study is to identify the genotypes of PARV4 circulating in Chinese plasma donors. By using a duplex quantitative polymerase chain reaction assay adapted for all genotypes of B19V and PARV4, 78 source plasma pools for fractionation were screened and quantified. Results showed that positive rates of B19V and PARV4 DNA in plasma pool samples were 25.64% and 14.10%, respectively. PARV4 sequences in two positive samples were next genotyped, and these two sequences belonged to PARV4 genotypes 1 and 2, respectively. In conclusion, the data present demonstrate the existence of PARV4 genotypes 1 and 2 in Chinese plasma donors for the first time and also show the relatively lower prevalence and level of PARV4 DNA in Chinese plasma donors in comparison with that of B19V DNA.

Development of a duplex SYBR Green I-based quantitative real-time PCR assay for the rapid differentiation of goose and Muscovy duck parvoviruses.

BACKGROUND: Waterfowl parvoviruses, including goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), can cause seriously diseases in geese and ducks. Developing a fast and precise diagnosis assay for these two parvoviruses is particularly important. RESULTS: A duplex SYBR Green I-based quantitative real-time PCR assay was developed for the simultaneous detection and differentiation of GPV and MDPV. The assay yielded melting curves with specific single peak (Tm = 87.3 ± 0.26 °C or Tm = 85.4 ± 0.23 °C) when GPV or MDPV was evaluated, respectively. When both parvoviruses were assessed in one reaction, melting curves with specific double peaks were yielded. CONCLUSION: This duplex quantitative RT-PCR can be used to rapid identify of GPV and MDPV in field cases and artificial trials, which make it a powerful tool for diagnosing, preventing and controlling waterfowl parvovirus infections.

A duplex PCR assay for the simultaneous detection and differentiation of Muscovy duck parvovirus and goose parvovirus.

Both Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) can cause high mortality and morbidity in Muscovy ducklings. MDPVs and GPVs share high nucleotide identity, which can cause errors during differential diagnosis. In this study, the NS genes of both MDPVs and GPVs were chosen for the design of specific primers after genetic comparison. Only three primers (GF1, MF1 and MGR1) were designed for the duplex PCR assay: GF1 is specific for GPV only; MF1 is specific for MDPV only; and MGR1 is highly conserved for both MDPV and GPV. After a series of optimization experiments, the duplex PCR assay amplified a 161-bp fragment specifically for GPV, a 1197-bp fragment specifically for MDPV, and two fragments (161-bp and 1197-bp) for both GPV and MDPV. The lowest detection limit was 103 copies/µl. No amplification was obtained using nucleic acids from other pathogens (including DAdV-A, DuCV, DEV, GHPV, R.A., E. coli., P.M. and S.S.) occurring in Muscovy ducks. Application of the duplex PCR assay in field samples showed that even one-day-old Muscovy ducklings were both MDPV-positive and GPV-positive. In conclusion, a duplex PCR assay for the simultaneous detection and differentiation of MDPV and GPV was established using only three highly specific primers. Our finding suggested that country-wide vaccination with MDPV and GPV vaccines in waterfowls are necessary.

Detection of adenovirus, papillomavirus and parvovirus in Brazilian bats of the species Artibeus lituratus and Sturnira lilium.

Bats play a significant role in maintaining their ecosystems through pollination, dispersal of seeds, and control of insect populations, but they are also known to host many microorganisms and have been described as natural reservoirs for viruses with zoonotic potential. The diversity of viruses in these animals remains largely unknown, however, because studies are limited by species, location, virus target, or sample type. Therefore, the aim of this study was to detect fragments of viral genomes in bat samples. We performed high-throughput sequencing analysis and specific PCR and RT-PCR on pools of anal and oropharyngeal swabs from Artibeus lituratus and Sturnira lilium collected in southern Brazil. As a result, a member of the family Adenoviridae related to human adenovirus C was detected in anal swabs from S. lilium. In addition, we detected a papillomavirus in an anal swab from A. lituratus. Our analyses also allowed the detection of adenoviruses and parvoviruses in oropharyngeal swabs collected from A. lituratus. These results increase our knowledge about viral diversity and illustrate the importance of conducting virus surveillance in bats.

Novel Parvovirus Related to Primate Bufaviruses in Dogs.

A novel protoparvovirus species, related genetically to human bufaviruses, was identified in dogs with respiratory signs. The canine bufavirus was distantly related to the well-known canine protoparvovirus, canine parvovirus type 2, sharing low amino acid identities in the nonstructural protein 1 (40.6%) and in the capsid protein 1 (33.4%). By screening collections of fecal, nasal, and oropharyngeal samples obtained from juvenile dogs (<1 year of age), canine bufavirus DNA appeared as a common component of canine virome. The virus was common in the stool samples of dogs with or without enteric disease and in the nasal and oropharyngeal swab samples of dogs with respiratory signs. However, the virus was not detected in nasal and oropharyngeal swab samples from animals without clinical signs.

Global Distribution of Human Protoparvoviruses.

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%-5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses.

Comparative genetic analysis and pathological characteristics of goose parvovirus isolated in Heilongjiang, China.

BACKGROUND: Goose parvovirus (GPV) causes acute enteritis, hepatitis, myocarditis and high morbidity and mortality in geese and ducks. GPV H strain was isolated from a Heilongjiang goose farm where the geese were showing signs of hemorrhage in the brain, liver, and intestinal tract. In this study, we explored the genetic diversity among waterfowl parvovirus isolates and the pathological characteristics of GPV H in Shaoxing ducklings. METHODS: The complete capsid protein (VP) and non-structural (NS) sequences of the isolated H strain were sequenced, and phylogenetic trees of VP and NS were constructed in MEGA version 5.05 using the neighbor-joining method. Three-day-old Shaoxing ducklings were inoculated with GPV and were euthanized at 1, 2, 4, 6, and 8 days post-inoculation (PI), and their organs were removed and collected. The organs of 6-day PI ducklings were fixed in formalin, embedded in paraffin, sectioned for histology, stained with HE and analyzed for pathological lesions. The distribution of the GPV H strain in the tissues of the inoculated ducklings was detected using the polymerase chain reaction (PCR) method. RESULTS: Genetic analysis of the NS and VP genes indicated that the H strain was closely related to strains circulating in China during 1999-2014, and the nucleic acid identity of those strains was 98%-99%. Classical symptoms were observed in the inoculated ducklings. GPV remained in many tissues and replicated in a majority of the tissues, leading to histopathological lesions in four tissues. CONCLUSIONS: We first reported the distribution and histopathological lesions of a Chinese strain of GPV in infected shaoxing ducklings. This H strain was moderate pathogenic for Shaoxing ducklings.

Avian parvovirus: classification, phylogeny, pathogenesis and diagnosis.

Poultry parvoviruses identified during the early 1980s are found worldwide in intestines from young birds with enteric disease syndromes as well as healthy birds. The chicken parvovirus (ChPV) and turkey parvovirus (TuPV) belong to the Aveparvovirus genus within the subfamily Parvovirinae. Poultry parvoviruses are small, non-enveloped, single-stranded DNA viruses consisting of three open reading frames, the first two encoding the non-structural protein (NS) and nuclear phosphoprotein (NP) and the third encoding the viral capsid proteins 1 (VP1 and VP2). In contrast to other parvoviruses, the VP1-unique region does not contain the phospholipase A2 sequence motif. Recent experimental studies suggested the parvoviruses to be the candidate pathogens in cases of enteric disease syndrome. Current diagnostic methods for poultry parvovirus detection include PCR, real-time PCR, enzyme linked immunosorbent assay using recombinant VP2 or VP1 capsid proteins. Moreover, sequence-independent amplification techniques combined with next-generation sequencing platforms have allowed rapid and simultaneous detection of the parvovirus from affected and healthy birds. There is no commercial vaccine; hence, the development of an effective vaccine to control the spread of infection should be of primary importance. This review presents the current knowledge on poultry parvoviruses with emphasis on taxonomy, phylogenetic relationship, genomic analysis, epidemiology, pathogenesis and diagnostic methods.

[Multicenter investigation of bufavirus in the etiology of viral central nervous system infections of adults and children]. / Bufavirusun çocuklar ve eriskinlerdeki viral santral sinir sistemi enfeksiyonlarinin etiyolojisi açisindan çok Mmerkezli olarak arastirilmasi.

Bufavirus (BuV) is a newly-identified parvovirus in the family of Parvoviridae. Metagenomic analysis of fecal samples from children in Burkina Faso with acute diarrhea showed a highly divergent parvovirus, which was named bufavirus (BuV). The global distribution, epidemiology and genetic characteristics of BuVs infections are obscure. It was first discovered as an agent causing gastroenteritis but the association of BuV infections with various clinical presentations mostly remain to be explored. The aims of this study were to investigate probable impact of BuV in central nervous system infections in a region where it was previously reported to cause human infections and to detect enteroviruses (EV) which are reported as a cause of central nervous system infections in our country. The study was undertaken in three institutions in Ankara province, Central Anatolia, Turkey. Patients, clinically diagnosed with febrile disease and/or central nervous system infections of presumed viral etiology, were enrolled in the study with informed consent. Cerebrospinal fluid specimens were collected from 93 children attended to Gazi University Hospital and Diskapi Yildirim Beyazit Hospital from October 2011-April 2015 and 33 adult patients, attended to Hacettepe University Hospital from June 2012 to March 2013. Clinical history and follow-up, physical examination and standard laboratory findings of the patients were recorded. Nucleic acid extraction was performed via commercially available spin-column assays and complementery DNA (cDNA) synthesis was performed by using commercially available cDNA synthesis kit with randomised hexamer primers. BuV detection was carried out by in house nested-polymerase chain reaction (PCR) utilized with previously-described primers. EV detection was carried out by in house PCR with pan-enterovirus primers. Seventy-four percent (93/126) and 26% (33/126) of the patients were children (0-18) and adults (19-86), respectively. In all patients, bacterial, mycobacterial and fungal cultures were negative, as well as PCR for herpes simplex virus (HSV) types 1 and 2. PCR results of all samples were negative for BuV and EV. This is the first study that evaluates a probable association of BuV and central nervous system infections. Although Parvovirus B19, a well-characterized human pathogen can rarely cause encephalitis, our findings did not confirm such an association for BuV in this preliminary investigation. However, long-term evaluation of individual cases with unknown etiology is required to reveal the relationship of the virus with specific environments.

Identification of Goose-Origin Parvovirus as a Cause of Newly Emerging Beak Atrophy and Dwarfism Syndrome in Ducklings.

A recent epizootic outbreak, in China, of duck beak atrophy and dwarfism syndrome (BADS) was investigated using electron microscopic, genetic, and virological studies, which identified a parvovirus with a greater similarity to goose parvovirus (GPV) (97% protein homology) than to Muscovy duck parvovirus (MDPV) (90% protein homology). The new virus, provisionally designated GPV-QH15, was found to be antigenically more closely related to GPV than to MDPV in a virus neutralization assay. These findings were further supported by phylogenetic analysis showing that GPV-QH15 evolved from goose lineage parvoviruses, rather than from Muscovy duck- or other duck species-related parvoviruses. In all, two genetic lineages (GPV I and GPV II) were identified from the GPV samples analyzed, and GPV-QH15 was found to be closely clustered with two known goose-origin parvoviruses (GPVa2006 and GPV1995), together forming a distinctive GPV IIa sublineage. Finally, structural modeling revealed that GPV-QH15 and the closely related viruses GPVa2006 and GPV1995 possessed identical clusters of receptor-interacting amino acid residues in the VP2 protein, a major determinant of viral receptor binding and host specificity. Significantly, these three viruses differed from MDPVs and other GPVs at these positions. Taken together, these results suggest that GPV-QH15 represents a new variant of goose-origin parvovirus that currently circulates in ducklings and causes BADS, a syndrome reported previously in Europe. This new finding highlights the need for future surveillance of GPV-QH15 in poultry in order to gain a better understanding of both the evolution and the biology of this emerging parvovirus.

Analysis of the genome sequence of the pathogenic Muscovy duck parvovirus strain YY reveals a 14-nucleotide-pair deletion in the inverted terminal repeats.

Genomic information about Muscovy duck parvovirus is still limited. In this study, the genome of the pathogenic MDPV strain YY was sequenced. The full-length genome of YY is 5075 nucleotides (nt) long, 57 nt shorter than that of strain FM. Sequence alignment indicates that the 5' and 3' inverted terminal repeats (ITR) of strain YY contain a 14-nucleotide-pair deletion in the stem of the palindromic hairpin structure in comparison to strain FM and FZ91-30. The deleted region contains one "E-box" site and one repeated motif with the sequence "TTCCGGT" or "ACCGGAA". Phylogenetic trees constructed based the protein coding genes concordantly showed that YY, together with nine other MDPV isolates from various places, clustered in a separate branch, distinct from the branch formed by goose parvovirus (GPV) strains. These results demonstrate that, despite the distinctive deletion, the YY strain still belongs to the classical MDPV group. Moreover, the deletion of ITR may contribute to the genome evolution of MDPV under immunization pressure.

Genetic and phylogenetic analysis of a novel parvovirus isolated from chickens in Guangxi, China.

A previously unidentified chicken parvovirus (ChPV) strain, associated with runting-stunting syndrome (RSS), is now endemic among chickens in China. To explore the genetic diversity of ChPV strains, we determined the first complete genome sequence of a novel ChPV isolate (GX-CH-PV-7) identified in chickens in Guang Xi, China, and showed moderate genome sequence similarity to reference strains. Analysis showed that the viral genome sequence is 86.4 %-93.9 % identical to those of other ChPVs. Genetic and phylogenetic analyses showed that this newly emergent GX-CH-PV-7 is closely related to Gallus gallus enteric parvovirus isolate ChPV 798 from the USA, indicating that they may share a common ancestor. The complete DNA sequence is 4612 bp long with an A+T content of 56.66 %. We determined the first complete genome sequence of a previously unidentified ChPV strain to elucidate its origin and evolutionary status.

Isolation and characterization of a distinct duck-origin goose parvovirus causing an outbreak of duckling short beak and dwarfism syndrome in China.

Many mule duck and Cherry Valley duck flocks in different duck-producing regions of China have shown signs of an apparently new disease designated "short beak and dwarfism syndrome" (SBDS) since 2015. The disease is characterized by dyspraxia, weight loss, a protruding tongue, and high morbidity and low mortality rates. In order to characterize the etiological agent, a virus designated SBDSV M15 was isolated from allantoic fluid of dead embryos following serial passage in duck embryos. This virus causes a cytopathic effect in duck embryo fibroblast (DEF) cells. Using monoclonal antibody diagnostic assays, the SBDSV M15 isolate was positive for the antigen of goose parvovirus but not Muscovy duck parvovirus. A 348-bp (2604-2951) VP1gene fragment was amplified, and its sequence indicated that the virus was most closely related to a Hungarian GPV strain that was also isolated from mule ducks with SBDS disease. A similar disease was reproduced by inoculating birds with SBDSV M15. Together, these data indicate that SBDSV M15 is a GPV-related parvovirus causing SBDS disease and that it is divergent from classical GPV isolates.

A metagenomic comparison of endemic viruses from broiler chickens with runting-stunting syndrome and from normal birds.

Runting-stunting syndrome (RSS) in broiler chickens is an enteric disease that causes significant economic losses to poultry producers worldwide due to elevated feed conversion ratios, decreased body weight during growth, and excessive culling. Of specific interest are the viral agents associated with RSS which have been difficult to fully characterize to date. Past research into the aetiology of RSS has implicated a wide variety of RNA and DNA viruses however, to date, no individual virus has been identified as the main agent of RSS and the current opinion is that it may be caused by a community of viruses, collectively known as the virome. This paper attempts to characterize the viral pathogens associated with 2-3-week-old RSS-affected and unaffected broiler chickens using next-generation sequencing and comparative metagenomics. Analysis of the viromes identified a total of 20 DNA and RNA viral families, along with 2 unidentified categories, comprised of 31 distinct viral genera and 7 unclassified genera. The most abundant viral families identified in this study were the Astroviridae, Caliciviridae, Picornaviridae, Parvoviridae, Coronaviridae, Siphoviridae, and Myoviridae. This study has identified historically significant viruses associated with the disease such as chicken astrovirus, avian nephritis virus, chicken parvovirus, and chicken calicivirus along with relatively novel viruses such as chicken megrivirus and sicinivirus 1 and will help expand the knowledge related to enteric disease in broiler chickens, provide insights into the viral constituents of a healthy avian gut, and identify a variety of enteric viruses and viral communities appropriate for further study.

Isolation and characterization of a goose parvovirus from Yan goose.

NO ABSTRACT Keywords: goose parvovirus; Yan goose; isolation; characterization.

Novel parvoviruses in reptiles and genome sequence of a lizard parvovirus shed light on Dependoparvovirus genus evolution.

Here, we report the detection and partial genome characterization of two novel reptilian parvoviruses derived from a short-tailed pygmy chameleon (Rampholeon brevicaudatus) and a corn snake (Pantherophis guttatus) along with the complete genome analysis of the first lizard parvovirus, obtained from four bearded dragons (Pogona vitticeps). Both homology searches and phylogenetic tree reconstructions demonstrated that all are members of the genus Dependoparvovirus. Even though most dependoparvoviruses replicate efficiently only in co-infections with large DNA viruses, no such agents could be detected in one of the bearded dragon samples, hence the possibility of autonomous replication was explored. The alternative ORF encoding the full assembly activating protein (AAP), typical for the genus, could be obtained from reptilian parvoviruses for the first time, with a structure that appears to be more ancient than that of avian and mammalian parvoviruses. All three viruses were found to harbour short introns as previously observed for snake adeno-associated virus, shorter than that of any non-reptilian dependoparvovirus. According to the phylogenetic calculations based on full non-structural protein (Rep) and AAP sequences, the monophyletic cluster of reptilian parvoviruses seems to be the most basal out of all lineages of genus Dependoparvovirus. The suspected ability for autonomous replication, results of phylogenetic tree reconstruction, intron lengths and the structure of the AAP suggested that a single Squamata origin instead of the earlier assumed diapsid (common avian-reptilian) origin is more likely for the genus Dependoparvovirus of the family Parvoviridae.

Parvovirus-derived endogenous viral elements in two South American rodent genomes.

We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the genomes of the long-tailed chinchilla (Chinchilla lanigera) and the degu (Octodon degus). The novel EVEs include dependovirus-related elements and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed in vivo, with increased expression in the liver.

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV.

Sesavirus: prototype of a new parvovirus genus in feces of a sea lion.

We describe the nearly complete genome of a highly divergent parvovirus, we tentatively name Sesavirus, from the feces of a California sea lion pup (Zalophus californianus) suffering from malnutrition and pneumonia. The 5,049-base-long genome contained two major ORFs encoding a 553-aa nonstructural protein and a 965-aa structural protein which shared closest amino acid identities of 25 and 28 %, respectively, with members of the copiparvovirus genus known to infect pigs and cows. Given the low degree of similarity, Sesavirus might be considered as prototype for a new genus with a proposed name of Marinoparvovirus in the subfamily Parvovirinae.

Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.