- Volume 73, Issue 11, 1992

Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.

We determined the susceptibility of monocytic cell lines to infection with viral strains derived from two infectious clones of simian immunodeficiency virus isolated from a mandrill. One of the strains, which replicates poorly in T cell lines, was found to grow more rapidly than the other in these cells. The viral determinant for this property was genetically mapped within the env gene encoding a surface protein. Six amino acid substitutions identified appeared to be located outside of the domains corresponding to human immunodeficiency virus type 1 env functional domains such as the CD4-binding and V3 loop regions.

The host ranges of the Rous sarcoma virus (RSV) pseudotype RSV(HPRS-103) of a novel avian leukosis virus (ALV), strain HPRS-103, and representative RSV pseudotypes of subgroups A to F, have been determined in embryo fibroblasts from 12 avian species. Domestic fowl, red jungle fowl, Sonnerat’s jungle fowl and turkey were susceptible to infection by RSV(HPRS-103); ring-necked pheasant, Japanese green pheasant, golden pheasant, Japanese quail, guinea-fowl, Peking duck, Muscovy duck and goose were resistant. The host range pattern of RSV(HPRS-103) differs from those of viruses of subgroups A to G and I, and provides support for placing the HPRS-103 strain of ALV in a new envelope subgroup, designated J.

Anti-dengue 2 virus core protein monoclonal antibodies (MAbs) reacted with antigens in the cytoplasm and in, or on, the nucleus of dengue 2 and dengue 4, but not dengue 1, dengue 3, Kunjin or Murray Valley encephalitis virus-infected cells. These MAbs also reacted with the core protein from dengue 1, 2 and 4 virions in Western blots. The antigens detected by these MAbs could not be detected in uninfected or heat-shocked cells, but were first detected in infected cells approximately 32 h post-infection. PEPSCAN epitope mapping suggested that all the MAbs react with a region of the dengue 2 virus core protein (9RNTPFNMLKRE19) which is adjacent to a putative nuclear localization sequence (6KKAR9) and spans a possible second site for the initiation of synthesis of core protein (12PFN↓MLKR18).

On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.

The pathogenicity of two rotavirus variants, 4F and 4S, obtained following adaptation to cell culture of rotavirus from a diarrhoeic pig in China, was compared by serial passage in 24 gnotobiotic piglets. The rotavirus variants have markedly different growth characteristics in vitro, and their genome profiles differ only in the relative migration of genes 4. Both cell culture-grown variants replicated to an equal extent in gnotobiotic piglets and neither caused disease, although weight gain was slightly affected in piglets inoculated with the 4F variant. During five serial pig-to-pig passages, variant 4F became highly pathogenic at the fourth and fifth passages, causing severe diarrhoea and weight loss, and premature death in two animals. Piglets inoculated with rotavirus variant 4S remained healthy during all passages although weight gain was slightly affected. Mean duration and peak infectivity titres of virus shedding were similar for both variants. Thus, variant 4F, which grew slowly and produced small plaques in vitro and had the faster migrating gene 4, was pathogenic in pigs, whereas variant 4S was apathogenic.

Three monoclonal antibodies (MAbs) obtained from inoculation of mice with either a serotype 1 human rotavirus or rotavirus SA11 (serotype 3) inhibited the in vitro transcription of rotavirus SA11. Two of the MAbs exhibited a biphasic inhibitory response. Removal of antibody from MAb preparations by adsorption with Sepharose-Protein G reduced the inhibitory activity completely for all three MAb preparations. Analysis by radioimmunoprecipitation and Western blotting indicated that all three MAbs reacted with VP6. All MAbs also reacted with four group A rotavirus serotypes by ELISA, but did not cross-react with reovirus type 1, poliovirus type 2 or MA-104 cell lysates. Transcription of four rotavirus serotypes as well as epizootic diarrhoea of infant mice rotavirus was inhibited when tested with two of the MAbs. Transcription of both purified single-shelled virus and purified heat-activated double-shelled SA11 rotavirus was inhibited by purified MAb. Our results indicate that these MAbs can be used effectively to study the events associated with rotavirus transcription.

Bluetongue virus (BTV) VP6 is often resolved into two closely migrating bands by SDS-PAGE (VP6 and VP6a). RNA segment 9 of BTV-serotype 1 South Africa (encoding VP6) has been cloned as cDNA, and the complete sequence has been determined. Expression of this clone both in vitro and in tissue culture produced the same polypeptide doublet as seen previously in extracts from BTV-infected cells. Modification of the cDNA, including the removal of the first initiation codon, demonstrated that the two forms of VP6 are derived from initiation of protein synthesis at two distinct sites and not by post-translational modification.

Scrapie strain replication in the nerve growth factor-induced, differentiated PC12 cell culture system was examined. Differences in replication between mouse-derived agents were demonstrated, with the 139A scrapie strain yielding 100- to 1000-fold higher levels of infectivity than the ME7 scrapie strain. Replication was not detected in PC12 cells infected with either the hamster-derived 263K or rat-derived 139R scrapie strains. Studies on the neurotransmitters in infected PC12 cells demonstrated that the adrenergic pathway was unchanged but the cholinergic pathway was altered. Furthermore, the degree of alteration correlated with the level of scrapie strain replication. Comparison of infectivity titres and enzymatic changes in ME7-infected PC12 cells with those in Chandler agent-infected mouse neuroblastoma cells suggests that the significant changes in neurotransmitter levels in cultures exhibiting low titres of infectivity involve factors in addition to strain replication. The variation in the range of scrapie strain replication in PC12 cells is discussed in relationship to species barrier, cell targeting, genetic susceptibility and species strain specificity. These studies further emphasize the value of the PC12 cell model system in examining the scrapie strain-host cell interaction and in addition support the concept of variation among scrapie strains.

The coding sequence for the entire 200K polyprotein of cowpea mosaic virus (CPMV) B-RNA was expressed in insect cells by using baculovirus expression vectors. The 200K polyprotein, which harbours all virus functions required for RNA replication, is completely cleaved into 170K and 32K products by the 24K protease activity contained within the polyprotein. Further processing of the 170K protein into CPMV-specific products of 60K, 84K, 87K, 110K and 112K occurred to a limited extent, similar to that observed in cowpea cells. Electron microscopy of insect cells in which the 200K protein was produced revealed the presence of membranous vesicles and electron-dense structures which were not seen in cells infected with wild-type baculovirus. Similar cytopathic structures develop in the cytoplasm of CPMV-infected cowpea cells and are thought to be the site of membrane-bound viral RNA replication. The electron-dense structures in insect cells could be preferentially labelled with several CPMV-specific antisera and Protein A-gold. Since electron-dense structures were not observed in cells in which the 170K protein only was produced, it seems that the 32K protein has a role in keeping the B-RNA-encoded proteins in these structures together. Membranous vesicles were also observed in insect cells in which the 60K protein only was produced. Use of specific antibodies and Protein A-gold showed that the 60K protein is associated with these vesicles, indicating that the 60K protein may induce the formation of vesicles. Although proteolytic processing of the 200K polyprotein and the induction of cytopathic structures indicate that the CPMV proteins produced in insect cells are functional, it has not been possible to demonstrate RNA polymerase activity in extracts of these cells using an oligo(U)-primed assay. The results indicate that in the assay an additional component is lacking and/or that the CPMV polymerase is not able to start RNA synthesis on an exogenous template.

The complete RNA genome of turnip mosaic potyvirus (TuMV) was amplified by seven consecutive reverse transcriptase-polymerase chain reactions and cloned into pUC9. The viral RNA is 9830 nucleotides long and contains a single open reading frame (ORF) of 9489 bases encoding a large polyprotein of 3863 amino acids with a calculated M r of 358000. The non-coding region (NCR) preceding the ORF is 129 nucleotides long and has a high AU content (70%). Its predicted secondary structure is characterized by a hairpin loop with a free energy loss of -69.9 kJ/mol. The termination codon is followed by an AU-rich NCR of 209 bases, excluding the poly(A) tail. Seven potential nuclear inclusion a proteinase (NIa-Pro) recognition heptapeptides are found in the polyprotein. Their sequences agree with consensus potyviral NIa-Pro cleavage sequences except for that at the 6K-VPg site, which is characterized by a glutamic acid residue preceding the hydrolysed peptide bond. The TuMV proteins are similar to their corresponding potyviral proteins.

The complete sequence of the tomato spotted wilt virus (TSWV) M RNA segment has been determined. The RNA is 4821 nucleotides long and has an ambisense coding strategy similar to that of the S RNA segment. The M RNA segment contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted size of 33.6K, and one in the viral complementary sense which encodes the precursor to the G1 and G2 glycoproteins, with a predicted size of 127.4K. Both ORFs are expressed via the synthesis of subgenomic mRNAs that possibly terminate at a stable hairpin structure, located in the intergenic region. The precursor for the glycoproteins contains a sequence motif (RGD) which is characteristic of cellular attachment domains. Significant sequence homology was found between the G1 glycoproteins of members of the genus Bunyavirus and a corresponding region in the glycoprotein precursor of TSWV, indicating a close evolutionary relationship between these viruses. With the elucidation of the M RNA sequence, the complete nucleotide sequence of TSWV has been determined. TSWV represents the first member of the Bunyaviridae shown to contain two ambisense RNA segments.

To determine the structural requirements for cucumber mosaic virus (CMV) satellites to elicit lethal tomato necrosis, three satellite variants D, S and Y were used in the construction and cloning of chimeric cDNAs. D and S are necrogenic and non-necrogenic ‘prototype’ variants, respectively, and Y possesses the 3′ conserved necrosis-determining region but does not cause lethal tomato necrosis. Its 5′ half harbours an insertion/deletion region that results in a molecule about 30 nucleotides longer than other variants. Tomato bioassays were conducted with RNA transcripts of all six chimeric combinations of the 5′ and 3′ halves of the three satellite variants divided by a common restriction site, as well as with a mutated chimera. None of the chimeras containing the 5′ half of Y induced lethal necrosis in tomato even when their 3′ halves were that of the D variant with the conserved necrogenic element. Chimeras with the 3′ half of Y elicited only partial or restricted necrosis which was much less severe than that induced by prototype variant D, and often was not lethal. Site-directed mutation of a single nucleotide in proximity to the necrogenic element of such a chimera containing the 3′ half of Y restored much lethal necrogenicity. The results revealed the presence of structural elements in CMV satellite variant Y that modulate or even suppress the expression of the 3′ conserved necrosis-determining element. They indicate that in CMV satellites widely separated sequence elements constituting a three-dimensional requirement are responsible for eliciting lethal necrosis in tomato.

An antiserum was raised against a fusion protein containing the C-terminal half of the protein (P3) encoded by the satellite RNA of grapevine fanleaf virus (GFLV; F13 isolate) and the N-terminal portion of the CI repressor of phage λ. This antiserum specifically recognized P3 synthesized in the in vitro wheatgerm translation system and also in infected Chenopodium quinoa plants. In these plants, the amount of virus increased for 10 days, then remained constant for up to 21 days, whereas P3 was detected transiently, reaching its maximum on day 10.

Leaves of plants infected by tobacco mosaic virus (TMV) strain U1, TMV strain M, tomato mosaic virus strain Dahlemense and tobacco mild green mosaic virus strain U2 were examined for the presence and intracellular distribution of their capsid and 126K/183K proteins by immunoblotting and immunogold electron microscopy. The bulk of the capsid protein was found in the virus bundles (crystals), although small amounts were found in the chloroplasts and nuclei of cells infected by some of these tobamo-viruses. The 126K protein of TMV-U1 and -M was localized in the X-bodies, whereas in cells infected by the other two viruses which induce no X-bodies, the 126K protein was found to be associated with virus bundles.

X-ray diffraction from oriented specimens of purified preparations of particles of heracleum latent clostero-virus (HLV) showed that they have a helical arrangement of protein subunits, and that the structure repeats in five helical turns in which there are 5q ± 1 protein subunits, where q is an integer. The pitch of the helix was estimated to be 3.26 (±0.10) nm. Optical diffraction patterns from electron micrographs of HLV particles give an estimated pitch of 3.3 (±0.2) nm and show that the number of subunits in the repeat period is 5q-1, where q has a value of 8 or 9. Optical diffraction from electron micrographs of particles of a second closterovirus, heracleum virus 6, shows that they too have a helical structure which repeats in five turns, in which there are 5q ± 1 protein subunits. The estimated pitch of the primary helix is 3.6 (± 0.2) nm, and the estimate of q is 9.