Surveillance of densoviruses and mesomycetozoans inhabiting grossly normal tissues of three Aotearoa New Zealand asteroid species.

Asteroid wasting events and mass mortality have occurred for over a century. We currently lack a fundamental understanding of the microbial ecology of asteroid disease, with disease investigations hindered by sparse information about the microorganisms associated with grossly normal specimens. We surveilled viruses and protists associated with grossly normal specimens of three asteroid species (Patiriella regularis, Stichaster australis, Coscinasterias muricata) on the North Island / Te Ika-a-Maui, Aotearoa New Zealand, using metagenomes prepared from virus and ribosome-sized material. We discovered several densovirus-like genome fragments in our RNA and DNA metagenomic libraries. Subsequent survey of their prevalence within populations by quantitative PCR (qPCR) demonstrated their occurrence in only a few (13%) specimens (n = 36). Survey of large and small subunit rRNAs in metagenomes revealed the presence of a mesomycete (most closely matching Ichthyosporea sp.). Survey of large subunit prevalence and load by qPCR revealed that it is widely detectable (80%) and present predominately in body wall tissues across all 3 species of asteroid. Our results raise interesting questions about the roles of these microbiome constituents in host ecology and pathogenesis under changing ocean conditions.

Variation in the susceptibility of urban Aedes mosquitoes infected with a densovirus.

Urban Aedes mosquitoes are vectors of many viruses affecting human health such as dengue, chikungunya and Zika viruses. Insecticide resistance and environmental toxicity risks hamper the effectiveness of chemical control against these mosquito vectors. Alternative control methods, such as the use of mosquito-specific entomopathogenic viruses should be explored. Numerous studies have focused on evaluating the potential of different densoviruses species as biological control agents. However, knowledge on the extent of inter- and intra-specific variations in the susceptibility of Aedes mosquitoes to infection by different densoviruses remains insufficient. In this study, we compared infection and mortality rates induced by the Aedes albopictus densovirus 2 in different strains of Aedes albopictus and Aedes aegypti mosquitoes. The two Aedes species were different in terms of susceptibility to viral infection. Under laboratory conditions, Aedes albopictus densovirus 2 appeared more virulent for the different strains of Aedes aegypti tested than for those of Aedes albopictus. In addition, we also found significant intra-specific variation in infection and mortality rates. Thus, although even if Aedes albopictus densoviruses could be powerful biocontrol agents used in the management of urban Aedes populations, our results also call into question the use of single viral isolate as biocontrol agents.

Insect-specific flaviviruses and densoviruses, suggested to have been transmitted vertically, found in mosquitoes collected in Angola: Genome detection and phylogenetic characterization of viral sequences.

This report describes a survey of RNA and DNA viruses carried out in adult mosquitoes from Angola, raised under laboratory conditions from field-collected immature forms. This viral genomic survey was performed using different sets of primers targeting groups of arboviruses with a considerable impact on human health, including flaviviruses, alphaviruses, and phleboviruses. Furthermore, the viral survey that was performed also included detection of densoviruses. The obtained results did not reveal the presence of recognizable pathogenic arboviruses but allowed the identification of insect-specific flaviviruses from two genetic lineages and a single lineage of brevidensoviruses. These viruses, collectivelly detected in Anopheles sp. and Culex pipiens s.l. mosquitoes, were most probably transmitted vertically.

A Highly Prevalent and Pervasive Densovirus Discovered among Sea Stars from the North American Atlantic Coast.

The etiology of sea star wasting syndrome is hypothesized to be caused by a densovirus, sea star-associated densovirus (SSaDV), that has previously been reported on the Pacific and Atlantic Coasts of the United States. In this study, we reevaluated the presence of SSaDV among sea stars from the North American Atlantic Coast and in doing so discovered a novel densovirus that we have named Asterias forbesi-associated densovirus (AfaDV), which shares 78% nucleotide pairwise identity with SSaDV. In contrast to previous studies, SSaDV was not detected in sea stars from the North American Atlantic Coast. Using a variety of PCR-based techniques, we investigated the tissue tropism, host specificity, and prevalence of AfaDV among populations of sea stars at five locations along the Atlantic Coast. AfaDV was detected in three sea star species (Asterias forbesi, Asterias rubens, and Henricia sp.) found in this region and was highly prevalent (>80% of individuals tested; n = 134), among sampled populations. AfaDV was detected in the body wall, gonads, and pyloric caeca (digestive gland) of specimens but was not detected in their coelomic fluid. A significant difference in viral load (copies mg-1) was found between tissue types, with the pyloric caeca having the highest viral loads. Further investigation of Asterias forbesi gonad tissue found germ line cells (oocytes) to be virus positive, suggesting a potential route of vertical transmission. Taken together, these observations show that the presence of AfaDV is not an indicator of sea star wasting syndrome because AfaDV is a common constituent of these animals' microbiome, regardless of health.IMPORTANCE Sea star wasting syndrome is a disease primarily observed on the Pacific and Atlantic Coasts of North America that has significantly impacted sea star populations. The etiology of this disease is unknown, although it is hypothesized to be caused by a densovirus, SSaDV. However, previous studies have not found a correlation between SSaDV and sea star wasting syndrome on the North American Atlantic Coast. This study suggests that this observation may be explained by the presence of a genetically similar densovirus, AfaDV, that may have confounded previous studies. SSaDV was not present in sea stars screened in this study, and instead, AfaDV was commonly found in sea star populations across the New England region, with no apparent signs of disease. These results suggest that sea star densoviruses may be common constituents of the animals' microbiome, and the diversity and extent of these viruses among wild populations may be greater than previously recognized.

Diaphorina citri densovirus is a persistently infecting virus with a hybrid genome organization and unique transcription strategy.

Diaphorina citri densovirus (DcDV) is an ambisense densovirus with a 5071 nt genome. Phylogenetic analysis places DcDV in an intermediate position between those in the Ambidensovirus and Iteradensovirus genera, a finding that is consistent with the observation that DcDV possesses an Iteradensoviris-like non-structural (NS) protein-gene cassette, but a capsid-protein (VP) gene cassette resembling those of other ambisense densoviruses. DcDV is maternally transmitted to 100 % of the progeny of infected female Diaphorina citri, and the progeny of infected females carry DcDV as a persistent infection without outward phenotypic effects. We were unable to infect naïve individuals by oral inoculation, however low levels of transient viral replication are detected following intrathoracic injection of DcDV virions into uninfected D. citri insects. Transcript mapping indicates that DcDV produces one transcript each from the NS and VP gene cassettes and that these transcripts are polyadenylated at internal sites to produce a ~2.2 kb transcript encoding the NS proteins and a ~2.4 kb transcript encoding the VP proteins. Additionally, we found that transcriptional readthrough leads to the production of longer non-canonical transcripts from both genomic strands.

Development of large-scale mosquito densovirus production by in vivo methods.

BACKGROUND: Mosquito-borne diseases (MBDs) cause a significant proportion of the global infectious disease burden. Vector control remains the primary strategy available to reduce the transmission of MBDs. However, long-term, wide-scale and large-scale traditional chemical pesticide application has caused significant and increased negative effects on ecosystems and broader emerging insecticide resistance in vectors; therefore, the development of a novel alternative approach is urgently needed. Mosquito densoviruses (MDVs) are entomopathogenic viruses that exhibit a narrow host range and multiple transmission patterns, making MDVs a great potential bioinsecticide. However, the application process has been relatively stagnant over the past three decades. The major obstacle has been that viruses must be produced in mosquito cell lines; therefore, the production process is both expensive and time-consuming. METHODS: In our study, two wild-type (wt) MDVs, AaeDV and AalDV-3, and a recombinant rAaeDV-210 were used to infect the Aag2 and C6/36 mosquito cell lines and the 1st-2nd-instar and 3rd-4th-instar larvae of Ae. albopictus, Ae. aegypti and Cx. quinquefasciatus. Viral titers and yields in cells, media, larvae and rearing water and total viral yield were evaluated. Three kinds of virus displayed higher maximum virus titers in vivo than in vitro, and they displayed higher maximum viral yields in rearing water. RESULTS: The three viruses displayed higher total maximum viral yields in C6/36 cells than in Aag2 cells. The three viruses displayed higher total maximum viral yields in Aedes mosquitoes than in Culex mosquitoes. Higher viral yields were produced by 1st-2nd-instar larvae compared to 3rd-4th-instar larvae. The recombinant viruses did not display significantly lower yields than wt viruses in nearly all samples. In summary, by using 100 1st-2nd-instar Aedes mosquito larvae in 200 ml of rearing water, more than 1013 genome equivalents (geq) MDV yield can be obtained. CONCLUSIONS: Considering the lower production cost, this in vivo method has great potential for the large-scale production of MDVs. MDVs exhibit promising prospects and great potential for mosquito control in the future.

A New Prevalent Densovirus Discovered in Acari. Insight from Metagenomics in Viral Communities Associated with Two-Spotted Mite (Tetranychus urticae) Populations.

Viral metagenomics and high throughput sequence mining have revealed unexpected diversity, and the potential presence, of parvoviruses in animals from all phyla. Among arthropods, this diversity highlights the poor knowledge that we have regarding the evolutionary history of densoviruses. The aim of this study was to explore densovirus diversity in a small arthropod pest belonging to Acari, the two-spotted spider mite Tetranychus urticae, while using viral metagenomics based on virus-enrichment. Here, we present the viromes obtained from T. urticae laboratory populations made of contigs that are attributed to nine new potential viral species, including the complete sequence of a novel densovirus. The genome of this densovirus has an ambisens genomic organization and an unusually compact size with particularly small non-structural proteins and a predicted major capsid protein that lacks the typical PLA2 motif that is common to all ambidensoviruses described so far. In addition, we showed that this new densovirus had a wide prevalence across populations of mite species tested and a genomic diversity that likely correlates with the host phylogeny. In particular, we observed a low densovirus genomic diversity between the laboratory and natural populations, which suggests that virus within-species evolution is probably slower than initially thought. Lastly, we showed that this novel densovirus can be inoculated to the host plant following feeding by infected mites, and circulate through the plant vascular system. These findings offer new insights into densovirus prevalence, evolution, and ecology.

Diagnostic protocols for the detection of Acheta domesticus densovirus (AdDV) in cricket frass.

The European house cricket (Acheta domesticus) is a species of interest for the emerging insect-as-food industry. Acheta domesticus densovirus (AdDV) is a member of the Parvoviridae virus family which infects A. domesticus, causing widespread mortality and even extinction of local cricket populations. Despite the well-known detrimental effects of AdDV in commercial rearing of A. domesticus there are no optimized protocols to accurately and non-destructively detect and quantify the virus. This study establishes a new protocol for the detection of AdDV in faecal material from A.domesticus. The protocol includes methodological improvements, such as upgrading from conventional PCR to quantitative real-time PCR and is much more sensitive than previously published protocols. Moreover, this study shows that cricket faeces are a suitable, non-destructive sample substrate to infer reliably if a cricket population is infected with AdDV or not. Early detection of lethal or economic threats, such as disease-causing viruses, is an essential part of commercial cricket management as well as for monitoring the risk of spread to wild cricket populations or to (human) consumers.

Presence of Penaeus monodon densovirus in the ovary of chronically infected P. monodon subadults.

Shrimp infected with Penaeus monodon densovirus (PmoDNV) usually display no specific gross signs, but heavy infections can kill postlarvae and retard juvenile growth. In the present study, samples of hepatopancreas, feces, gonads and hemolymph were isolated from male and female P. monodon subadults chronically infected by PmoDNV. Each sample of hepatopancreas and gonad was divided into 2 parts: one for PmoDNV detection by polymerase chain reaction (PCR), and the other for routine histology and immunohistochemistry. The frequency of positive findings via PCR assays was 92% in the hepatopancreas, 57% in feces, 50% in ovary, 35% in hemolymph and 0% in the testis. Using the densitometric value (DV) of the specific band for PmoDNV relative to that of the ß-actin gene as an index of the viral load in the samples, no significant differences were observed among sample types and sexes. Hematoxylin-eosin staining of infected hepatopancreas revealed typical PmoDNV inclusions in the nuclei of infected cells. The ovaries with high DV (>1) contained various types of inclusions along the row of the follicular cells or possibly in the connective tissue cells surrounding the oocytes. Using immunohistochemistry with specific probes to detect PmoDNV proteins, a positive reaction was observed in viral inclusions found in infected hepatopancreas and in ovaries with high DV, specifically in the ovarian capsule, hemolymph, oocytes and nuclear inclusions. These results suggest that the localization of PmoDNV in P. monodon is not confined to the hepatopancreas, but rather that the virus can also occur in the ovary; hence, trans-ovarian, vertical transmission of the virus is highly possible.

Prevalence, diversity and co-occurrence of the white spot syndrome virus, monodon baculovirus and Penaeus stylirostris densovirus in wild populations of Penaeus monodon in the Philippines.

The farming of the black tiger shrimp Penaeus monodon in the Philippines relies on wild broodstock. PCR was thus used to determine the prevalence of white spot syndrome virus (WSSV), monodon baculovirus (MBV) and Penaeus stylirostris densovirus (PstDV) in a total of 178 shrimp from 6 geographically disparate locations where broodstock are captured for use in hatcheries. PCR amplicons were also sequenced to identify phylogenetic relationships of the virus haplotypes detected. Shrimp from southeastern Luzon (Camarines Norte) had the highest prevalence of each of the 3 viruses and were frequently co-infected with 2 or more viruses. No viruses were detected in shrimp from northwestern Luzon (Pangasinan). MBV was most prevalent and PstDV strains displayed the most genetic diversity. WSSV was detected at 3 sites, and a VP28 gene sequence examined was invariant and consistent with strains found in many countries, including Thailand, China, Japan, Korea, Indonesia, Iran, Brazil and Mexico. WSSV open reading frame 94 gene sequence analysis identified location-specific repeat types. MBV sequences were dissimilar to haplotypes detected in India. PstDV sequences were diverse and included 2 lineages detected either in Australia or in the United States, Ecuador, Taiwan, China and Vietnam. The PCR data confirmed that WSSV, MBV and PstDV are endemic in P. monodon in the Philippines but that populations at some locations might remain free of infection.

Densoviruses in oyster Crassostrea ariakensis.

Densoviruses have short ssDNA genomes and mainly infect arthropods. To characterize viral nucleic acid in shellfish, oysters (Crassostrea ariakensis) were analyzed using viral metagenomics. Two large de novo assembled contigs, CaaDV1 and CaaDV2, consisting of nearly complete densovirus genomes (5860 nucleotides (nt) and 4034 nt) with two major ambisense protein coding regions were identified. Several potential non-structural proteins and capsid proteins were encoded by these genomes, but these were divergent from the existing densoviral species. The NS1 protein of the two CaaDVs shared 43.3%~61.5% amino acid identities with the sea star-associated densovirus and cherax quadricarinatus densovirus, with the four species clustering by phylogenetic analysis. This is the first report of densovirus detection in shellfish, increasing the potential host range of densoviruses and the genetic diversity of the genus Ambidensovirus.

A new densovirus in cerebrospinal fluid from a case of anti-NMDA-receptor encephalitis.

We characterized the genome of a densovirus, tentatively called human CSF-associated densovirus 1 (HuCSFDV1), in cerebrospinal fluid (CSF) from a human case of encephalitis with antibodies against the N-methyl D-aspartate receptor. The presence of the viral genome in CSF was independently confirmed. This virus, which is proposed to be a member of a new species in the genus Iteradensovirus of the subfamily Densovirinae, showed the typical two ORFs encoding nonstructural and structural proteins with low-level identities of 22 and 16 % to the closest known densovirus relative. No other eukaryotic viral sequences were detected using deep sequencing. The replication and pathogenicity in humans of this virus, which belongs to a viral subfamily whose members are only known to replicate in invertebrates, remain to be demonstrated. Alternative explanations for the detection of densovirus DNA in CSF are discussed.

A gradient-free method for the purification of infective dengue virus for protein-level investigations.

Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins - including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis.

Genetic characterization of a densovirus isolated from great tit (Parus major) in China.

During a study of ornithophilous viruses in China, a new densovirus (DNV) was isolated from the lung tissue of Parus major (PmDNV-JL). The complete genome of PmDNV-JL was cloned and sequenced. Five open reading frames (ORFs) were identified in the 5166nt sequence, on the basis of deduced amino acids. It was further shown that this virus caused cytopathic effects (CPE) in Feline kidney cells. The NS1 gene sequence of PmDNV-JL shares 70-99% nucleotide sequence identity with isolates of the Blattella germanica densovirus (BgDNV) and BgDNV-like virus. Phylogenetic analysis indicated that the predicted amino acid sequences of capsid (VP) and non-structural domain (NS1) of PmDNV-JL clustered with the BgDNV and were similar to BgDNV-HB within the genus Densovirus.

Effects of recombination on densovirus phylogeny.

Densoviruses are a group of arthropod-infecting viruses with a small single-stranded linear DNA genome. These viruses constitute the subfamily Densovirinae of the family Parvoviridae. While recombination in between vertebrate-infecting parvoviruses has been investigated, to date, no systematic analysis of recombination has been carried out for densoviruses. The aim of the present work was to study possible recombination events in the evolutionary history of densoviruses and to assess possible effects of recombination on phylogenies inferred using amino acid sequences of nonstructural (NS) and capsid (viral protein, VP) proteins. For this purpose, the complete or nearly complete genome nucleotide sequences of 40 densoviruses from the GenBank database were used to construct a phylogenetic cladogram. The viruses under study clustered into five distinct groups corresponding to the five currently accepted genera. Recombination within each group was studied independently. The RDP4 software revealed three statistically highly credible recombination events, two of which involved viruses of the genus Ambidensovirus, and the other, viruses from the genus Iteradensovirus. These recombination events led to mismatches between phylogenetic trees constructed using comparison of amino acid sequences of proteins encoded by genome regions of recombinant and non-recombinant origin (regulatory NS1 and NS3 proteins and capsid VP protein).

An improved validated SYBR green-based real-time quantitative PCR assay for the detection of the Penaeus stylirostris densovirus in penaeid shrimp.

The Penaeus stylirostris densovirus (PstDV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), one of the major shrimp pathogens, has a worldwide distribution in farmed and wild shrimp populations. Outbreaks of IHHNV have been associated with substantial economic losses which are accompanied by a negative social impact. Current diagnostic PCR tests may result in false-positive results as several parts of PstDV genome may be endogenized in the nuclear genome of the shrimp P. stylirostris. A one-step qPCR SYBR-Green based quantitative real-time polymerase chain reaction (qPCR) assay to detect different isolates of the IHHNV in shrimp samples was developed. The detection limit of the assay was 81 viral copies of targeted DNA per reaction. The specificity of the assay was evaluated by melting curve analysis, which showed that the IHHNV product generated a single melt peak at 81.4±0.044°C. The assay was more sensitive than conventional PCR. The standardized PCR was shown to be highly sensible, specific, robust, and reproducible, which makes it an economical and powerful tool for both diagnostic applications and general research of IHHNV.

Recombinase polymerase amplification combined with a lateral flow dipstick for discriminating between infectious Penaeus stylirostris densovirus and virus-related sequences in shrimp genome.

Penaeus stylirostris densovirus (PstDV) is an important shrimp pathogen that causes mortality in P. stylirostris and runt deformity syndrome (RDS) in Penaeus vannamei and Penaeus monodon. Recently, PstDV-related sequences were found in the genome of P. monodon and P. vannamei. This led to false positive results by PCR-based detection system. Here, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed for detecting PstDV. Under the optimal conditions, 30 min at 37°C for RPA followed by 5 min at room temperature for LFD, the protocol was 10 times more sensitive than the Saksmerphrome et al's interim 3-tube nested PCR and showed no cross-reaction with other shrimp viruses. It also reduced false positive results arising from viral inserts to ∼5% compared to 76-78% by the IQ2000™ nested PCR kit and the 309F/R PCR protocol currently recommended by World Organization for Animal Health (OIE) for PstDV detection. Together with simplicity and portability, the protocol serves as an alternative tool to PCR for primarily screening PstDV, which is suitable for both laboratory and field application.

Separation of endogenous viral elements from infectious Penaeus stylirostris densovirus using recombinase polymerase amplification.

Non-infectious Penaeus stylirostris densovirus (PstDV)-related sequences in the shrimp genome cause false positive results with current PCR protocols. Here, we examined and mapped PstDV insertion profile in the genome of Australian Penaeus monodon. A DNA sequence which is likely to represent infectious PstDV was also identified and used as a target sequence for recombinase polymerase amplification (RPA)-based approach, developed for specifically detecting PstDV. The RPA protocol at 37 °C for 30 min showed no cross-reaction with other shrimp viruses, and was 10 times more sensitive than the 309F/R PCR protocol currently recommended by the World Organization for Animal Health (OIE) for PstDV diagnosis. These features, together with the simplicity of the protocol, requiring only a heating block for the reaction, offer opportunities for rapid and efficient detection of PstDV.

Application of quantitative PCR for quantization of densovirus genome.

Densovirus is classified as invertebrate virus belonging to the subfamily Densovirinae of Parvoviridae family. This group of viruses infects only insects and several densoviruses have been isolated from indigenous mosquitoes and mosquito cell lines. A number of mosquitoes, especially Aedes aegypti and Ae. albopictus are important vectors of viruses, which are the major causes of dengue hemorrhagic fever and yellow fever in humans. As densoviruses do not cause any pathology in humans, these viruses have been proposed to be a potential vector for use in biological control of mosquitoes and insects. We report the application of quantitative (q)PCR to determine the amount of densovirus genome in mosquito cell culture supernatant and mosquito. This method is simple, rapid and has a wide dynamic range, and therefore is likely to be useful and applicable in the determination of viral load of other viruses in a variety of biological specimens.

Discovery of urchin-associated densoviruses (family Parvoviridae) in coastal waters of the Big Island, Hawaii.

Echinoderms are important constituents of marine ecosystems, where they may influence the recruitment success of benthic flora and fauna, and are important consumers of detritus and plant materials. There are currently no described viruses of echinoderms. We used a viral metagenomic approach to examine viral consortia within three urchins - Colobocentrotus atratus, Tripneustes gratilla and Echinometra mathaei - which are common constituents of reef communities in the Hawaiian archipelago. Metagenomic libraries revealed the presence of bacteriophages and densoviruses (family Parvoviridae) in tissues of all three urchins. Densoviruses are known typically to infect terrestrial and aquatic arthropods. Urchin-associated densoviruses were detected by quantitative PCR in all tissues tested, and were also detected in filtered suspended matter (>0.2 µm) from plankton and in sediments at several locations near to where the urchins were collected for metagenomic analysis. To the best of our knowledge, this is the first report of echinoderm-associated viruses, which extends the known host range of parvoviruses.