- Volume 78, Issue 9, 1997

Recently, gene fragments of several novel variants of GB virus A were isolated from the serum of distinct monkey species that had not been experimentally inoculated with an infectious agent. These variants appeared to be species-specific in that sequences isolated within a species were virtually identical, though sequences were strikingly different when compared between each species. In the present study, the nucleotide sequence of one of these variants, GBV-Alab, was extended to neargenome length. Similar to the other GB viruses, GBV-Alab appears to encode a single large polyprotein of 2967 amino acids that is post-trans-lationally cleaved by cellular and viral proteases into the individual viral proteins. The structural proteins are found at the N-terminal end of the polyprotein, while the nonstructural proteins are found at the C teminus. Amino acid sequence comparisons of the large polyprotein demonstrate that GBV-Alab is 74% identical to GBV-A and 48% identical to GBV-C, sharing only marginal identity with GBV-B and HCV-1 at 27%. Examination of the GBV-Alab polyprotein reveals that structural motifs are conserved for a protease, a helicase and a replicase. Phylogenetic analysis of the polyprotein confirms previous results that GBV-Alab is a member of the Flaviviridae, distinct from GBV-B and HCV, though more closely related to GBV-A and GBV-C.

The gene coding for the major capsid protein (VP60) from rabbit haemorrhagic disease virus was expressed in Saccharomyces cerevisiae under the phosphoglycerate kinase promoter. The recombinant VP60 produced in yeast was antigenically similar to the viral polypeptide as determined with a polyclonal serum. Electron microscopic observation of the recombinant yeast-derived antigen revealed the presence of virus-like particles similar in size and appearance to native capsids. Subcutaneous vaccination of rabbits with a single dose of this antigen in the absence of commercial adjuvants conferred complete protection against the haemorrhagic disease.

Forty-seven European bat lyssaviruses (EBL) and two African insectivorous bat lyssaviruses (Duvenhage viruses) were selected for a comparison to be made of their evolutionary relationships. Studies were based on direct sequencing of the PCR-amplified products of the 400 nucleotides coding for the amino terminus of the nucleoprotein. Phylogenetic relationships were analysed after bootstrap resampling using the maximum parsimony and the neighbour-joining methods. Analyses of both the nucleotide and amino acid sequences placed these viruses in three separate clusters, namely genotype 4 (Duvenhage), genotype 5 (EBL1) and genotype 6 (EBL2). Evolutionary analysis of the nucleoprotein gene of EBL1 and EBL2 indicated low intrinsic heterogeneity mainly due to synonymous substitutions. In addition, both EBL1 and EBL2 evolved into at least two genetically distinguishable lineages (a and b) following geographical drifting. We can speculate that subsequently the lineages EBL1a and EBL1b were introduced into parts of northern Europe from two different geographical directions; EBL1b was probably introduced most recently and was from North Africa. Eptesicus serotinus appears to be the principal reservoir for EBL1 and Myotis dasycneme and M. daubentonii the reservoirs for EBL2.

All gene segments of influenza virus share a common feature at their respective termini. Both the 5′- and 3′-terminal sequences are highly conserved and possess partial inverted complementarity. This allows for the formation of a double-stranded duplex, which plays a major role in transcription, replication and packaging of the viral genome. In vitro studies have shown that the viral polymerase binds to short RNA molecules containing these termini. In this study, attempts were made to test whether mini-RNA decoys containing either or both termini can inhibit the activity of the viral polymerase in vivo. RNA molecules containing either the 5′ or the 3′ noncoding sequences were unable to inhibit NS-CAT RNA replication, while mini-RNA decoys consisting of both the 5′ and 3′ noncoding sequences of vRNA or cRNA were able to efficiently inhibit the activity of the viral polymerases expressed from vaccinia virus vectors.

A recombinant baculovirus expressing the nucleocapsid gene (NP) of Newcastle disease virus (NDV), a member of the genus Rubulavirus, has been generated and shown to express the native protein to high levels in insect cells. In contrast to the NP protein of the rubulavirus human parainfluenza virus 2, the NDV protein has been demonstrated by electron microscopy and caesium chloride gradient analysis to be capable of self-assembly in vivo to form nucleocapsid-like structures in the absence of other NDV proteins. These structures, which contained RNA that was resistant to micrococcal nuclease digestion, were also observed when the protein was expressed in E. coli, a phenomenon which was not inhibited by the presence of a 40 amino acid fusion region at the amino terminus of the protein. Further, the formation of these structures was inhibited by the co-expression of the phosphoprotein (P). Therefore, we conclude that the P protein acts as a chaperone, preventing uncontrolled encapsidation of non-viral RNA by NP protein.

Comparison of nonstructural glycoprotein NSP4 gene sequences from 22 rotavirus strains originating from six host species and of 14 different combinations of G and P types revealed the presence of three distinct NSP4 alleles, represented by strains Wa, KUN and AU-1. Genetic distances between any of these alleles (18·0%) were significantly greater than those within each allele (5·5%) and phylogenetic analysis suggested that divergence into three distinct alleles had occurred at about the same time during evolution. While amino acid variation among strains was minimal in the amino-terminal two-thirds of the protein (aa 1–130), variability increased toward the carboxy terminus of the enterotoxic peptide region (aa 114–135) and was greatest between residues 135 and 141. Comparison of the amino acid sequences corresponding to the enterotoxic peptide region between strains isolated from asymptomatic neonates and those from children with diarrhoea failed to identify any conserved changes that correlated with the capacity of the virus to cause disease. Amino acids were relatively conserved in the domains important for viral morphogenesis.

A full-length in vivo infectious cDNA clone of strawberry mild yellow edge-associated potexvirus (SMYEaV) was constructed and used to inoculate Fragaria vesca ‘Alpine’ seedlings and Rubus rosifolius. Both host plants could be infected using particle bombardment or agroinoculation, but not by mechanical inoculation. A method that used potted strawberry plants for particle bombardment resulted in high survival and infection rates. The plants developed systemic infection and virus particles were detected by ELISA and immuno-electron microscopy. Mechanical inoculation of Chenopodium quinoa and C. foetidum with the 35S construct resulted in localized infections. F. vesca ‘Alpine’ indicator plants produced symptoms that were indistinguishable from control plants inoculated with a naturally occurring isolate of strawberry mild yellow edge by graft or aphid transmission. These results suggest that SMYE potexvirus is the causal agent of strawberry mild yellow edge disease. As this virus is capable of causing the disease, we propose the name strawberry mild yellow edge potexvirus, with the acronym SMYEPV, to replace the name strawberry mild yellow edge-associated potexvirus.

The genome of rice grassy stunt virus (RGSV) consists of six RNA segments. The nucleotide (nt) sequences of the two smallest segments, RNAs 5 and 6, were determined and found to comprise 2704 and 2584 nt, respectively. The 5′ - and 3′ -terminal sequences of both RNAs were identical over a length of 21 nt and could potentially form a panhandle-like structure due to intramolecular complementarity. Each RNA segment contained a virus (v) sense open reading frame (ORF) in the 5′-proximate region, and a virus complementary (vc) ORF in the 3′-proximate region, indicating an ambisense coding strategy. The protein encoded by the ORF on the vc strand of RNA5 was identified as the viral nucleocapsid protein (M r 35927). The ORF on the v strand of RNA6 encoded a protein of M r 20581 which represented the major nonstructural protein, previously shown to be produced in RGSV-infected rice tissues. The predicted proteins encoded by RGSV RNAs 5 and 6 were only distantly similar in sequence to the four proteins encoded by RNAs 3 and 4 of other viruses belonging to the genus Tenuivirus. These low sequence similarities, together with the apparently distinct number of genome segments, set RGSV apart from the other tenuiviruses and indicate that it should be placed in a taxonomically separate genus.

In the genomes of two baculoviruses, Spodoptera exigua and S. littoralis multicapsid nucleopoly-hedroviruses (SeMNPV and SpliMNPV, respectively), an open reading frame (ORF) encoding the large subunit of ribonucleotide reductase (RR1) was identified. The predicted amino acid sequences of SeMNPVand SpliMNPV RR1 showed high homology to RR1 proteins from eukaryotes (ca. 70% and 80% similarity, respectively). The amino acid residues thought to be involved in catalytic function were conserved in the baculoviral RR1 ORFs. The RR1 ORFs in SeMNPV and SpliMNPV were located in different genomic positions. In SeMNPV, the RR1 ORF was located upstream of the polyhedrin gene, in an anti-genomic orientation. In SpliMNPV, the RR1 ORF preceded the p74 gene. By searching databanks, sequences homologous to the N terminus of RR1 were also detected upstream of the polyhedrin gene of three other baculoviruses, Mamestra brassicae multicapsid NPV, Panolis flammea multicapsid NPV and Orgyia pseudotsugata single nucleocapsid NPV. The baculovirus type species, Autographica californica multicapsid NPV, however, does not encode RR. A 2·7 kb transcript could be detected throughout infection with SeMNPV, classifying SeMNPV rr1 as an early gene. Primer extension analysis revealed several early and late start sites. None of the major start sites showed similarity to previously characterized baculoviral transcriptional start motifs. Phylogenetic analysis of prokaryotic, eukaryotic and viral RR1 proteins suggested that SeMNPV and SpliMNPV acquired the gene for RR1 from a eukaryotic source, but independently from each other.

The endogenous retrovirus gypsy is controlled by the Drosophila gene flamenco (flam). New insertions of gypsy occur in any individual Drosophila if its mother is homozygous for the flam 1 permissive allele and contains functional gypsy proviruses. The ovaries of flam 1 females also contain high amounts of gypsy RNAs. Unexpectedly however, gypsy derepression does not occur in the flam 1 female germline proper but in the somatic follicular epithelium of the ovary. Since extracts from these females are able to efficiently infect the germ-line of a strain devoid of active gypsy proviruses, we assume that a similar kind of germ-line infection, which would occur inside the flam 1 females themselves, could be required for gypsy insertions to occur in their progeny. This hypothesis was confirmed by electron microscopy observations showing that non-enve-loped intracytoplasmic particles containing gypsy RNAs accumulate in the apical region of the flam 1 follicle cells, close to specific membrane domains to which the gypsy envelope proteins are targeted, whereas both are absent in the flam controls. Low amounts of similar virus-like particles were also observed in flam 1 oocytes, but it is not yet known whether they entered passively or as a result of membrane fusion. This is the first report of the beginning of a retrovirus cycle in invertebrates and these observations should be taken into account when explaining the maternal effect of the flamenco gene on the multiplication of gypsy proviruses.

The development of diagnostic tools for transmissible spongiform encephalopathies (TSEs) would greatly assist their study and may provide assistance in controlling the disease. The detection of an abnormal form of the host protein PrP in noncentral nervous system tissues may form the basis for diagnosis of TSEs. Using a new antibody reagent to PrP produced in chickens, PrP can be readily detected in crude tissue extracts. PrP from uninfected spleen had a lower molecular mass range than PrP from brain, suggesting a lower degree of glycosylation. A simple method for detecting the abnormal form of the protein, PrPSc, in ruminant brain and spleen has been developed. PrPSc was detected in sheep spleen extracts from a flock affected by natural scrapie and was also found in spleens from some, but not all, experimental TSE cases. In spleens from cattle with bovine spongiform encephalopathy (BSE) no PrPSc was detected. It is therefore suggested that there is differential targeting of PrPSc deposition between organs in these different types of TSE infection which, with other factors, depends on strain of infecting agent.