Complete Genomic and Ultrastructural Analysis of a Nam Dinh Virus Isolated from Culex pipiens quinquefasciatus in China.

The Nam Dinh virus (NDiV) was isolated from Culex quinquefasciatus in Shenzhen, China, for the first time, in 2011. In this study, we characterized the ultrastructure of NDiV, determined its complete genome sequence and made comparisons with other known nidoviruses. Electron microscopic observation revealed that the NDiV strain isolated in China produced viral nucleocapsid-like particles and vesicles in host cells. The extracellular virions were enveloped and were spherical with short spikes. The complete genome sequence of the newly isolated NDiV was submitted to the GenBank database (GenBank accession number KF522691). Sequencing of the viral genome showed that the homologies of NDiV isolated in China and Vietnam were greater than 94.0% and 89.0% at the nucleotide and amino acid sequence levels, respectively. Moreover, gene substitution was detected, whereas insertions and deletions were not. A phylogenetic tree analysis showed that these viruses belong to the genus Alphamesonivirus1 of the family Mesoniviridae. The similarity between the two viruses regarding morphological and molecular biological characteristics indicates that the molecular genetics of NDiV are conservative and that the regional differences are unlikely to have a significant effect. This is the first report of the isolation and complete sequencing of a mesonivirus in mainland China.

[Isolation and Identification of the Nam Dinh Virus from Mosquitoes on the China-Laos-Myanmar Border].

Three strains of an insect nidovirus, the Nam Dinh virus (NDiV), isolated in Yunnan Province, China, have been identified. Aedes albopictus C6/36 cells were used to isolate NDiV from mosquitoes collected in Yunnan Province in 2012.Culture supernatants with a positive cytopathic effect were harvested for virus identification by sequence-independent single primer amplification. Transmission electron microscopy revealed virion structure to be spherical with a diameter of 60~80nm.Reverse transcription-polymerase chain reaction was applied to amplify sequences of RNA-dependent RNA-polymerase (RdRp), HEL1(superfamily 1helicase)and spike protein. The amino-acid sequences of three isolates from Yunnan Province showed>98% homology with NDiV strains. Phylogenetic analyses showed that these three isolates, along with NDiV, could be classified into the family Mesoniviridae.

Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease.

The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9×10(8)/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12h post-infection. Maximum levels of cell-associated viral RNA were detected at 24h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus.

Mesoniviruses are mosquito-specific viruses with extensive geographic distribution and host range.

BACKGROUND: The family Mesoniviridae (order Nidovirales) comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes. FINDINGS: Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d'Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 - 588 nt) of unknown function in the 5' region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication. CONCLUSIONS: In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species (Alphamesonivirus-5, Alphamesonivirus-6 and Alphamesonivirus-7). The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.

A new nidovirus (NamDinh virus NDiV): Its ultrastructural characterization in the C6/36 mosquito cell line.

We describe the ultrastructure of the NamDinh virus (NDiV), a new member of the order Nidovirales grown in the C6/36 mosquito cell line. Uninfected and NDiV-infected cells were investigated by electron microscopy 24-48 h after infection. The results show that the viral nucleocapsid-like particles form clusters concentrated in the vacuoles, the endoplasmic reticulum, and are scattered in the cytoplasm. Mature virions of NDiV were released as budding particles on the cell surface where viral components appear to lie beneath and along the plasma membrane. Free homogeneous virus particles were obtained by ultracentrifugation on sucrose gradients of culture fluids. The size of the round-shaped particles with a complete internal structure was 80 nm in diameter. This is the first study to provide information on the morphogenesis and ultrastructure of the first insect nidovirus NDiV, a missing evolutionary link in the emergence of the viruses with the largest RNA genomes.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Vertical transmission of gill-associated virus (GAV) in the black tiger prawn Penaeus monodon.

Chronic gill-associated virus (GAV) infection is endemic in Penaeus monodon broodstock captured from north-east Queensland in Australia and in farmed shrimp produced from these. We investigated the role of vertical transmission in perpetuating the high prevalence of these chronic GAV infections. Reverse transcription (RT)-nested PCR detected GAV in spermatophores and mature ovarian tissue from broodstock and in fertilized eggs and nauplii spawned from wild-fertilized females. In laboratory-reared P. monodon (> 12 mo old) that had a high mortality rate, RT-nested PCR detected GAV in male spermatophores at levels significantly higher than that detected in the lymphoid organ. By transmission electron microscopy (TEM), GAV virions were detected in spermatophore seminal fluid, but not sperm cells. Histological evidence of hypertrophied cell foci (spheroids) and TEM observation of GAV nucleocapsids and virions in spheroid cells was also found in 100% of lymphoid organs of approximately 1.2 g juvenile P. monodon reared in the laboratory from postlarvae collected from commercial hatcheries. Sequence analysis of PCR amplicons from parental P. monodon and fertilized eggs of artificially inseminated broodstock indicated that GAV associated with eggs can originate from both the male and female parents. Although the female GAV genotype was predominant in eggs, there was some dependence on infection levels in the male and female shrimp as indicated by RT-PCR. RT-nested PCR data on GAV levels in eggs, nauplii, protozoea and PL5 progeny of the artificial matings suggests that vertically transmitted virus is most probably associated with the egg surface.