- Volume 71, Issue 4, 1990

The basic protein of Autographa californica nuclear polyhedrosis virus (AcMNPV) is associated with virus DNA in virion nucleocapsids and is produced in infected cells during the late phase of gene expression. A transfer vector was constructed containing the β-galactosidase gene, under the control of a copy of the putative bask protein promoter, in place of the polyhedrin gene within the AcMNPV EcoRI fragment I. After cotransfection of Spodoptera frugiperda cells with the transfer vector and infectious AcMNPV DNA, polyhedrin-negative recombinant viruses were selected which expressed high levels of β-galactosidase. Radiolabelling of infected cell proteins showed that β-galactosidase was expressed at the same time as the viral basic protein, between 8 to 24 h post-infection, with a peak synthesis at 12 to 15 h. These results demonstrated that the temporal regulation of the basic protein promoter was not affected by its position within the virus genome. Furthermore, a new baculovirus vector system is now available for high level expression of foreign genes at earlier times in infected cells.

The putative viral transcriptase p90 in infectious bursal disease virus (IBDV) was shown to form an enzyme- guanylate intermediate which is indicative of guanylyl- transferase activity. The p90-nucleotide bond is most likely to be a phosphodiester linkage, as it resisted treatment with HC1 and NH2OH but was sensitive to NaOH. This is in contrast to phosphoamide bonds formed by reovirus cores. Methyltransferase activity was also demonstrated in IBDV, and is closely associated with transcription, suggesting that p90 may be a multifunctional enzyme.

The genome DNAs of 12 strains of human parvovirus B19, isolated in Japan at two different times, 1981 and 1986 to 1987, were molecularly cloned in the plasmid pUC18. The cloned B19 DNAs were analysed by cleaving with restriction endonucleases, and were classified into several groups by genome type. The restriction endonuclease cutting patterns of B19 strains isolated during 1981 were similar to that of the group IV genome type, and the patterns of those isolated later were similar to that of group II, suggesting a correlation between the genome type and the prevalence. We conclude that the prevalences of B19 infection in Japan during 1981 and in 1986 to 1987 were caused by viruses differing in genome type, and that B19 viruses with similar genome types disseminated widely in Japan during each prevalence.

Parts of the F gene from 16 mumps viruses derived from vaccines and clinical isolates were amplified using the polymerase chain reaction and their nucleotide sequences were determined. Over a region of 111 nucleotides, eight regions of variability were detected with a maximum of six (5·4%) changes occurring between any two virus strains. The Jeryl Lynn and Urabe vaccine strains were clearly different from each other and from wild virus isolated from cases of nonvaccine-associated mumps. In contrast, viruses isolated from the cerebrospinal fluid and throat in cases of meningitis and parotitis following vaccination with the Urabe strain were identical to this strain. We conclude that the vaccine was the source of these infections.

TsO82, a spontaneous temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV) isolated in chick embryo fibroblasts (CEFs), complements the five prototype ts mutants of the virus. The data presented here indicate that the defect in tsO82 is localized in the M gene. The mutation changed a methionine to an arginine at position 51 of the M protein. Only true revertants could be isolated, and their frequency was low, perhaps due to the type of substitution required to return to the wild-type phenotype. TsO82 does not exhibit hypertranscription, in contrast to the data reported for all of the other ts mutants affected in the M protein. Moreover, tsO82 is conditionally ts, since it grows normally in BHK-21 cells at all temperatures. It exhibits no c.p.e. at the non-permissive temperature in CEFs. Our data argue for multiple functions of the M protein of VSV, the domain affected by the tsO82 mutation possibly being implicated both in the shut-off of cellular RNA synthesis, and for the recognition of a cellular factor required for efficient viral RNA synthesis.

Previous studies have shown that the molecules of P protein associated with transcriptionally active Sendai virus nucleocapsids are arranged in discrete clusters. Our study investigates whether or not this localized distribution is due to the existence of only a few P protein binding sites on the nucleocapsid core. We used immunoelectron microscopy to examine whether additional P proteins could bind at locations between the groups of endogenous P proteins. To differentiate between endogenous and added proteins, we constructed a recombinant gene which instructs the in vitro synthesis of a chimeric protein containing the carboxyl-terminal nucleocapsid-binding region of P protein, fused to chloramphenicol acetyltransferase (CAT). Immunogold labelling, using an antibody to the CAT moiety, revealed at the electron microscope level, that the chimeric product bound to nucleocapsids at many sites located over the entire length of the nucleocapsid. This indicated that the localized distribution of P protein molecules is not due to a limited number of P protein binding sites on the nucleocapsid core.

cDNA clones complementary to the 3′-terminal regions of the genomic RNAs of the carlavirus lily symptomless virus and the potexvirus lily virus X (LVX) have been sequenced. The carlavirus RNA sequence contains five open reading frames (ORFs) coding for proteins of Mr 25374, 11631, 6960, 32041 (coat protein) and 16121, which are all very similar in size, amino acid sequence and relative position in the genome to proteins encoded by two different potato carlaviruses. The first four of these proteins also show considerable amino acid sequence similarity to proteins encoded by RNA of potexviruses, and the relative position of the ORFs on the carlavirus genome strongly resembles that in the potexvirus genomes. The LVX cDNA clone contains three ORFs encoding proteins of M r 23574, 11767 and 21569 (coat protein). A small ORF immediately 5′ of the coat protein ORF, which has been found in other potexvirus genomes, is not present in the LVX genome. Thus, the data confirm the close taxonomic relationship between carlaviruses and potexviruses and reveal some differences in genome organization among the potexviruses.

The nucleotide sequence of the genomic RNA of kennedya yellow mosaic tymovirus-Jervis Bay isolate (KYMV-JB) has been determined. The genome of KYMV-JB is 6362 nucleotide residues long and encodes three major open reading frames. The genomic organization and the encoded proteins of KYMV-JB are very similar to those of three other tymoviruses that have recently been reported. Sequence comparisons revealed that the possible replicase proteins of tymoviruses are closely related to those of potexviruses and carlaviruses, suggesting a close evolutionary relationship among these viruses, despite differences in their genome organization and particle morphology.

A serologically distinct member of the tomato spotted wilt virus (TSWV) group was isolated from the hybrid flower crop New Guinea impatiens (Impatiens sp.) and termed TSWV-I. TSVW-I type isolates have frequently been detected in a wide variety of flower crops throughout the United States. TSWV-shares many characteristics with TSWV, such as symptomatology and possession of three ssRNA species (L, M and S of 8·3 kb, 5·2 kb and 3·4 kb, respectively) and three structural proteins (G1, G2 and N of 78K, 52K and 28K respectively). The TSWV-I G1 and G2 glycoproteins were serologically related to the respective proteins of TSWV, but the TSWV-I nucleocapsid or N protein was serologically unrelated to that of TSWV. Hybridization analysis under high stringency conditions revealed no hybridization between clones of TSWV-1 S and M and the S and M RNAs of TSWV, respectively and in addition, a TSWV S clone hybridized only with TSWV S RNA. The cytopathology of TSWV-1 also differed from that of TSWV. TSWV-1- infected tissue primarily contained filamentous structures arranged in paracrystalline arrays, which were also observed by immunosorbent electron microscopy of tissue extracts. The filamentous structures were only trapped by TSWV-1 antibodies. The conserved serological relatedness between TSWV types for G1 and G2, but not N, is consistent with serological analyses of the nairovirus and phlebovirus genera of the Bunyaviridae, the virus family that TSWV most closely resembles.

Virus particles were reassembled in vitro from the proteins of two cucumoviruses, the aphid-transmissible V strain of tomato aspermy virus (VTAV) or the non aphid-transmissible M strain of cucumber mosaic virus (MCMV) with the RNAs of either virus or that of tobacco mosaic virus. Particles reassembled from VTAV protein and RNAs from any of the three viruses were transmitted by the aphid, Myzus persicae, following virus acquisition through membranes. However particles reassembled from MCMV protein and RNAs from any of the three viruses were not transmitted by the aphids. These results indicate that the transmission of cucumoviruses by aphids is determined solely by properties of their protein coats.

Red clover necrotic mosaic dianthovirus (RCNMV) has a genome of two ssRNA species, RNA 1 (4.0 kb) and RNA 2 (1.4 kb). Double-stranded RNA was isolated from Phaseolus vulgaris plants infected separately with five isolates of RCNMV from Czechoslovakia, Sweden, Scotland and England. In each case three species of dsRNA, designated A, B and C in order of increasing mobility, were resolved by gel electrophoresis in non-denaturing conditions. The mobilities of dsRNA species A and B were similar for all the isolates, but the mobilities of the different species of dsRNA C showed some variation. Southern blotting followed by hybridization with cloned probes showed that dsRNA species A and B are related to RNA 1, whereas dsRNA C is related to RNA 2. In gel electrophoresis in denaturing conditions, RNA from dsRNA A had the same mobility as ssRNA 1, whereas RNA from dsRNA species B and C had the same mobility as ssRNA 2. It is concluded that dsRNAs A and C are the doublestranded forms of the genomic RNA 1 and RNA 2 respectively and that dsRNA B is the double-stranded form of a subgenomic RNA which is derived from RNA 1 and has the same size as RNA 2.