Cryphonectria hypovirus 3, a virus species in the family hypoviridae with a single open reading frame

Isolate Grand Haven (GH) 2 is a naturally occurring isolate of the chestnut blight fungus, Cryphonectria parasitica, that is greatly reduced in virulence due to the presence of a double-stranded RNA virus. Unlike many other virus-infected, hypovirulent isolates, GH2 is not substantially reduced in pigmentation, conidiation, or laccase expression compared to its virus-free counterpart. The dsRNA genome of the GH2 virus was cloned, sequenced, and compared to hypovirulence-associated viruses of the family Hypoviridae. GH2 dsRNA is considerably smaller than previously characterized members of the family, 9.8 kb compared to 12.5-12.7 kb for other members. The genome organization of GH2 dsRNA reflected the substantial difference in genome size. Like other members of the family, one strand contained a poly(A)(+) tail at the 3' end and a long sequence with several minicistrons at the 5' end of the same strand. Only a single open reading frame (ORF) of 8622 nucleotides was predicted from deduced translations of the poly(A)(+)-containing strand, however. This contrasts with the two-ORF structures of previously characterized members. Analysis of the deduced ORF of GH2 dsRNA revealed putative proteinase, RNA polymerase, and helicase domains similar to those previously identified in confirmed members of the virus family Hypoviridae. GH2 dsRNA was more distantly related to Cryphonectria hypovirus (CHV) 1-EP713 and CHV2-NB58 than the latter two were to each other but has features in common with each of those viruses. We propose that the GH2 virus be included in this taxon as a member of the genus Hypovirus, representing a strain of a new species, CHV3.     

Polyacrylamide gel electrophoresis of the Kemerovo virus RNA

Ribonuclease (RNase)-resistant RNA was isolated from partially purified Kemerovo virus by gel chromatography and or sucrose density gradient centrifugation. Double-stranded (ds) RNA only was found in the purified viral cores. The RNAs from both sources exhibited the same pattern of distribution in polyacrylamide gels. Ten dsRNA segments were identified. According to the results of coelectrophoresis of the Kemerovo virus and reovirus dsRNAs, the size of Kemerovo virus genome was estimated to be of about 11.7 X 10(6). The grouping of Kemerovo virus double-stranded segments according to their size in polyacrylamide gels corresponded to the 2:4:3:1 pattern.     

Synthesis of Drosophila X virus proteins in cultured Drosophila cells

The genome of Drosophila X virus (DXV) is made up of two segments of dsRNA with molecular weights of 2.3 X 10(6) (A) and 2.2 X 10(6) (B). Agarose gel electrophoresis of RNA fragments produced by S1 nuclease digestion of partially denatured, purified segment A and B indicated that the two genome segments have different nucleotide sequences. The dose-response curve of virus infectivity was linear, indicating that the two genome segments reside in the same particle. The virions contained five polypeptides (VPs) that fell into three molecular weight size classes; large, 110K; medium, 49K and 45K; and small, 34K and 27K. Virus infection of Drosophila cells induced the synthesis of five infected cell polypeptides (ICPs); 110K, 67K, 49K, 34K, and 27K. Pulse-chase experiments and peptide mapping revealed that four of these (110K, 67K, 34K, and 27K) were primary gene products and that ICP 49 was generated by post-translational cleavage of ICP 67. The major capsid protein, VP 45, was cleaved from ICP 49; however, this cleavage was incomplete because both polypeptides were present in purified virus. The results suggest that the strategy for protein synthesis of DXV differs from that of other dsRNA viruses.     

Isolation of a recombinant between simian and bovine rotaviruses

A recombinant between simian rotavirus, simian agent 11 (SA-11) and bovine rotavirus, neonatal calf diarrhoea virus (NCDV), was obtained by mixed infection of MA-104 cells with NCDV and u.v.-irradiated SA-11 virus, and isolation of a plaque formed in the presence of anti-NCDV serum. The genome of the recombinant contained dsRNA segments 4, 5 and 10 derived from SA-11 virus and segments 1, 2, 3, 6 and 11 derived from NCDV, and segments 7, 8 and 9 of undetermined origin. Polypeptides VP4, VP5, VP7a, VP7b, NCVP1 and MCVP3 were derived from SA-11 virus and polypeptides VP1, VP2, VP3, VP6, VP8, NCVP2a and NCVP2b from NCDV. Haemagglutination of the recombinant was inhibited and its infectivity neutralized by the antiserum against SA-11 virus but not by anti-NCDV serum.     

The housefly virus contains double-stranded RNA

The housefly virus (HFV) was shown to contain double-stranded RNA (dsRNA) when examined by fluorescent staining with acridine orange (AO). The pale green color observed was not affected by digestion with single-stranded specific nuclease. In contrast, denatured wet, but not dried virus samples fluoresced red. Viral RNA, extracted and analyzed by polyacrylamide gel electrophoresis, was resolved into 10 segments, which were not affected by predigestion with RNase, confirming the ds nature of the genome. The HFV thus belongs to the family Reoviridae. By comparison with dsRNA segments from reovirus type 3, Dearing strain, the housefly virus genome is estimated to have a molecular weight of 17 to 18 x 10(6) daltons.     

Analysis of the terminal sequences of the genome segments of four orbiviruses

The dsRNA genome segments of bluetongue virus (BTV) types 1 and 20 and Ibaraki virus (a member of the epizootic haemorrhagic disease (EHD) serogroup) have conserved sequences of six bases at both of their 3' termini. One strand of all the genome segments analysed ends in 3'CAUUCA ... 5' while the other strand ends in 3'CAAUUU ... 5'. These conserved sequences are identical to those previously reported for BTV types 10 and 11 (A. Kiuchi, C. D. Rao, and P. Roy (1983), "Double-Stranded RNA Viruses" (R. W. Compans and D. H. L. Bishop, eds.), pp. 55-64. Elsevier, New York; C. D. Rao, A. Kiuchi, and P. Roy (1983), J. Virol. 46, 378-383). The 3' terminal sequences of segments 3 and 10 of the BTV type 1 genome were confirmed by the detection of exactly complementary sequences at the 5' termini of the ssRNA strands of opposite polarity. This also confirmed for these dsRNA segments (and by analogy for all the genome segments of these viruses) that the dsRNA molecules are fully base paired end to end. Using in vitro synthesised mRNA of BTV type 1 in annealing experiments with the two ssRNAs separated from each of the individual genome segments, it was shown that in each case the strand ending in 3'CAUUCA ... 5' is of the same polarity as the mRNA (+ve), while the strand ending in 3'CAAUUU ... 5' is of the opposite (-ve) polarity. The fourth virus analysed (Tilligerry virus, a member of the Eubenangee serogroup) only had five conserved bases at the 3' termini of one strand of its genome segments (3'CAU-CA ... 5') and three conserved bases at the 3' termini of the other strand (3'CA--U ... 5'). Considerable sequence homology was found in the near-terminal nonconserved regions of comparable genome segments from the different viruses, particularly between the different BTV types. There was little evidence, however, for absolute conservation of "segment specific" sequences in these regions of the RNA.     

The complete genomic sequence of a novel mycovirus from Rhizoctonia solani AG-1 IA strain B275

The complete genome of a novel mycovirus, Rhizoctonia solani dsRNA virus 1 (RsRV1) was sequenced and analyzed. It is composed of two dsRNA genome segments, 2379 bp and 1811 bp in length, which were referred to as RsRV1-1 and RsRV1-2, respectively. RsRV1-1 contains a single open reading frame (ORF1), which has a conserved RNA-dependent RNA polymerase (RdRp) domain, whereas RsRV1-2 contains a single ORF2, which might encode a multifunctional protein. The genome organization of RsRV1 is similar to that of members of the family Partitiviridae. However, phylogenetic analysis indicated that RsRV1 formed a distinct clade together with three other unclassified viruses, suggesting that RsRV1 may belong to a new family of dsRNA mycoviruses. This is the first report of the full-length nucleotide sequence of a novel dsRNA mycovirus, RsRV1, infecting R. solani AG-1 IA strain B275, the causal agent of rice sheath blight.     

The H1 double-stranded RNA genome of Ustilago maydis virus-H1 encodes a polyprotein that contains structural motifs for capsid polypeptide, papain-like protease, and RNA-dependent RNA polymerase.

The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light). The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences. As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype. The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins. The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa. The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses. The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomyces cerevisiae viruses ScV-L1 and -La.     

Construction of carrier state viruses with partial genomes of the segmented dsRNA bacteriophages

The cystoviridae are bacteriophages with genomes of three segments of dsRNA enclosed within a polyhedral capsid. Two members of this family, Phi6 and Phi8, have been shown to form carrier states in which the virus replicates as a stable episome in the host bacterium while expressing reporter genes such as kanamycin resistance or lacalpha. The carrier state does not require the activity of all the genes necessary for phage production. It is possible to generate carrier states by infecting cells with virus or by electroporating nonreplicating plasmids containing cDNA copies of the viral genomes into the host cells. We have found that carrier states in both Phi6 and Phi8 can be formed at high frequency with all three genomic segments or with only the large and small segments. The large genomic segment codes for the proteins that constitute the inner core of the virus, which is the structure responsible for the packaging and replication of the genome. In Phi6, a carrier state can be formed with the large and middle segment if mutations occur in the gene for the major structural protein of the inner core. In Phi8, carrier state formation requires the activity of genes 8 and 12 of segment S.     

Characterization of different viruses infecting the marine harmful algal bloom species Phaeocystis globosa

Twelve lytic viruses (PgV) infecting the marine unicellular eukaryotic harmful algal bloom species Phaeocystis globosa were isolated from the southern North Sea in 2000-2001 and partially characterized. All PgV isolates shared common phenotypic features with other algal viruses belonging to the family Phycodnaviridae and could be categorized in four different groups. Two main groups (PgV Group I and II) were discriminated based on particle size (150 and 100 nm respectively), genome size (466 and 177 kb) and structural protein composition. The lytic cycle showed a latent period of 10 h for PgV Group I and latent periods of 12 h and 16 h for PgV Group IIA and IIB. Host specificity and temperature sensitivity finally defined a fourth group (PgV Group IIC). Our results imply that viral infection plays an important role not only in P. globosa dynamics but also in the diversity of both host and virus community.     

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses

An aquareovirus was isolated from several fish species in the USA (including healthy golden shiners) that is not closely related to members of species Aquareovirus A, B and C. The virus, which is atypical (does not cause syncytia in cell cultures at neutral pH), was implicated in a winter die-off of grass carp fingerlings and has therefore been called 'American grass carp reovirus' (AGCRV). Complete nucleotide sequence analysis of the AGCRV genome and comparisons to the other aquareoviruses showed that it is closely related to golden ide reovirus (GIRV) (>92% amino acid [aa] identity in VP5(NTPase) and VP2(Pol)). However, comparisons with grass carp reovirus (Aquareovirus C) and chum salmon reovirus (Aquareovirus A) showed only 22% to 76% aa identity in different viral proteins. These findings have formed the basis for the recognition of AGCRV and GIRV as members of a new Aquareovirus species 'Aquareovirus G' by ICTV. Further sequence comparisons to other members of the family Reoviridae suggest that there has been an 'evolutionary jump,' involving a change in the number of genome segments, between the aquareoviruses (11 segments) and coltiviruses (12 segments). Segment 7 of AGRCV encodes two proteins, from two distinct ORFs, which are homologues of two Coltivirus proteins encoded by genome segments 9 and 12. A similar model has previously been reported for the rotaviruses and seadornaviruses.     

Electrophoretic separation of dsRNA genome segments from Fiji disease and maize rough dwarf viruses

By employing two different buffer solutions for polyacrylamide gel electrophoresis, all genome segments of Fiji disease and maize rough dwarf virus were separated. Fiji disease virus contains ten genome segments with approximate genome molecular weights of 19.26 x 10(6) and 19.85 x 10(6), depending on the buffer employed for electrophoresis. Maize rough dwarf virus possesses ten dsRNA segments and according to the buffer employed for electrophoresis the approximate molecular weights of the genome were 18.91 x 10(6) and 19.61 x 10(6). In the samples of maize rough dwarf virus analysed, the evidence indicates that two types of virions were present which were distinguished by a slight difference in molecular weight for segment 10. The electrophoretic patterns for all dsRNA components of Fiji disease and maize rough dwarf viruses were very similar suggesting a close relationship between them.     

A large inverted duplicated DNA region associated with an amplified oncogene is stably maintained in a YAC

In the polyoma virus (Py) transformed 3B rat cell line the Pyoncogene and adjacent cellular DNA are amplified in arrays ofvery large inverted duplications. A region of the 3B amplifiedDNA was cloned as a 550 kb insert in a Yeast Artificial Chromosome(YAC) vector, designated y3B01. Analysis of the y3B01 clonedinsert revealed it contained a large inverted duplicated DNAregion which was approximately 400 kb in size (two palindromicarms of about 200 kb). At least 420 kb of the 550 kb YAC inserthas been identified as being derived from the 3B amplified DNAand the amplicon in 3B cells is at least 220 kb in size. NoDNA instability of the y3B01 YAC clone was detected. The y3B01DNA replicated as efficiently as yeast chromosomes and was structurallystable in yeast cells during more than 30 cell divisions. Comparisonof the restriction endonuclease maps of the inverted duplicatedregion of the y3B01 DNA insert and the amplified 3B genomicDNA did not reveal any gross differences suggesting that norearrangements had occurred during or after the cloning intothe YAC vector. These results suggest that large inverted duplications,which can show instability in prokaryotic cloning systems, canbe stably cloned and maintained in YAC vectors in yeast.     

Double-stranded RNAs in mitochondrial extracts of stem rusts and leaf rusts of cereals

Summary Double-stranded ribonucleic acids (dsRNA) were isolated from mitochondria in urediosporelings of three cereal stem rusts, Puccinia graminis f. sp. tritici, f. sp. secalis and f. sp. avenae, and two cereal leaf rusts, P. recondita f. sp. tritici and P. coronata f. sp. avenae, and analyzed by agarose gel electrophoresis. The double strandedness of the RNA molecules was characterized by nuclease treatments (RNase A, DNase 1 and S1 nuclease) and CF-11 cellulose column chromatography. No interspecific variation in multisegments of dsRNA was observed among races of each forma specialis. As to the interspecific variation, although each of three forma specialis of Puccinia graminis had similar dsRNA segments, 4.8, 5.0 and 5.2 kb, wheat leaf rust and oat crown rust had additional dsRNA segments of 2.7, 2.8, 5.8 and 6.0 kb. The presence of a dsRNA segment of 5 kb size in all isolates and species examined indicates that this unique segment can be a molecular marker for the rust family, Uredinales. Dot-blot hybridization indicated that there is no sequence homology between dsRNA segments and mitochondrial DNA.