Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China.

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

The XVIII International Parvovirus Workshop Rimini, Italy, 14-17 June 2022.

The XVIII International Parvovirus Workshop took place in Rimini, Italy, from 14 to 17 June 2022 as an on-site event, continuing the series of meetings started in 1985 and continuously held every two years. The communications dealt with all aspects of research in the field, from evolution and structure to receptors, from replication to trafficking, from virus-host interactions to clinical and veterinarian virology, including translational issues related to viral vectors, gene therapy and oncolytic parvoviruses. The oral communications were complemented by a poster exhibition available for view and discussion during the whole meeting. The XVIII International Parvovirus Workshop was dedicated to the memory of our dearest colleague Mavis Agbandje-McKenna (1963-2021).

Deciphering the Origin of DNA Viruses (Replication-Associated Parvo-NS1) That Infect Vertebrates from Invertebrate-Infecting Viruses.

DNA replication is a standard and essential function among DNA viruses; however, this functional domain's common ancestor, origin, and evolutionary path in invertebrate- and vertebrate-infecting viruses are not yet fully understood. Here, we present evidence, using a combination of phylogenetic relationships, coevolution, and CLANS (cluster analysis of sequences) analysis, that the parvo-NS1 domain (nonstructural protein NS1, DNA helicase domain) of these DNA viruses that infect vertebrates potentially originated from the invertebrate (Platyhelminthes) parvo-NS1 domain of parvovirus-related sequences (PRSs). Our results suggest that papillomaviruses and the parvovirus subfamilies Densovirinae and Hamaparvovirinae DNA helicase evolved directly from the Platyhelminthes NS1 domain (PRSs). Similarly, the parvovirus subfamily Parvovirinae NS1 domain displayed evolutionary heritage from the PRSs through Hamaparvovirinae. Further, our analysis also clarified that herpesviruses and adenoviruses independently obtained the parvo-NS1 domain from Dependoparvovirus (Parvovirinae). Furthermore, virus-host coevolution analysis revealed that the parvovirus NS1 domain has coevolved with hosts, from flatworms to humans, and it appears that the papillomavirus may have obtained the DNA helicase during the early stages of parvovirus evolution and later led to the development of the DNA helicase of adomavirus and polyomavirus. Finally, herpesviruses and adenoviruses likely inherited the parvo-NS1 domain from Dependoparvovirus in the later stages of evolution. To the best of our knowledge, this is the first evolutionary evidence to suggest that the DNA helicase of viruses that infect vertebrates originated from the invertebrate PRSs. IMPORTANCE DNA replication of DNA viruses is an essential function. This allows DNA replication of viruses to form virus particles. The DNA helicase domain is responsible for this primary function. This domain is present in parvoviruses, papillomaviruses, polyomaviruses, herpesviruses, and adenoviruses. But little is known about the common ancestor, origin, and evolutionary path of DNA helicase in invertebrate- and vertebrate-infecting viruses. Here, we report the possibility of the origin of DNA viruses (DNA helicase) infecting vertebrates from Platyhelminthes (invertebrate) PRSs. Our study established that the parvovirus subfamily Parvovirinae NS1 domain displayed evolutionary heritage from the Platyhelminthes PRSs through Hamaparvovirinae. Furthermore, our study suggests that the papillomavirus DNA helicase may have evolved in the early stages of parvovirus evolution and then led to the development of the adomavirus and polyomavirus. Our study suggests that the herpesviruses and adenoviruses likely inherited the parvo-NS1 domain through gene capture from Dependoparvovirus in the later stages of parvovirus evolution in their hosts.

Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion.

The oncolytic autonomous parvovirus Minute Virus of Mice (MVM) establishes infection in the nuclear environment by usurping host DNA damage signaling proteins in the vicinity of cellular DNA break sites. MVM replication induces a global cellular DNA Damage Response (DDR) that is dependent on signaling by the ATM kinase and inactivates the cellular ATR-kinase pathway. However, the mechanism of how MVM generates cellular DNA breaks remains unknown. Using single molecule DNA Fiber Analysis, we have discovered that MVM infection leads to a shortening of host replication forks as infection progresses, as well as induction of replication stress prior to the initiation of virus replication. Ectopically expressed viral non-structural proteins NS1 and NS2 are sufficient to cause host-cell replication stress, as is the presence of UV-inactivated non-replicative MVM genomes. The host single-stranded DNA binding protein Replication Protein A (RPA) associates with the UV-inactivated MVM genomes, suggesting MVM genomes might serve as a sink for cellular stores of RPA. Overexpressing RPA in host cells prior to UV-MVM infection rescues DNA fiber lengths and increases MVM replication, confirming that MVM genomes deplete RPA stores to cause replication stress. Together, these results indicate that parvovirus genomes induce replication stress through RPA exhaustion, rendering the host genome vulnerable to additional DNA breaks.

Herpesviruses, polyomaviruses, parvoviruses, papillomaviruses, and anelloviruses in vestibular schwannoma.

Etiology of vestibular schwannoma (VS) is unknown. Viruses can infect and reside in neural tissues for decades, and new viruses with unknown tumorigenic potential have been discovered. The presence of herpesvirus, polyomavirus, parvovirus, and anellovirus DNA was analyzed by quantitative PCR in 46 formalin-fixed paraffin-embedded VS samples. Five samples were analyzed by targeted next-generation sequencing. Viral DNA was detected altogether in 24/46 (52%) tumor samples, mostly representing anelloviruses (46%). Our findings show frequent persistence of anelloviruses, considered normal virome, in VS. None of the other viruses showed an extensive presence, thereby suggesting insignificant role in VS.

Design and Characterization of Mutated Variants of the Oncotoxic Parvoviral Protein NS1.

Oncotoxic proteins such as the non-structural protein 1 (NS1), a constituent of the rodent parvovirus H1 (H1-PV), offer a novel approach for treatment of tumors that are refractory to other treatments. In the present study, mutated NS1 variants were designed and tested with respect to their oncotoxic potential in human hepatocellular carcinoma cell lines. We introduced single point mutations of previously described important residues of the wild-type NS1 protein and a deletion of 114 base pairs localized within the N-terminal domain of NS1. Cell-viability screening with HepG2 and Hep3B hepatocarcinoma cells transfected with the constructed NS1-mutants led to identification of the single-amino acid NS1-mutant NS1-T585E, which led to a 30% decrease in cell viability as compared to NS1 wildtype. Using proteomics analysis, we could identify new interaction partners and signaling pathways of NS1. We could thus identify new oncotoxic NS1 variants and gain insight into the modes of action of NS1, which is exclusively toxic to human cancer cells. Our in-vitro studies provide mechanistic explanations for the observed oncolytic effects. Expression of NS1 variants had no effect on cell viability in NS1 unresponsive control HepG2 cells or primary mouse hepatocytes. The availability of new NS1 variants in combination with a better understanding of their modes of action offers new possibilities for the design of innovative cancer treatment strategies.

Rapid sample preparation and low-resource molecular detection of hepatopancreatic parvoviruses (HPV) by recombinase polymerase amplification lateral flow detection assay in shrimps (Fenneropenaeus merguiensis).

BACKGROUND: Viral diseases are a major problem in shrimp aquaculture facilities as these diseases reduce growth rates, which inevitably lead to production and profit losses. Hepatopancreatic parvoviruses (HPV) are common diseases in shrimp that appear to be associated with high or low levels of replication in specific genetic lineages. Selective breeding may result in resistance to HPV and improved body traits such as body weight, meat yield and shrimp colour, facilitating shrimp farming. HPV virus titre is commonly determined by quantitative PCR (qPCR), which is a time-consuming method requiring laboratory equipment unsuitable for field implementation. The aim of this study was to develop a simple, robust, rapid and reliable method to detect HPV in low-resource environments. METHODS: We developed a rapid shrimp HPV test that uses (1) a simple three-step sample preparation protocol, followed by (2) isothermal recombinase polymerase amplification (RPA) and lateral flow strip detection (LFD). Analytical sensitivity testing was performed in a background banana shrimp sample matrix, and retrospective testing of Fenneropenaeus merguiensis hepatopancreas tissues (n = 33) with known qPCR viral titres was used to determine diagnostic sensitivity and specificity. RESULTS: The rapid shrimp HPV test could detect as little as 35 genome-equivalent copies per reaction in homogenized F. merguiensis banana shrimp. Retrospective testing of stored tissues (n = 33) indicated 100% diagnostic sensitivity (95% confidence interval, CI: 86-100%) and 100% specificity (95% CI: 66-100%) for detection of HPV. CONCLUSION: The rapid shrimp HPV test could be completed in only 40 minutes, and required only homogenization pestles, some pipettors, and a small heating block for single temperature incubation at 39°C. Critically, our procedure eliminated the time-consuming purification of nucleic acids from samples and when combined with RPA-LFD offers a user-friendly HPV detection format that can potentially be performed on-site. Our approach represents a major step forward in the development of a simple and sensitive end-point method for quick determination of unfavourable HPV virus numbers in shrimp, and has great potential to advance on-site management of shrimps in aquaculture.

Comparative analysis reveals the long-term coevolutionary history of parvoviruses and vertebrates.

Parvoviruses (family Parvoviridae) are small DNA viruses that cause numerous diseases of medical, veterinary, and agricultural significance and have important applications in gene and anticancer therapy. DNA sequences derived from ancient parvoviruses are common in animal genomes and analysis of these endogenous parvoviral elements (EPVs) has demonstrated that the family, which includes twelve vertebrate-specific genera, arose in the distant evolutionary past. So far, however, such "paleovirological" analysis has only provided glimpses into the biology of ancient parvoviruses and their long-term evolutionary interactions with hosts. Here, we comprehensively map EPV diversity in 752 published vertebrate genomes, revealing defining aspects of ecology and evolution within individual parvovirus genera. We identify 364 distinct EPV sequences and show these represent approximately 200 unique germline incorporation events, involving at least five distinct parvovirus genera, which took place at points throughout the Cenozoic Era. We use the spatiotemporal and host range calibrations provided by these sequences to infer defining aspects of long-term evolution within individual parvovirus genera, including mammalian vicariance for genus Protoparvovirus, and interclass transmission for genus Dependoparvovirus. Moreover, our findings support a model of virus evolution in which the long-term cocirculation of multiple parvovirus genera in vertebrates reflects the adaptation of each viral genus to fill a distinct ecological niche. Our findings show that efforts to develop parvoviruses as therapeutic tools can be approached from a rational foundation based on comparative evolutionary analysis. To support this, we published our data in the form of an open, extensible, and cross-platform database designed to facilitate the wider utilisation of evolution-related domain knowledge in parvovirus research.

Shrimp genome sequence contains independent clusters of ancient and current Endogenous Viral Elements (EVE) of the parvovirus IHHNV.

BACKGROUND: Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a genome sequence (GenBank record JABERT000000000) of the giant tiger shrimp (Penaeus monodon for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). RESULTS: The shrimp genome sequence contained three piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in pseudochromosome 35 (PC35). Both PC35 clusters contained multiple sequences with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called "non-infectious IHHNV Type A" (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two PC35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in pseudochromosome 7 (PC7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against current types of infectious IHHNV. One disadvantage was that some EVE in PC7 can give false positive PCR test results for infectious IHHNV. CONCLUSIONS: Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important because whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against the disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus.

Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (>99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.

Co-circulation of five species of dog parvoviruses and canine adenovirus type 1 among gray wolves (Canis lupus) in northern Canada.

Several viruses can infect wild carnivores but their impact on wildlife health is poorly understood. We investigated the presence, diversity and distribution of various DNA viruses in 303 wolves inhabiting a vast area of the Northwest Territories, Canada, over a period of 13 years. We found evidence for the presence of canine bufavirus (CBuV, 42.6%), canine parvovirus 2 (CPV-2, 34.0%), canine bocavirus 2 (CBoV-2, 5.0%), cachavirus (CachaV-1, 2.6%), canine adenovirus 1 (CAdV-1, 1%) and minute virus of canines (MVC, 0.3%). To our knowledge, this is the first detection of CBoV-2, MVC and CachV-1 in wild animals. We also demonstrate that CBuV and CachaV-1 were already circulating among wild animals at least 11 and 10 years, respectively, before their discoveries. Although CBuV prevalence was higher, CPV-2 was the most prevalent virus among juveniles, while CBuV infection was associated with poor nutrition conditions. Even if its prevalence was low, CachaV-1 had the highest multiple infection rate (87.5%). CadV-1 and MVC sequences were highly identical to reference strains, but we observed a high diversity among the other viruses and detected three new variants. One CPV-2 variant and one CBuV variant were endemic since the beginning of the 2000s in the entire investigated region, whereas one CBuV variant and two CBoV-2 variants were found in a more restricted area over multiple years and CachaV-1 was found only in one region. Two CPV-2 variants and one CachaV-1 variant were observed only once, indicating sporadic introductions or limited circulation. Different patterns of endemicity might indicate that viruses were introduced in the wolf population at different timepoints and that mixing between wolf packs may not be constant. Different epidemiological behaviors depend on viral factors like infectivity, transmission routes, pathogenicity and tissue-tropism, and on host factors like proximity to densely populated areas, carnivory and pack density and mixing.

Characterizing two novel goose parvoviruses with different origins.

Outbreaks of short beak dwarf syndrome caused by novel goose parvovirus (NGPV) have been prevalent in China since 2015, resulting in a high mortality rate of ducks. Herein we evaluated differences between two NGPV strains: Muscovy duck-origin (AH190917-RP: MD17) and Cherry Valley duck-origin (JS191021-RP: CVD21) NGPV. Both of them showed certain level of pathogenicity to primary duck embryo fibroblasts, Cherry Valley duck embryos and ducklings. CVD21 showed comparatively stronger pathogenicity than MD17. Only CVD21 caused obvious cytopathic effect (CPE), characterized by cell shedding; further, the virus titer of MD17 and CVD21 was 102.571 ELD50 (i.e. median embryo lethal dose)/0.2 ml and 106.156 ELD50 /0.2 ml, respectively, and the mortality rate of CVD21- and MD17-infected Cherry Valley ducklings was 100% and 80%, respectively. In addition, CVD21 had a greater influence on the growth and development of ducklings. Furthermore, we found that MD17 could infect Muscovy duck embryos and produce lesions similar to Cherry Valley duck embryos, but it could not infect Muscovy duck embryo fibroblasts (MDEFs,) and Muscovy ducklings. MDV21 had no infection to MDEFs, Muscovy duck embryo and Muscovy ducklings. We then sequenced the complete genome of the two isolates to enable genomic characterization. The complete genome of MD17 and CVD21 was 5046 and 5050 nucleotides in length, respectively. Nucleotide alignment, amino acid analysis and phylogenetic tree analysis revealed that MD17 showed higher homology to goose parvovirus (GPV), while CVD21 demonstrated stronger similarity with NGPV. Moreover, the two isolates shared 95.8% homology, with encoded proteins showing multiple amino acid variations. Our findings indicate that Muscovy ducks seem to have played a crucial role in the evolution of GPV to NGPV. We believe that our data should serve as a foundation for further studying the genetic evolution of waterfowl parvoviruses and their pathogenic mechanisms.

Molecular Detection of Parvovirus in Manchurian Chipmunks (Tamias sibiricus asiaticus) Captured in Korea.

Cross-species transmission of viral diseases alarms our global community for its potential of novel pandemic events. Of various viral pathogens noted recently, parvoviruses have posed public health threats not only to humans but also to wild animals. To investigate the prevalence of parvoviruses in wild Manchurian chipmunks, here we detected genetic fragments of the nonstructural protein of parvovirus by polymerase chain reaction in wild Manchurian chipmunk specimens captured in the central and southern regions of South Korea and compared their sequence homology with references. Of a total of 348 specimens examined, chipmunk parvovirus (ChpPV)-specific gene fragments were detected with a 31.32% rate (109 chipmunks of 348) in their kidney, liver, lung, and spleen samples, and the chipmunks captured in Gangwon Province exhibited the highest positive rate (45.37%), followed by Gyeongsang (35.29%), Gyeonggi (31.03%), Chungcheong (20.00%), and Jeolla (19.70%). When compared with the reference sequences, a partial ChpPV sequence showed 97.70% identity to the previously reported Korean strain at the nucleic acid level. In the phylogenetic analysis, ChpPV exhibited closer relationship to primate parvoviruses, erythroviruses, and bovine parvovirus than to adeno-associated viruses. Despite limited sample size and genetic sequences examined in this study, our results underline the prevalence of ChpPV in Korea and emphasize the need of close surveillance of parvoviruses in wild animals.

Small but mighty: old and new parvoviruses of veterinary significance.

In line with the Latin expression "sed parva forti" meaning "small but mighty," the family Parvoviridae contains many of the smallest known viruses, some of which result in fatal or debilitating infections. In recent years, advances in metagenomic viral discovery techniques have dramatically increased the identification of novel parvoviruses in both diseased and healthy individuals. While some of these discoveries have solved etiologic mysteries of well-described diseases in animals, many of the newly discovered parvoviruses appear to cause mild or no disease, or disease associations remain to be established. With the increased use of animal parvoviruses as vectors for gene therapy and oncolytic treatments in humans, it becomes all the more important to understand the diversity, pathogenic potential, and evolution of this diverse family of viruses. In this review, we discuss parvoviruses infecting vertebrate animals, with a special focus on pathogens of veterinary significance and viruses discovered within the last four years.

Clinical Course of Infection and Cross-Species Detection of Equine Parvovirus-Hepatitis.

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler's disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler's disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013-2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013-2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.

Hairpin Transfer-Independent Parvovirus DNA Replication Produces Infectious Virus.

Parvoviruses package a linear single-stranded DNA genome with hairpin structures at both ends. It has been thought that terminal hairpin sequences are indispensable for viral DNA replication. Here, we provide evidence that the hairpin-deleted duplex genomes of human bocavirus 1 (HBoV1) replicate in human embryonic kidney 293 (HEK293) cells. We propose an alternative model for HBoV1 DNA replication in which the leading strand can initiate strand displacement without hairpin transfer. The transfection of the HBoV1 duplex genomes that retain a minimal replication origin at the right end (OriR) but with extensive deletions in the right-end hairpin (REH) generated viruses in HEK293 cells at a level 10 to 20 times lower than that of the wild-type (WT) duplex genome. Importantly, these viruses that have a genome with various deletions after the OriR but not the one retaining only the OriR replicated in polarized human airway epithelia. We discovered that the 18-nucleotide (nt) sequence (nt 5403 to 5420) beyond the OriR was sufficient to confer virus replication in polarized human airway epithelia, although its progeny virus production was ∼5 times lower than that of the WT virus. Thus, our study demonstrates that hairpin transfer-independent productive parvovirus DNA replication can occur. IMPORTANCE Hairpin transfer-independent parvovirus replication was modeled with human bocavirus 1 (HBoV1) duplex genomes whose 5' hairpin structure was ablated by various deletions. In HEK293 cells, these duplex viral genomes with ablated 5' hairpin sequence replicated efficiently and generated viruses that productively infected polarized human airway epithelium. Thus, for the first time, we reveal a previously unknown phenomenon that productive parvovirus DNA replication does not depend on the hairpin sequence at REH to initiate rolling-hairpin DNA replication. Notably, the intermediates of viral DNA replication, as revealed by two-dimensional electrophoresis, from transfections of hairpin sequence-deleted duplex genome and full-length genome in HEK293 cells as well as from virus infection of polarized human airway epithelia are similar. Thus, the establishment of the hairpin transfer-independent parvoviral DNA replication deepens our understanding of viral DNA replication and may have implications in the development of parvovirus-based viral vectors with alternative properties.

Equine Parvovirus-Hepatitis Screening in Horses and Donkeys with Histopathologic Liver Abnormalities.

There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler's disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases (n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 103 and 9.5 × 103 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler's disease.

Emergence of a novel pegivirus species in southwest China showing a high rate of coinfection with parvovirus and circovirus in geese.

Previously, we isolated a novel strain of goose pegivirus (GPgV) that infects geese and shows high levels of lymphotropism. This novel pegivirus strain is phylogenetically distinct from previously known Pegivirus species, Pegivirus A-K, and qualifies as a candidate new Pegivirus species, GPgV. GPgV is tentatively named Pegivirus M. Here, to better understand the epidemic of GPgV infection and the coinfection of this virus with other viruses in Southwest China, 25 geese in poor health from Sichuan Province and 24 geese in poor health from the municipality of Chongqing were collected. The geese were tested for 9 types of goose viruses (goose hemorrhagic polyomavirus, GPgV, astrovirus, parvovirus, circovirus, reovirus, coronavirus, paramyxovirus, and avian influenza virus) by RT-PCR or nested RT-PCR. GPgV RNA was detected in 2 out of 25 monoinfections and 8 out of 25 coinfections with other viruses on Sichuan farms and 2 out of 24 monoinfections and 10 out of 24 coinfections on Chongqing farms. Overall, 22 of the 49 (44.9%) geese were positive for GPgV, which indicated a high infection rate. To the best of our knowledge, this is the first report of GPgV coinfection with other epidemic viruses. This study enhances our understanding of the emergence and epidemiology of Pegivirus.

Human Protoparvovirus DNA and IgG in Children and Adults with and without Respiratory or Gastrointestinal Infections.

Three human protoparvoviruses, bufavirus (BuV), tusavirus (TuV) and cutavirus (CuV), have recently been discovered in diarrheal stool. BuV has been associated with diarrhea and CuV with cutaneous T-cell lymphoma, but there are hardly any data for TuV or CuV in stool or respiratory samples. Hence, using qPCR and IgG enzyme immunoassays, we analyzed 1072 stool, 316 respiratory and 445 serum or plasma samples from 1098 patients with and without gastroenteritis (GE) or respiratory-tract infections (RTI) from Finland, Latvia and Malawi. The overall CuV-DNA prevalences in stool samples ranged between 0-6.1% among our six patient cohorts. In Finland, CuV DNA was significantly more prevalent in GE patients above rather than below 60 years of age (5.1% vs 0.2%). CuV DNA was more prevalent in stools among Latvian and Malawian children compared with Finnish children. In 10/11 CuV DNA-positive adults and 4/6 CuV DNA-positive children with GE, no known causal pathogens were detected. Interestingly, for the first time, CuV DNA was observed in two nasopharyngeal aspirates from children with RTI and the rare TuV in diarrheal stools of two adults. Our results provide new insights on the occurrence of human protoparvoviruses in GE and RTI in different countries.

Best of most possible worlds: Hybrid gene therapy vectors based on parvoviruses and heterologous viruses.

Parvoviruses and especially the adeno-associated virus (AAV) species provide an exciting and versatile platform for the rational design or molecular evolution of human gene-therapy vectors, documented by literature from over half a century, hundreds of clinical trials, and the recent commercialization of multiple AAV gene therapeutics. For the last three decades, the power of these vectors has been further potentiated through various types of hybrid vectors created by intra- or inter-genus juxtaposition of viral DNA and protein cis elements or by synergistic complementation of parvoviral features with those of heterologous, prokaryotic, or eukaryotic viruses. Here, we provide an overview of the history and promise of this rapidly expanding field of hybrid parvoviral gene-therapy vectors, starting with early generations of chimeric particles composed of a recombinant AAV genome encapsidated in shells of synthetic AAVs or of adeno-, herpes-, baculo-, or protoparvoviruses. We then dedicate our attention to two newer, highly promising types of hybrid vectors created via (1) pseudotyping of AAV genomes with bocaviral serotypes and capsid mutants or (2) packaging of AAV DNA into, or tethering of entire vector particles to, bacteriophages. Finally, we conclude with an outlook summarizing critical requirements and improvements toward clinical translation of these original concepts.