- Volume 68, Issue 1, 1987

The DNA sequence was determined of the HindIII l fragment of herpes simplex virus type 2 (HSV-2), which is located in the short unique region of the HSV-2 genome. HindIII l was found to comprise 9629 base pairs. Comparison with the previously determined corresponding sequence for herpes simplex virus type 1 (HSV-1), and limited mRNA mapping, showed that HindIII l contained six genes (termed US2 to US7) and part of another (US8). The HSV-1 and HSV-2 sequences were found to be generally colinear, with one major exception: the HSV-2 DNA contained an extra sequence of about 1460 base pairs, in the coding region of gene US4. By use of an antiserum raised against an oligopeptide representing amino acids near the C terminus of the predicted HSV-2 US4 polypeptide it demonstrated that this gene encodes the virion glycoprotein gG-2, while HSV-1 US4 encodes a much smaller virion glycoprotein with homology to the C-terminal portion of gG-2. Quantitative comparisons of the HSV-2 HindIII l and corresponding HSV-1 sequences showed that they had diverged by point mutation and by local addition and deletion, as well as by the major change in genes US4. It was found that within the HSV-2-specific part of gG-2 there was a locality showing sequence similarity to a glycoprotein of pseudorabies virus (gX), and weaker similarity to glycoproteins D of HSV-1 and HSV-2. These data were interpreted to suggest, first, that HSV-2 US4 represents an ancient gene of alphaherpesviruses, and, more tentatively, that the evolution of the genes for gG and gD may have proceeded through a duplication event.

SUMMARY

The thymidine kinase (TK) gene from herpes simplex virus type 1 strain SC 16 was cloned into bacteriophage M13 mp8 so that functional HSV-1 TK was expressed in bacteria infected with the recombinant bacteriophage, M13/TK. Oligonucleotide site- directed mutagenesis was then employed to introduce single nucleotide changes into the TK gene in M13/TK in order to alter the codon for cysteine 171 in the wild-type enzyme to a codon specifying either serine or glycine. Analysis of the mutant enzymes in bacterial extracts showed that these substitutions had little effect on the activity of the enzyme, indicating that the side chain of this residue is not involved in nucleoside binding and is not essential for the catalytic activity of the enzyme.

SUMMARY

Sequences encoding the surface projection glycoprotein of the coronavirus, murine hepatitis virus (MHV), strain JHM, have been cloned into pAT153 using cDNA produced by priming with specific oligonucleotides on infected cell RNA. The regions of three clones pJMS1010, pJS112 and pJS92, which together encompass the surface protein gene have been sequenced by the chain termination method. The sequence of the primary translation product, deduced from the DNA sequence, predicts a polypeptide of 1235 amino acids with a molecular weight of 136600. This polypeptide displays the features characteristic of a group 1 membrane protein; an amino-terminal signal sequence and carboxy-terminal membrane and cytoplasmic domains. There are 21 potential glycosylation sites in the polypeptide and a cysteine-rich region in the vicinity of the transmembrane domain. During maturation proteolytic processing of the polypeptide occurs and at positions 624 to 628 the sequence Arg-Arg-Ala-Arg- Arg is found, which is similar to a number of basic sequences involved in the cleavage of enveloped RNA virus glycoproteins. The fusogenic properties of the MHV surface protein do not appear to correlate with a strongly hydrophobic region at the putative amino terminus of the carboxy-terminal cleavage product.

The nucleotide sequence determination of the genome of the Beaudette strain of the coronavirus avian infectious bronchitis virus (IBV) has been completed. The complete sequence has been obtained from 17 overlapping cDNA clones, the 5′-most of which contains the leader sequence (as determined by direct sequencing of the genome) and the 3′-most of which contains the poly(A) tail. Approximately 8 kilobases at the 3′ end of this sequence have already been published. These contain the sequences of mRNAs A to E within which are the genes for the spike, the membrane and the nucleocapsid polypeptides: the main structural components of the virion. The remainder of the sequence, equivalent to the ‘unique’ region of mRNA F, is some 20 kilobases in length and is thought to code for a polymerase or polymerases which are involved in the replication of the genome and the production of the subgenomic messenger RNAs. This sequence contains two large open reading frames, potentially coding for polypeptides of molecular weights 441000 and 300000. Unlike other large open reading frames in the virus, the 300000 open reading frame appears to have no subgenomic RNA associated with it which would allow it to be at the 5′ end of an mRNA species. Because of this, and because of the characteristics of the sequence in the region immediately upstream of its start codon, other mechanisms of translation, such as ribosome slippage, must be postulated.

SUMMARY

There are many distinct strains of scrapie agent, identified by their relative incubation periods and quantitative and qualitative neuropathological properties in inbred mice of particular genotypes. When serially passaged under specified conditions of mouse strain, route of infection and dose of infectivity these properties are stable. However, they may change in a predictable manner if the passage strategy is altered.

The scrapie strain 87A shows what has previously been defined as Class III stability; it is stable when passaged at low dose in C57BL mice, but often suddenly changes its properties in the course of a single passage if high doses are used, always resulting in the same new strain. The latter, designated 7D, has shorter incubation periods and more extensive pathology than 87A, properties which are subsequently stable on serial passage even at high dose. This phenomenon has been seen repeatedly using scrapie isolates from six different natural cases in five different breeds of sheep. These isolates are closely similar in all their properties, showing them to be independent isolations of the 87A strain; there have been no isolations of 87A in which the phenomenon did not occur. On the other hand, none of the many other scrapie strains used in the same laboratory have shown this change. 87A brain samples consistently behave as if they contain 87A together with a smaller amount of 7D. This is so even after 87A has previously been passaged at high dilution, well beyond the limiting dilution for 7D, a procedure which would eliminate any minor agent strain originally present in the isolate. Therefore it is highly likely that the 7D in tissues of mice infected with 87A is generated de novo at each passage by mutational change from 87A during the incubation period. The established fact that many different strains exist and the considerable evidence that mutation can occur lead to the conclusion that scrapie agent has its own independently replicating genome.

SUMMARY

Nineteen monoclonal antibodies produced to dengue type 4 virus (DEN-4) strain 4328-S were tested for their ability to mediate antibody-dependent infection enhancement (ADE) with seven DEN-2 strains in P-388D1 mouse macrophage-like cells. In this first study of the distribution of enhancing epitopes on multiple DEN-2 strains reacted with monoclonal antibodies to a different serotype (DEN-4), DEN-4 monospecific antibodies produced ADE with DEN-2 viruses, indicating the presence of DEN-4-like determinants on DEN-2 viruses. Analysis differentiated at least one and possibly more DEN-2 strain subgroups, one of which (isolates AHF-110 and AHF-191) was previously identified by DEN-2 monoclonal antibody analysis. The study demonstrates the heterogeneous distribution of dengue complex and DEN-4 epitopes on DEN-2 strains. Monoclonal antibodies are valuable tools for study of the biology of ADE and its relation to dengue shock syndrome.

SUMMARY

Primary brain cell cultures prepared from newborn mice were infected with Semliki Forest virus (SFV). The effects of interferon (IFN-α β) treatment on SFV replication, SFV and major histocompatibility complex (MHC) class I antigen expression, and susceptibility to lysis by SFV-specific cytotoxic T lymphocytes (CTL) were determined.

The IFN-αβ treatment prevented replication of SFV as determined by incorporation of [3H]uridine into SFV RN A and very markedly reduced the expression of SFV antigens on the cell surface as determined by lysis with antibody and complement or indirect immunofluorescence. However, IFN-aJ? increased expression of MHC class I antigens, measured by indirect immunofluorescence and as assessed indirectly by susceptibility to killing by alloreactive T cell lines. SFV infection had no effect on MHC class I expression in either IFN-αβ -treated or -untreated cells. The infected IFN-αβ-untreated brain cells were susceptible to killing by the CTL at effector/target ratios in the range 3 to 30. The killing was MHC antigen-restricted, and uninfected cells were not killed. A target cell (YAC) highly susceptible to natural killer cell cytotoxicity was not killed by the CTL. IFN-αβ treatment prior to SFV infection resulted in an augmentation of lysis by the CTL, indicating that even where SFV antigen expression is reduced, in the context of enhanced MHC class I expression brain cells remain susceptible to CTL killing.

SUMMARY

Four temperature-sensitive(ts) mutants of the A7 strain of Semliki Forest virus (SFV) have been isolated. All mutants were defective in RNA synthesis at the restrictive temperature (39 °C) compared to the permissive temperature (30 °C). Since the body temperature of mice fluctuates between 37 °C and 39 °C, multiplication was also examined at 37 °C; only the multiplication of ts4 was restricted. After intraperitoneal infection of 8-day pregnant mice, the wild-type induced rapid abortion.Ts4 and ts26 had no effect on embryonic development. Litters born to ts4-infected mothers developed no postnatal immunity whereas 50 % of litters from ts26-infected mothers were immune. Unlike the wild-type, tsl4 induced the same or higher virus titres in placental tissue in most mice than in foet al tissue. Ts22 and ts14 induced a range of development defects, including developmental arrest, mummification, abortion and postnatal death. Most surviving offspring were immune. Although ts4 induced no viraemia, tsl4, ts22 and ts26 induced a lower titre but longer lasting viraemia than the wild-type. It is concluded that infections of pregnant mice with tsl4 and ts22 in particular are good models for analysis of the mechanism of virus-induced developmental defects.

SUMMARY

Mixed infections of secondary rhesus monkey kidney cells with human rotaviruses carrying rearranged genomes and with bovine rotavirus yielded a high percentage of reassortants. The genotypes of 511 plaque-purified clones raised in either MA104 or BSC-1 cells have been determined and the frequencies of different genotypes have been calculated. It was found that (i) reassortants did not emerge at random; (ii) there was non-random association of certain genes; (iii) the cell line used to isolate reassortants influenced the result, i.e. host cell factors had a selective effect on a recombinational mixture.

SUMMARY

The nucleocapsid protein of bovine rotavirus was shown to exist in trimeric units in both the virus particle and in infected cells, with the subunits linked by non-covalent interactions. These trimeric units complex further by disulphide bridges into larger units which may represent the hexameric structures observed by electron microscopy. Visualization of various nucleocapsid protein complexes was also achieved on polyacrylamide gels by treating virus preparations with urea at 37 °C or boiling in the presence and absence of 2-mercaptoethanol. Since virus particles devoid of nucleic acid were also broken down into trimeric subunits by such treatments, assembly of virus particles appears not to require an RNA-protein interaction. Four nucleocapsid-specific monoclonal antibodies with low neutralizing ability reacted with the monomeric (45000 mol. wt., 45K), dimeric (90K), trimeric (135K) and trimeric pair (270K) subunits, indicating that a site responsible for neutralization is probably exposed after assembly of these subunits. Analysis of radiolabelled virus revealed that a high proportion (80 %) of infectious particles could be immunqprecipitated by these monoclonal antibodies, suggesting that the virus particles are either partially doubleshelled or have the nucleocapsid exposed on the surface. The monoclonal antibodies also cross-reacted with the nucleocapsid proteins of simian (SA11), pig (OSU), bovine (NCDV and UK) and human (Wa and ST4) rotaviruses in an immunoblot ELISA reaction. Since these six viruses belong to two different subgroups, it is likely that the antibodies did not recognize the subgroup-specific site, but a shared exposed antigenic determinant. Due to the hexameric configuration of the nucleocapsid in virus particles the neutralizing epitope may be repeatedly presented and, therefore, may contribute to the immunogenicity of this protein.

SUMMARY

The reovirus S1 gene codes for two polypeptides: σl and σs. In order to characterize the structure and function of the σl polypeptide, we have expressed the σ1 protein in Escherichia coli. The SI gene from mammalian reovirus type 3 (Dearing strain) and the variant K strain were subcloned into an expresssion vector containing the tac (trp-lac) promoter designed to express foreign gene products in E. coli efficiently. The hybrid plasmids, upon induction with isopropyl-β-d-thiogalactopyranoside, expressed two polypeptides that were detected by [35S]methionine labelling. One of the induced proteins had a relative molecular mass (M r) of approx. 46000 and corresponded to σl, as shown by immunoprecipitation with goat anti-reovirus antibody and a monoclonal antibody against σl, The second induced protein had a M r of approx. 12000 and was very similar to σs as judged by comparative tryptic peptide map analysis. Protein σl produced in E. coli was shown to be functional as judged from its ability to bind to mouse L fibroblasts.

SUMMARY

We have investigated the interaction between bovine parvovirus (BPV) capsid and non-capsid proteins and restriction fragments of the BPV genome by a combined DN A hybridization and immunodetection assay. 32P-labelled DNA was bound to nitrocellulose membranes bearing lysates of mock-infected and virus-infected cells whose proteins had been separated by SDS-polyacrylamide gel electrophoresis. The position of bound DNA was determined by autoradiography. The proteins on the membrane were still accessible to specific antibodies, allowing confirmation of the DNA-binding species by an immunodetection reaction. In 0·2 m-NaCl, BPV capsid proteins VP2 (72000 daltons) and VP3 (62000 daltons) bound the 0 to 16 map unit EcoRI fragment of BPV DNA which contained label in either the minus or plus strand. At higher salt concentration (0·5 m), only VP2 still bound DNA. Within this fragment, the capsid protein binding was restricted to those nucleotides between map units 0 and 4. No binding to capsid proteins was seen with the fragment spanning the middle of the genome and minor binding to VP3 was seen with the 5' end. Binding to the BPV noncapsid protein NP-1 was observed with the 0 to 16 map unit fragment when label was in the virion strand and to other possibly BPV-coded proteins when label was in the plus strand. The NP-1 binding was localized to map units 4 to 16. We did not detect binding to the BPV homologue(s) of the autonomous parvovirus non-capsid protein NS1, due in part to its low concentration in the cell lysates used. Points of the parvovirus replication cycle at which DNA-binding proteins may serve controlling functions are discussed.

Large numbers of virus particles were identified by electron microscopy in the epithelial cells of the ventriculus of the bedbug, Cimex lectularius. The morphology of the virus particles and the presence of a segmented double-stranded RNA genome imply that this isolate should be included in the Reoviridae.

In tissues of the sheep blowfly, Lucilia cuprina, which contain a previously undescribed virus-like particle (VLP) the mitochondria are invariably transformed into vesicle-containing bodies. The relationship between the presence of VLP and transformed mitochondria is confirmed by the production of similar vesiculated mitochondria in a Drosophila cell line inoculated with the VLP. The vesicles appear to be invaginations of the outer mitochondrial membrane and contain fibrillar material. The transformed mitochondria have many similarities with those described in virus- infected plant cells.

Regions of homology on the physical maps of Spodoptera exempta multiple-nucleocapsid nuclear polyhedrosis virus (SeMNPV-25), an Autographa californica MNPV genomic variant, and S. frugiperda (SfMNPV-2) baculovirus DNAs were identified by reciprocal DNA-DNA blot hybridization under conditions of an effective temperature of T m − 25 °C. In addition, cloned fragments of the viral genome which contained the homologous regions were used in hybridization experiments to confirm, refine and correlate the regions of the two physical maps. Five homologous regions conserved between the two physical maps were identified. When the stringency of the hybridization was increased (T m − 20 °C), only two of the original regions were identified by blot hybridization. One of the two regions contained the polyhedrin gene, and the other region was not associated with any known viral function. The five regions did not overlap with the intragenic homologous sequence (hrl to hr5) regions on the SeMNPV-25 map or with restriction endonuclease variant (vl to vIV) regions on the SeMNPV-25 and SfMNPV-2 maps. The degree of similarity in the genomic organization of these two baculoviruses is discussed.

A DNA clone of human T cell leukaemia virus type I (HTLV-I) was isolated from extrachromosomal closed circular copies in chronically infected promyelocytic leukaemia HL60 cells. The new HTLV-I isolate had an intact reading frame in the gag-pol region which could encode protein of 234 amino acids. This open reading frame has not been observed in previous HTLV-I isolates, although similar open reading frames have been reported in the corresponding locations in the related bovine leukaemia virus and HTLV type II. We consider that this open reading frame codes for the virus-encoded protease, on the basis of the homology of the predicted amino acid sequence with those of previously identified retrovirus proteases.

The scrapie-infected hamster has been considered an excellent model for the study of slow virus diseases of man (Creutzfeldt-Jakob disease) and animals. At the moment no therapy is available for the cure of these fatal central nervous system diseases, although several drugs have been tested. We found that amphotericin B (AmB), a polyene antibiotic, increased the incubation time of scrapie disease in animals infected by either the intraperitoneal or intracerebral route. Hamsters inoculated with a 10% brain suspension of the 263K strain of scrapie showed clinical signs of disease in 54·6 ± 4·7 days. Under AmB treatment (1 mg/kg for 6 days a week) the incubation time increased with the length of treatment, up to a maximum delay of 45 days. AmB may interact with the scrapie agent on cell plasma membranes and may thereby decrease the rate of scrapie replication. However, AmB did not have any effect when administered after the clinical onset of scrapie disease.

High levels of scrapie infectivity were found in detergent-insoluble residues of hamster brain purified by either repeated pelleting in 10% NaCl or by separation in Nycodenz® gradients. Titres determined by the method of incubation interval assay were 100-fold higher than titres measured by endpoint dilution assay. The protein profiles and end-labelled RNA examined by one-dimensional polyacrylamide gel electrophoresis were not different from samples prepared from uninfected brain. Preparations produced by repeated pelleting were treated with RNase A and/or 7 m-urea with no loss of scrapie infectivity. However, the infectivity of samples prepared by gradient centrifugation in Nycodenz® were reduced by 2 to 3 log10 LD50 by treatment with RNase A alone but not in combination with SDS. These results suggest that the scrapie agent may be aggregated by methods of purification employing pelleting in high concentrations of salt, or by adding polycations to disaggregated samples.

Earlier results have demonstrated a mutagenic activity of simian virus 40 (SV40) in mammalian cells. To analyse this ability further, the effect of SV40 DNA fragments, introduced into Chinese hamster cells, on the frequency of mutations at the hypoxanthine phosphoribosyltransferase locus and other loci was studied. It was found that the mutagenic effect (i) was substantially maintained when the viral genome had been replaced by a fragment comprising the T antigen-coding region and the early promoter-enhancer region, (ii) was strongly reduced or abolished when the promoter region including upstream sequences in this fragment had been replaced by the chicken lysozyme gene promoter or both enhancer elements were deleted, and (iii) was abolished in an SV40 replication origin-defective mutant in which the structure of the T antigen-binding site II was affected. It may be concluded that SV40-induced mutagenesis depends on the expression of the early region of the genome and on a function involved in specific binding of large T antigen to viral DNA. Since origin- defective mutants of SV40 were reported as being able to transform cells, the functions of transformation and mutation do not seem to correlate.