Genetic characterization of Yokose virus, a flavivirus isolated from the bat in Japan

Yokose virus (strain Oita-36) was isolated from the bat in Japan in 1971. In the present study, we determined complete nucleotide sequences of Yokose virus using RT-PCR and RACE techniques. Yokose virus genome consists of 10,857 nucleotides in length (accession no. AB114858), containing a single open reading frame (3425 amino acids) encoding 11 viral proteins. We deduced the boundaries of each protein in the polyprotein sequence according to the protein cleavage sites of other flaviviruses. The nucleotide sequences of the 5' and 3' nontranslated region (NTR) and amino acid sequences of individual proteins of the virus were compared with those of six other flaviviruses including Japanese encephalitis virus, dengue-2 virus, yellow fever virus, West Nile virus, tick-borne encephalitis virus, and Rio Bravo virus or Modoc virus. Yokose virus demonstrated the highest similarity to yellow fever virus. Yokose virus also has CS1 motif, which are well-conserved specifically in mosquito-born flaviviruses, in its 3' NTR. When a part of the NS5 amino acid sequence (345 amino acids) was compared with those of other four flaviviruses, Entebbe bat virus, Sokuluk virus, Sepik virus, and yellow fever virus, the three former viruses are more closely related to Yokose virus than yellow fever virus. Human sera from dengue-virus-infected case and yellow fever vaccine reacted with the viral proteins. Moreover, human serum from a yellow fever vaccine weakly neutralized Yokose virus. Our results suggest that there are cross-reactive antigenicities among Yokose virus and other flaviviruses.     

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.     

Genetic, biochemical, and structural characterization of a new densovirus isolated from a chronically infected Aedes albopictus C6/36 cell line

We report the isolation, sequencing, biochemical, and structural characterization of a previously undescribed virus in a chronically infected Aedes albopictus C6/36 cell line. This virus is identified as a new densovirus under the Densovirinae subfamily of the Parvoviridae based on its biological and morphologic properties as well as sequence homologies, and is tentatively designated A. albopictus C6/36 cell densovirus (C6/36 DNV). Analysis of the 4094 nt of the C6/36 DNV genome revealed that the plus strand had three large open reading frames (ORFs): a left ORF, a right ORF, and a mid-ORF (within the left ORF), whose potential coding capacities are 91.0, 40.8, and 41.2 kDa, respectively. The left ORF likely encodes the nonstructural protein NS-1, which contains NTP-binding and helicase domains. The right ORF likely encodes structural proteins, VP1 and VP2. Our analyses revealed that C6/36 DNV has a similar genomic organization and shares very high homology in nucleotide sequence and amino acid sequences with Aedes aegypti densovirus (AaeDNV) and A. albopictus densovirus (AalDNV), members of the genus Brevidensovirus of the Densovirinae. Similar to other densoviruses, C6/36 DNV has a different genomic organization and no recognizable sequence homology with viruses in the Parvovirinae. The three-dimensional (3D) reconstruction of the C6/36 DNV at 15.6-A resolution by electron cryomicroscopy (cryoEM) revealed distinctive outer surface features not previously seen in other parvoviruses, indicating structural divergence of densoviruses, in addition to its genomic differences, while the inner surface of the C6/36 DNV capsid exhibits features that are conserved among parvoviruses.     

Complete genome analysis and molecular characterization of Usutu virus that emerged in Austria in 2001: comparison with the South African strain SAAR-1776 and other flaviviruses

Here we describe the complete genome sequences of two strains of Usutu virus (USUV), a mosquito-borne member of the genus Flavivirus in the Japanese encephalitis virus (JEV) serogroup. USUV was detected in Austria in 2001 causing a high mortality rate in blackbirds; the reference strain (SAAR-1776) was isolated in 1958 from mosquitoes in South Africa and has never been associated with avian mortality. The Austrian and South African isolates exhibited 97% nucleotide and 99% amino acid identity. Phylogenetic trees were constructed displaying the genetic relationships of USUV with other members of the genus Flavivirus. When comparing USUV with other JEV serogroup viruses, the closest lineage was Murray Valley encephalitis virus (nt: 73%, aa: 82%) followed by JEV (nt: 71%, aa: 81%) and West Nile virus (nt: 68%, aa: 75%). Comparison of the genomes showed that the conserved structural elements and putative enzyme motifs were homologous in the two USUV strains and the JEV serogroup. The factors that determine the severe clinical symptoms caused by the Austrian USUV strain in Eurasian blackbirds are discussed. We also offer a possible explanation for the origins and dispersal of USUV, JEV, and MVEV out of Africa.     

Isolation and characterization of a new Vesivirus from rabbits

This report describes the isolation, cDNA cloning, complete genome nucleotide sequence, and partial characterization of a new cultivable calicivirus isolated from juvenile feeder European rabbits (Oryctolagus cuniculus) showing symptoms of diarrhea. Absence of neutralization by type-specific neutralizing antibodies for 40 caliciviruses and phylogenetic sequence comparisons of the open reading frame 1-encoded polyprotein with those of other caliciviruses demonstrate that this new calicivirus is a putative novel member of the Vesivirus genus which is closely related to the marine calicivirus subgroup. According to its putative classification, this new virus has been named rabbit vesivirus.     

Characterization of a small plaque variant of West Nile virus isolated in New York in 2000

A small-plaque variant (SP) of West Nile virus (WNV) was isolated in Vero cell culture from kidney tissue of an American crow collected in New York in 2000. The in vitro growth of the SP and parental (WT) strains was characterized in mammalian (Vero), avian (DF-1 and PDE), and mosquito (C6/36) cells. The SP variant replicated less efficiently than did the WT in Vero cells. In avian cells, SP growth was severely restricted at high temperatures, suggesting that the variant is temperature sensitive. In mosquito cells, growth of SP and WT was similar, but in vivo in Culex pipiens (L.) there were substantial differences. Relative to WT, SP exhibited reduced replication following intrathoracic inoculation and lower infection, dissemination, and transmission rates following oral infection. Analysis of the full length sequence of the SP variant identified sequence differences which led to only two amino acid substitutions relative to WT, prM P54S and NS2A V61A.     

Genetic characterization of an insect-specific flavivirus isolated from Culex theileri mosquitoes collected in southern Portugal

We describe the full genetic characterization of an insect-specific flavivirus (ISF) from Culex theileri (Theobald) mosquitoes collected in Portugal. This represents the first isolation and full characterization of an ISF from Portuguese mosquitoes. The virus, designated CTFV, for Culex theileri flavivirus, was isolated in the C6/36 Stegomyia albopicta (=Aedes albopictus) cell line, and failed to replicate in vertebrate (Vero) cells in common with other ISFs. The CTFV genome encodes a single polyprotein with 3357 residues showing all the features expected for those of flaviviruses. Phylogenetic analyses based on all ISF sequences available to date, place CTFV among Culex-associated flaviviruses, grouping with recently published NS5 partial sequences documented from mosquitoes collected in the Iberian Peninsula, and with Quang Binh virus (isolated in Vietnam) as a close relative. No CTFV sequences were found integrated in their host's genome using a range of specific PCR primers designed to the prM/E, NS3, and NS5 region.     

Providence virus: a new member of the Tetraviridae that infects cultured insect cells

We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea). Virus purified from these cells also productively infected a H. zea fat body cell line, and a cell line from whole embryos of the beet armyworm, Spodoptera exigua. PrV is thus the first tetravirus shown to replicate in cell culture. PrV virions are isometric particles composed of two structural proteins (60 and 7.4 kDa) that encapsidate both the genomic (6.4 kb) and the subgenomic (2.5 kb) RNAs. The monopartite organization of the PrV genome resembles that of Nudaurelia beta virus and Thosea asigna virus, members of the genus Betatetravirus. The predicted sequence of the PrV structural proteins demonstrates homology to tetraviruses in both genera. The infectivity of PrV for cultured cells uniquely permitted examination of tetravirus RNA and protein synthesis during synchronous infection. The discovery of PrV greatly facilitates studies of tetravirus molecular biology.     

Identification and characterization of prohibitin as a receptor protein mediating DENV-2 entry into insect cells

Dengue is transmitted primarily by mosquitoes of the Aedes genus. Despite a number of studies, no insect dengue virus receptor protein has been clearly identified and characterized. Using a number of separation methodologies and virus overlay protein binding assays we identified a 35 kDa protein that segregated with susceptibility to dengue serotype 2 (DENV-2) infection in two mosquito species and two mosquito cell lines. Mass spectroscopy identified the protein to be prohibitin, a strongly conserved and ubiquitously expressed protein in eukaryotic cells. Antibody mediated inhibition of infection and siRNA mediated knockdown of prohibitin expression significantly reduced infection levels and subsequent virus production in both Aedes aegypti and Aedes albopictus cell lines. Confocal microscopy showed a significant degree of intracellular colocalization between prohibitin and DENV-2 E protein, and coimmunoprecipitation confirmed that prohibitin interacts with dengue E. Prohibitin is the first characterized insect cell expressed dengue virus receptor protein.     

Genome sequence of the lytic bacteriophage P1201 from Corynebacterium glutamicum NCHU 87078: evolutionary relationships to phages from Corynebacterineae

P1201 is a lytic corynephage of Corynebacterium glutamicum NCHU 87078. Its genome consists of a linear double-stranded DNA molecule of 70,579 base pairs, with 3′-protruding cohesive ends of ten nucleotides. We have identified 69 putative open reading frames, including three apparent genes (thymidylate synthase, terminase, and RNR α subunit genes) that are interrupted by an intein. Protein-splicing activities of these inteins were demonstrated in Escherichia coli. Three structural proteins including major capsid and major tail proteins were separated by SDS-PAGE and identified by both LC-MS-MS and N-terminal sequence analyses. Bioinformatics analysis indicated that only about 8.7% of its putative gene products shared substantial protein sequence similarity with the lytic corynephage BFK20 from Brevibacterium flavum, the only corynephage whose genome had been sequenced to date, revealing that the P1201 genome is distinct from BFK20. The mosaic-like genome of P1201 indicates extensive horizontal gene transfer among P1201, Gordonia terrae phage GTE5, mycobacteriophages, and several regions of Corynebacterium spp. genomes.     

Genome sequence and molecular characterization of Homalodisca coagulata virus-1, a novel virus discovered in the glassy-winged sharpshooter (Hemiptera: Cicadellidae)

The complete nucleotide sequence of a novel single-stranded RNA virus infecting the glassy-winged sharpshooter, Homalodisca coagulata, has been determined. In silico analysis of H. coagulata virus-1 (HoCV-1) revealed a 9321-nt polyadenylated genome encoding two large open reading frames (ORF1 and ORF2) separated by a 182-nt intergenic region (IGR). The deduced amino acid sequence of the 5'-proximal ORF (ORF1, nt 420-5807) exhibited conserved core motifs characteristic of the helicases, cysteine proteases, and RNA-dependent RNA polymerases of other insect-infecting picorna-like viruses. A structural model created using Mfold exposed a series of stem loop (SL) structures immediately preceding the second ORF which are analogous to an internal ribosome entry site (IRES), suggesting that ORF2 begins with a noncognate GCA triplet rather than the canonical AUG. This 3' ORF2 (5990-8740) showed significant similarity to the structural proteins of members of the family Dicistroviridae, particularly those belonging to the genus Cripavirus. Evidence demonstrating relatedness of these viruses regarding genome organization, amino acid sequence similarity, and putative replication strategy substantiate inclusion of HoCV-1 into this taxonomic position.     

Full-length genome sequence of Mossman virus, a novel paramyxovirus isolated from rodents in Australia

Mossman virus (MoV) was isolated on two occasions from wild rats trapped in Queensland, Australia, during the early 1970s. Together with Nariva virus and J-virus MoV belongs to a group of novel paramyxoviruses isolated from rodents during the last 40 years, none of which had been characterized at the molecular level until now. cDNA subtraction strategies used to isolate virus-specific cDNA derived from both MoV-infected cells and crude MoV pellets were pivotal steps in rapid characterization of the complete genome sequence. Analysis of the full-length genome and its encoded proteins confirmed that MoV is a novel member of the subfamily Paramyxovirinae which cannot be assigned to an existing genus. MoV appears to be more closely related to another unclassified paramyxovirus Tupaia paramyxovirus (TPMV), isolated from the tree shrew Tupaia belangeri. Together with Salem virus (SalV), a further unclassified paramyxovirus that was isolated from a horse, MoV and TPMV make up a new collection of paramyxoviruses situated evolutionally between the genus Morbillivirus and the newly established genus Henipavirus.     

Full genomic analysis of human rotavirus strain TB-Chen isolated in China

A G2P[4]/NSP4[A] rotavirus strain TB-Chen was isolated from a 2-year-old patient hospitalized with acute gastroenteritis in Kunming, China. The strain TB-Chen was demonstrated having group A-specific antigenicity, a “short” (subgroup II) electropherotype. To investigate its overall genomic relatedness and to determine which group it belonged, the complete genome of strain TB-Chen was determined. Genomic comparison based on amino acid sequence identity and phylogenetic analysis revealed that all 11 gene segments of strain TB-Chen were highly identical (> 91.80%) with the representative G2P[4]/NSP4[A] human strains DS-1, S2, NR1 and IS2, suggesting that this rotavirus strain was derived from human host. Besides, almost all the available representative rotavirus gene segments among group A were analyzed and identified within 15 G-types, 28 P-types, and 6 NSP4 genotypes. This is the first report of group A rotavirus genomic analyses in China and the findings have important implications for rotavirus vaccine development.     

Molecular evolution of West Nile virus in a northern temperate region: Connecticut, USA 1999-2008

West Nile virus (WNV) has become firmly established in northeastern US, reemerging every summer since its introduction into North America in 1999. To determine whether WNV overwinters locally or is reseeded annually, we examined the patterns of viral lineage persistence and replacement in Connecticut over 10 consecutive transmission seasons by phylogenetic analysis. In addition, we compared the full protein coding sequence among WNV isolates to search for evidence of convergent and adaptive evolution. Viruses sampled from Connecticut segregated into a number of well-supported subclades by year of isolation with few clades persisting ≥ 2 years. Similar viral strains were dispersed in different locations across the state and divergent strains appeared within a single location during a single transmission season, implying widespread movement and rapid colonization of virus. Numerous amino acid substitutions arose in the population but only one change, V → A at position 159 of the envelope protein, became permanently fixed. Several instances of parallel evolution were identified in independent lineages, including one amino acid change in the NS4A protein that appears to be positively selected. Our results suggest that annual reemergence of WNV is driven by both reintroduction and local-overwintering of virus. Despite ongoing evolution of WNV, most amino acid variants occurred at low frequencies and were transient in the virus population.     

Isolation and characterization of a novel indigenous intestinal N4-related coliphage vB_EcoP_G7C

Lytic coliphage vB_EcoP_G7C and several other highly related isolates were obtained repeatedly from the samples of horse feces held in the same stable thus representing a component of the normal indigenous intestinal communities in this population of animals. The genome of G7C consists of 71,759 bp with terminal repeats of about 1160 bp, yielding approximately 73 kbp packed DNA size. Seventy-eight potential open reading frames, most of them unique to N4-like viruses, were identified and annotated. The overall layout of functional gene groups was close to that of the original N4 phage, with some important changes in late gene area including new tail fiber proteins containing hydrolytic domains. Structural proteome analysis confirmed all the predicted subunits of the viral particle. Unlike N4 itself, phage G7C did not exhibit a lysis-inhibited phenotype.     

TTSV1, a new virus-like particle isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax

A new virus-like particle TTSV1 was isolated from the hyperthermophilic crenarchaeote Thermoproteus tenax sampled at a hot spring region in Indonesia. TTSV1 had a spherical shape with a diameter of approximately 70 nm and was morphologically similar to the PSV isolated from a strain of Pyrobaculum. The 21.6 kb linear double-stranded DNA genome of TTSV1 had 38 open reading frames (ORFs), of which 15 ORFs were most similar to those of PSV. The remaining 23 ORFs showed little similarity to proteins in the public databases. Southern blot analysis demonstrated that the viral genome is not integrated into the host chromosome. TTSV1 consisted of three putative structural proteins of 10, 20, and 35 kDa in size, and the 10-kDa major protein was identified by mass spectrometry as a TTSV1 gene product. TTSV1 could be assigned as a new member of the newly emerged Globuloviridae family that includes so far only one recently characterized virus PSV.