- Volume 76, Issue 10, 1995

To understand the mechanism of regulation of bovine herpesvirus 4 (BHV-4) early gene expression by immediate-early (IE) gene products, we studied activation of expression of the BHV-4 early gene encoding thymidine kinase (TK). BHV-4 encodes two IE RNAs. IE RNA 2 encodes a protein with predicted amino acid sequence similarity to the Epstein—Barr virus (EBV) R transactivator. The BHV-4 TK promoter-regulatory region was specifically transactivated approximately 9-fold by the IE2 gene product in transient expression cotransfection assays. Deletion analysis localized the IE2 responsive element(s) within a 72 bp fragment 5′ to the start of transcription. Multiple copies of this 72 bp fragment conferred IE2-responsiveness on an enhancer-less simian virus 40 (SV40) promoter and mediated IE2 transactivation in either orientation. Gel retardation assays demonstrated a sequence-specific complex between the IE2 gene product and the 72 bp DNA fragment, and indicated that the IE2 gene product binds DNA as a dimer. Sequences contained in a 25 bp subfragment were sufficient for IE2-DNA complex formation. No nucleotide sequence similarity was noted between the TK IE2-binding fragment and the consensus binding site for the EBV R transactivator. A single amino acid change in the putative DNA-binding domain of the IE2 gene product abolished both complex formation in vitro and transactivation activity in vivo.

The ecdysteroid UDP-glucosyltransferase (egt) gene isolated from a plaque-purified isolate of Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus (CfMNPV) was compared to its homologue from a defective MNPV virus (CfDEF) present in wild-type virus populations infecting the eastern spruce budworm, C. fumiferana. The egt genes were located in the same relative position within the virus genomes and their genomic location and arrangement were similar to that found in Autographa californica MNPV (AcMNPV) and Orgyia pseudotsugata MNPV (OpMNPV). The genes encoded 491 and 494 amino acid open reading frames respectively, and were 67% identical at the amino acid level and 74% identical at the nucleotide level. Transcripts of the egt of CfMNPV peaked around 12 h post-infection (p.i.) and disappeared after 36 h p.i. Transcripts of the egt of CfDEF peaked between 6 and 9 h p.i. and were not detected 24 h p.i. The egt from CfMNPV was more similar to the partially sequenced egt identified from OpMNPV, at the nucleotide and amino acid levels, than it was to the egt from the CfDEF, AcMNPV, Bombyx mori NPV, Lymantria dispar MNPV or Spodoptera exigua MNPV. Phylogenetic analysis of egt supported the baculovirus evolution scheme suggested by polyhedrin sequence analysis.

The nucleocapsid protein NCp15 of human immunodeficiency virus type 1 (HIV-1) is a small basic protein with two zinc fingers. It is required for virion morphogenesis and synthesis of proviral DNA. As the first step in our study of the structural domains involved in the various functions of this protein, 18 monoclonal antibodies (MAbs) were isolated. The epitopes of NCp7 recognized by the MAbs were mapped using synthetic peptides representing overlapping sequences and truncated forms of NCp7. These anti-NCp7 MAbs were investigated by ELISA and real-time biospecific interaction analysis (BIAcore). Five classes of anti-NCp7 MAbs were characterized. Three classes (14 MAbs) were directed against continuous epitopes, one in the N-terminal part, another next to the second zinc finger and the third in the C-terminal part of the protein. Two other classes comprised four MAbs reacting only with the entire NCp7 and not with any of the small overlapping peptides used, suggesting that these MAbs were directed against conformational epitopes. The anti-NCp7 MAbs directed against linear epitopes were able to react efficiently with both NCp7 and NCp15, the NCp7 precursor, whereas the anti-NCp7 MAbs directed against conformational epitopes were capable of inhibiting the tight interaction between NCp7 and the HIV-1 replication primer tRNALys,3. In contrast, most of the MAbs directed against linear epitopes did not inhibit this interaction.

We previously reported that pigtailed macaques (Macaca nemestrina) became infected after intravenous inoculation with the LAI strain of human immunodeficiency virus type 1 (HIV-1), an isolate that replicates in both M. nemestrina lymphocytes and blood-derived monocyte/macrophages. In the current study we investigated the presence of HIV-1 and pathology in the postmortem brains of four of these macaques. Histopathological findings revealed focal lesions in white matter in the frontal and occipital lobes of one macaque, with myelin loss, nerve fibre loss, and gliosis within these lesions. Semi-quantitative, solution-based PCR revealed HIV-1 DNA in the brains of two of the other macaques. Using slide-based PCR-driven in situ hybridization, we studied these two macaques further and detected intranuclear, circular HIV-1 DNA in vascular endothelia and other non-neuronal brain cells. These findings indicate that M. nemestrina brain can be infected with HIV-1 in vivo and may provide a useful animal model for understanding early HIV-1 brain infection in humans.

Bovine rotavirus VP8*, N-terminal trypsin cleavage product of VP4, was produced in Escherichia coli. To examine if this antigen could induce neutralizing antibody responses, different species of animals were immunized with the recombinant VP8* protein. The VP8* antigen was found to stimulate a neutralizing immune response in rabbits. When VP8*-immunized mice were exposed to bovine rotavirus strain C486, significantly higher antibody responses were observed than if they were only exposed to C486. To simulate a current vaccination protocol in the field with livestock, mice were exposed to live C486 virus first and then to VP8*. These mice had the elevated immune responses indicating that VP8* could boost immunity in primed mice. The immune response to VP8* was also tested in pregnant cows. The efficacy of VP8* in stimulating milk antibody was compared with a commercial inactivated vaccine. Differences in colostral antibody titres between VP8*-vaccinated and unvaccinated cows were statistically significant (P < 0.05) and equivalent to the commercial vaccine (P = 0.0569). The milk antibody titres on day 10 were comparable between VP8*- and commercial vaccine-vaccinated animals and were significantly higher (P < 0.05) than in unvaccinated controls. Furthermore, rabbit and bovine antibodies induced by VP8* were able to neutralize different P types of bovine rotaviruses to varying degrees, suggesting that serotype-specific and cross-reactive epitope(s) are present on the VP8* protein of rotavirus. Taken together, E. coli-expressed VP8* may be useful as a subunit vaccine candidate for bovine rotavirus.

To study the in vitro susceptibility of peripheral blood mononuclear cells (PBMC) to hepatitis C virus (HCV), we incubated cells from healthy donors with HCV-positive sera. Using RT-PCR and in situ hybridization, the genomic viral RNA was detected in PBMC and in their supernatants until 25 days post-incubation. The PBMC of the different donors were not all permissive to HCV, but results were more constantly positive when cells from four donors were pooled. Quantification of the genomic viral RNA by the branched-DNA assay showed a decrease in the HCV RNA concentration during the first week of culture followed by a peak during the second or third week, and also an increase in the total amount of viral RNA in the inoculated cells. Although HCV RNA could be detected in the supernatants by RT-PCR, the concentration was very low. Using a sense-specific RT-PCR method, the HCV negative-strand was also detected in the cells but not in the supernatants. In two experiments PBMC were successfully infected using HCV-positive culture supernatants, therefore suggesting that infectious particles can be produced in this system. Our findings demonstrate that PBMC are permissive for HCV replication in vitro but the replication level is very low. The HCV RNA concentration measured in PBMC of 10 chronically infected patients was not significantly higher than the maximal concentration obtained in PBMC infected in vitro.

Genotypes of hepatitis C virus (HCV) present within 104 samples from HCV-infected individuals from Africa, the Middle East, the Indian subcontinent and South-East Asia were identified by sequence comparisons in the core and NS-5 regions. Relatively short sequences (such as the 222 bp fragment of NS-5) provided effective discrimination of types, subtypes and isolates, and produced equivalent relationships between genotypes as were found upon comparison of longer sequences of NS-5, of the core region, and by comparison of the limited number of complete genomic sequences currently available. Measurement of evolutionary distances in the core and NS-5 regions allowed 79 of the 104 samples to be identified as examples of known genotypes, while 17 of the remainder could be provisionally classified as new subtypes of types 1 (Nigeria), 2 (Gambia), 3 (India, Pakistan and Bangladesh) and 4 (Middle East) on the basis of sequence comparison in core and NS-5 (n = 9) or provisionally using core alone (n = 8). The remaining sequences from Thailand made up two groups showing no close similarity to any of the six major genotypes classified to date, although one corresponded to an as yet unclassified variant of HCV also found in Thailand. However, phylogenetic analysis of the core and NS-5 regions indicated a distant relationship between these sequences with variants found in Vietnam and with type 6a, and collectively they formed a diverse single phylogenetic group. The existence of great diversity within a single genotype was also found amongst type 3 sequences in the Indian subcontinent, amongst type 4 variants in Central Africa and the Middle East, and amongst type variants in Nigeria. These findings may provide clues for understanding the origins and mechanisms of transmission of HCV.

The molecular biology of Mapuera virus was studied at both the protein and nucleic acid levels. Seven virus-encoded proteins were detected in infected Vero cells. The sizes and characteristics of each of the proteins determined from various radiolabelling experiments allowed preliminary identification of the proteins as the large (L; 190 kDa), haemagglutinin neuraminidase (HN; 74 kDa), nucleocapsid (N; 66 kDa), fusion (F0; 63 kDa), phosphoprotein (P; 49 kDa), matrix (M; 43 kDa) and non-structural (V; 35 kDa) proteins. Western blot analysis showed that the HN, N and P proteins were major antigens recognized in the mouse. A cDNA library of total virus-infected cellular mRNA was created and screening of the library resulted in the detection of cDNA sequences representing the N mRNA transcript of Mapuera virus. The N mRNA sequence determined from the clones was 1731 nt in length and contained an ORF that encoded 537 amino acids, the complete 3′ untranslated region and part of the 5′ non-coding region. The calculated M r of the N protein was 59 kDa, which is close to the 66 kDa protein observed by SDS-PAGE.

The complete nucleotide sequence of the genome of the fish rhabdovirus infectious haematopoietic necrosis virus (IHNV) has been determined after cDNA cloning of the viral genomic RNA. Sequence analysis showed the presence of six open reading frames encoding the nucleoprotein N, the matrix proteins M1 and M2, the glycoprotein G, a so-called non-structural protein NV, and the RNA polymerase L. The genome organization is 3′ N-M1-M2-G-NV-L 5′. The extreme 5′ and 3′ ends of the genome were sequenced after RNA ligation or RACE. Prokaryotic expression products of the open reading frames predicted to encode the matrix proteins M1 and M2, the glycoprotein G and the NV protein reacted with rabbit anti-IHNV serum thereby confirming their identity. This is the first complete nucleotide sequence of a fish rhabdovirus. Knowledge of the complete sequence is an essential prerequisite for future manipulation of the genome and also serves to provide gene- and protein-specific reagents for use in further examination of the replication of the fish rhabdoviruses.

Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-accetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-κB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex.

Two antigenically distinct H1N1 influenza A viruses were isolated during an outbreak of respiratory disease in Quebec swine in 1990/91. Analysis of haemagglutinin and partial nucleoprotein sequences indicated that one was a variant of the swine H1N1 influenza virus circulating in the American Midwest whereas the other was very similar to virus isolated from swine in 1930. The existence of this latter isolate supports the concept that influenza viruses can be maintained for long periods in swine, perhaps in geographically limited pockets. Serological evidence indicates that these distinct strains continued to circulate widely in south-central Quebec until at least 1993.

We have analysed, by PCR using consensus primers followed by sequencing, 12 human rhinoviruses (HRVs) in a genomic region including that corresponding to the immunogenic site NIm-II. Together with published information, 21 sequences are available for comparison. In the region analysed, which encodes 112 amino acids, the majority (18) of the serotypes exhibited at least 70% amino acid identity to one another and some serotypes are very closely related. These include HRV-36, -58 and -89, known to exhibit antigenic cross-reactivity, which were shown to differ at only three amino acid positions. Three serotypes, HRV-3, -14 and -72, share at least 84% identity with one another but are less than 66% identical to the majority group. Interestingly, membership of these two molecular clusters correlates with the groupings determined by sensitivity to antivirus drugs, suggesting that they reflect a fundamental division of HRVs. In contrast, there is no correlation with receptor grouping, since the majority group contains members belonging to both HRV receptor groups.

The development of diabetes in mice induced by encephalomyocarditis (EMC) virus provides the best experimental evidence that viruses have an aetiological role in the pathogenesis of this disease. The major capsid protein (VP1) of EMC virus is important for both the attachment of the virus to pancreatic beta cells and for the determination of antigenicity. This experiment was initiated to clone the gene for the major capsid protein, VP1, of the diabetogenic EMC (EMC-D) virus, express the VP1 protein, and test whether the recombinant VP1 protein can prevent development of EMC-D virus-induced diabetes in mice. We successfully cloned the VP1 gene of the EMC-D virus in the expression vector pRSET and subsequently expressed the protein in Escherichia coli. The recombinant VP1 protein was then purified by affinity chromatography. Five- to six-week-old male SJL/J mice were immunized intraperitoneally with purified VP1 protein and then challenged after various intervals with highly diabetogenic EMC-D virus. None of the VP1-immunized mice developed diabetes, irrespective of the interval between immunization and virus challenge, whereas 80 to 95% of the EMC-D virus-infected control mice did develop diabetes. All of the VP1-immunized mice showed intact pancreatic islet architecture, whereas most of the infected control mice showed severe beta cell necrosis and lymphocytic infiltration of their pancreatic islets. On the basis of these observations, we conclude that the recombinant VP1 protein of EMC-D virus can completely prevent the development of EMC-D virus-induced diabetes in mice.

SAF-protein, an amyloid, is the main constituent of scrapie-associated fibrils (SAF) and a specific marker for transmissible spongiform encephalopathies (TSE). Using an improved extraction method and Western blot detection, the disease-specific amyloid was found in various parts of the central nervous system of hamsters orally infected with scrapie, of squirrel monkeys orally infected with kuru, sporadic Creutzfeldt-Jakob disease (CJD) and scrapie, of human patients with sporadic CJD, of a sheep with natural scrapie and of a cow with bovine spongiform encephalopathy (BSE). In human CJD samples, the concentration of TSE-specific amyloid was estimated to be 1000- to 10000-fold lower than in the central nervous system of hamsters with scrapie. The extraction method has a yield of 70% and allows Western blot detection of the TSE-specific amyloid in samples representing 1–10 µg of brain tissue from intracerebrally infected hamsters, as well as in individual spleens from hamsters with terminal scrapie infected by the intracerebral, oral or intraperitoneal route. A 20–100 mg sample of material is sufficient for the extraction of the pathological protein from different rodent, monkey, ovine, bovine and human tissues. The results reported here demonstrate the potential suitability of the method for the routine diagnosis of TSE as well as for the detailed analysis of distribution patterns of the TSE-specific amyloid in experimental approaches to the investigation of these diseases.

We investigated the relationships between amino acid polymorphisms of the prion protein (PrP), restriction fragment length polymorphisms (RFLP) of the PrP gene and the incidence of natural scrapie in Japan. Six variant alleles of the PrP gene were found in healthy sheep. Based on the substitutions at codons 112, 136, 154 and 171, these allelic variants were designated PrPMARQ, PrPTARQ, PrPMVRQ, PrPMAHQ, PrPMARR and PrPMARH. Each RFLP haplotype (e1h1, e1h2 or e3h1) consisted of multiple alleles including PrPMARQ. Three of these variant alleles were found in scrapie-affected Suffolk sheep. PrPMARQ was associated with high disease incidence, PrPTARQ and PrPMARR were associated with low disease incidence. We found that one scrapie-affected Suffolk was homozygous for PrPMARR and four PrPSc-positive Suffolks carried PrPMVRQ. Both of two scrapie-affected Corriedales and two out of three scrapie-affected cross-breeds between Suffolk and Corriedale carried PrPMARH, suggesting that this allele associates with high incidence of scrapie in Corriedale and its cross-breeds.

A cellular form of the prion protein (PrPC) is thought to be a substrate for an abnormal isoform of the prion protein (PrPSc) in scrapie. PrPC is abundant in tissues of the central nervous system, but little is known about the distribution of PrPC in non-neuronal tissues of sheep, the natural host of scrapie. This study investigated the tissue distribution of PrPC in sheep. Although PrPC was abundant in neuronal tissues, it was also detected in non-neuronal tissues such as spleen, lymph node, lung, heart, kidney, skeletal muscle, uterus, adrenal gland, parotid gland, intestine, proventriculus, abomasum and mammary gland. Neither PrPC nor PrP mRNA was detected in the liver. The tissue distribution of PrPC appears to be inconsistent with the tissues which possess scrapie infectivity, suggesting that factor(s) specific to certain cell types may be required to support multiplication of the scrapie agent.

Using a PCR strategy which detects disruptions in the E2 reading frame we have analysed the progression of human papillomavirus type 16 (HPV-16)-positive cervical lesions. From a total of 192 samples analysed, we detected HPV-16 in 74. In samples from the spectrum of inflammatory states and cervical intraepithelial neoplasia (CIN) grade I lesions we detected episomal forms of the virus. In invasive tumours and in samples from CIN III lesions there were no episomes detected, suggesting that lesions with integrated HPV-16 precede the invasive stage. The RT-PCR analysis demonstrated the presence of E6 transcripts at all stages and E2 transcripts in all early lesions. The E2 transcripts were not detected in 26 out of 29 CIN III lesions and tumours in which there was a disruption in the E2 gene. In tumours with E2 gene disruptions, we used single-primer PCR to demonstrate the presence of E2 gene sequences.

The gene for the major capsid protein (hexon) of fowl adenovirus serotype 10 (FAV-10) has been identified by the use of the expression vector pGEX and rabbit polyclonal antisera raised against FAV-10. The nucleotide sequence of the entire hexon gene has been determined. Sequence analysis revealed an open reading frame of 2808 bp coding for a putative polypeptide 936 amino acids long with a molecular mass of 105.5 kDa. The translation initiation codon has a local sequence which conforms with the optimal translation start sequence of CC(A/G)CCATGG. The location of the hexon gene in the FAV genome was from 46.85 to 52.81 map units, which is to the left of the hexon gene in the genomes of both bovine and human adenoviruses (52.4 to 60.5 map units). A splice acceptor site was identified 12 bp upstream of the initiation codon by using mRNA and PCR. It had the sequence TAGG which conforms to the consensus sequence of (C/T)AGG. Comparison of the amino acid sequence of the FAV-10 hexon with those of the bovine, human and murine hexon gene products revealed highest levels of identity occurring in the regions corresponding to the pedestals which form the base region of the hexon, and the lowest levels of identity in the regions corresponding to the loops which are exposed to the external environment.

We have attempted to relate genetic recombination involving human immunodeficiency virus type 1 (HIV-1) to multiple drug resistance by using PEG to fuse subclones of U937 cells that carried HIV-1 recombinants resistant to either 3′-azido-3′-deoxythymidine (AZT) or the (-) enantiomer of 2′,3′-dideoxy-3′-thiacytidine (3TC). The parental viruses employed contained well-defined mutations in the pol gene. Fused cells were cocultured with the MT4 lymphocyte cell line for virus amplification to yield progeny that, in some cases, possessed different patterns of drug resistance from parental viruses. Mutational analyses were performed by PCR to substantiate these observations, which were also confirmed by direct sequencing of single strands of DNA segments, obtained from plaque-purified viruses. These studies indicate that viral recombination had occurred, and establish a theoretical basis on which to conclude that the acquisition of multiple drug resistance on the part of HIV-1 may be related to its ability to promote cell fusion.