- Volume 71, Issue 7, 1990

Cervical biopsies were collected from Birmingham women having cervical intraepithelial neoplasia or invasive cervical carcinoma and normal controls, and examined for the presence of human papillomavirus type 16 (HPV-16) E6-E7 DNA and mRNA using an adaptation of the polymerase chain reaction. HPV-16 E6-E7 sequences were detected in all abnormal biopsies and in 90% of the normal biopsies examined, confirming previous studies describing the high prevalence of cervical HPV-16 infection. While we were unable to identify any qualitative differences in RNA transcripts from the p97 promoter, substantial quantitative differences in HPV-16-specific early region transcripts between normal and cytologically abnormal cervices were observed. These results suggest that although the level of E6-E7 transcription may contribute to the malignant phenotype, additional factors are likely to be important in the development of cervical neoplasia.

Epstein-Barr virus (EBV)-positive Burkitt’s lymphoma (BL) biopsy cells and early passage BL cell lines have been reported as showing an unusual type of virus-cell interaction; at least two EBV latent proteins appear not to be expressed. Serial passage of such lines is often accompanied by a broadening of virus latent gene expression and a corresponding change in the cell surface/growth phenotype towards that shown by in vitro transformed lymphoblastoid cell lines (LCLs). The sequence of events, both viral and cellular, involved in this transition needs to be defined properly. In the present work, phenotypically distinct cell clones have been derived from early passage cultures of a BL cell line in phenotypic transition, thereby giving access to relatively stable cell populations through which the different EBV-B cell interactions within the parental line can be studied. Clones retaining the original BL biopsy cell phenotype (CD10/CD77-positive, activation antigen/adhesion molecule-negative) expressed the virus-encoded nuclear antigen EBNA 1 but not any of the other known latent proteins, EBNAs 2, 3a, 3b, 3c, -LP and latent membrane protein (LMP). Other clones which had developed an LCL-like phenotype (CD10/CD77-negative, activation antigen/adhesion molecule-positive) now expressed all the above latent proteins and also contained significant numbers of cells in lytic cycle. Phenotypic change occurring within the parental BL cell line itself was initiated in a small subpopulation of cells in which the virus-encoded proteins EBNA 2 and LMP were transiently induced to an unusually high level of expression; this was accompanied by the first detectable changes in cell surface phenotype, namely the increase of cellular adhesion molecules. Some control over EBNA 2/LMP expression then appeared to be re-imposed since the presumed clonal descendents of these cells stably expressed EBNA 2 and LMP at much reduced levels typical of those seen in conventional LCLs.

Herpes simplex virus type 2 (HSV-2) causes lethal illness after intravaginal (IVAG) inoculation into BALB/cJ mice. In the present studies, we demonstrated in mice that primary IVAG vaccination with an attenuated strain of HSV-2 induced humoral immunity in sera and in vaginal secretions. Secondary genital exposure to HSV-2 enhanced this response. However, intraperitoneal exposure to attenuated HSV-2 elicited an antiviral antibody response in sera but not in vaginal secretions. In both sera and vaginal secretions, antiviral IgG antibodies were the major isotype. Systemic exposure to HSV-2 elicited antibodies only in sera that were specific for the major viral antigens whereas IVAG inoculation with HSV-2 stimulated both serum and vaginal antibody responses. Intravenous transfer of antiviral monoclonal antibodies protected against systemic HSV-2 infection but were ineffective against vaginal infection due to a lack of transudation into vaginal secretions. These results suggested that local humoral immunity in the genital tract is important in resistance to HSV-2.

Cloned cDNA and synthetic deoxyoligonucleotides, complementary to various parts of the genomic RNA of tick-borne encephalitis virus (TBEV), strain Soljin, were used to distinguish between strains of TBEV and other flaviviruses. The cDNA probe hybridized with strains of TBEV and related flaviviruses of the TBE complex except for Powassan virus, and it did not react with flaviviruses of the Japanese encephalitis and dengue subgroups. Viruses of the TBE complex and some strains of TBEV were differentiated from TBEV strain Soljin by the thermal stability of RNA-DNA hybrids. Negishi and louping-ill viruses were the most closely related to TBEV strain Soljin, among viruses of the TBE complex. Eight strains of TBEV isolated in different geographical areas from different sources were tested by dot-hybridization with 11 deoxyoligonu-cleotide probes. The probes revealed genetic variations among strains of TBEV. The pattern of hybridization correlated with the source of virus strains: TBEV strains isolated from TBE patients reacted with more probes than strains isolated from ticks. Within a group of epidemic strains of TBEV there was a correlation between the geographical area of isolation and similarity to TBEV strain Soljin.

The nucleotide sequence of a 10465 bp HindIII genomic fragment from fowlpox virux (FPV) is presented. Analysis of the nucleotide sequence revealed 10 potential major open reading frames (ORFs). Five of these ORFs are predicted to encode polypeptides with significant homology to hypothetical polypeptides derived from nucleotide sequence analysis of the vaccinia virus (VV) HindIII D region. Interestingly, these homologous ORFs do not occur in the same tandem arrangement in the FPV genome as they do in the VV genome. These results are discussed.

The consequences of locating the polyhedrin gene coding sequences and the p10 promoter at heterologous positions within the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome were investigated. Positioning the polyhedrin or β-galactosidase coding sequences under the control of the p10 gene promoter via the use of the new transfer vector, pAcUW 1, resulted in viable recombinant viruses able to produce high levels of each non-fused gene product at the appropriate time. Polyhedra were also produced by the virus with the p10 promoter-polyhedrin hybrid gene and appeared normal in thin sections. Therefore the combination of polyhedrin promoter and coding sequences is evidently not essential for efficient expression of this protein. The p10 promoter can serve this function equally well. Viruses with the p10 promoter and β-galactosidase coding sequences placed upstream from the polyhedrin gene in either orientation produced large amounts of β-galactosidase protein in infected cells, thus demonstrating that the p10 promoter can function at an alternative position within the virus genome. A second transfer vector, pAc- UW2B, was constructed, with a copy of the p10 gene promoter placed upstream and in opposition to the polyhedrin gene. This mediates the insertion of any foreign gene under the control of the p10 promoter while preserving normal p10 gene expression. The advantages of these constructs over the conventional vectors presently used to express foreign genes in insect cell systems and their utilization in the production of virus insecticides are discussed.

The δ-endotoxin gene from Bacillus thuringiensis subsp. kurstaki HD-73 was inserted into Autographa californica nuclear polyhedrosis virus (AcMNPV) using two transfer vector systems. In the first, the δ- endotoxin gene was placed under the control of the polyhedrin gene promoter in lieu of the polyhedrin coding sequences, thus deriving a polyhedrin-negative virus. In the second, it was inserted under the control of a copy of the AcMNPV p10 promoter positioned upstream of the polyhedrin gene to produce a poly- hedrin-positive virus. Analysis of infected cell extracts showed that the δ-endotoxin was expressed in insect cells as 130K, 62K and 44K proteins, with peak syntheses at 18 h post-infection. Each of these products reacted with antisera specific for the complete protoxin and the cleaved, active form. When extracts from the cells infected with the polyhedrin-negative virus were fed to Trichoplusia ni larvae, feeding by the insects was inhibited and deaths occurred that were inconsistent with virus infection. This effect was also observed after the inoculum had been treated with detergents to inactivate virus particles prior to feeding to the larvae. These data indicate that the expression of the B. thuringiensis δ-endotoxin gene by a baculovirus in insect cells produces material with insecticidal activity. The biological activities of the two recombinant viruses were assessed in conventional bioassay tests by feeding virus particles or occlusion bodies to the insects. The polyhedrin-negative virus preparation appeared to be contaminated with endotoxin which inhibited feeding of the insects and prevented determination of the LD50 value. The polyhedrin-positive virus had an LD50 value about twofold higher than that of unmodified AcMNPV. The significance of these data for the genetic engineering of virus insecticides is discussed.

Full-length cDNA clones of the nucleoprotein (NP) genes of influenza A/Ann Arbor/6/60 and B/Ann Arbor/1/86 viruses were constructed from virion RNA and subsequently expressed in Spodoptera frugiperda (Sf9) cells using the baculovirus vector, Autographa californica nuclear polyhedrosis virus. Western blot analysis of lysates prepared from Sf9 cells infected with the recombinant viruses confirmed that the baculo- virus-expressed NP antigens were reactive with monoclonal antibodies specific for either type A or B NP and with anti-NP antibodies in human serum samples. Electrophoretic analysis indicated that the expressed NP antigens comigrated with NP purified from influenza A or B virions and that the recombinant NP antigens represented greater than 10% of total protein in infected cells. Dilutions of clarified Sf9 cell lysates were used as antigens in a standard enzyme immunoassay to detect serum antibody specific for influenza A or B viruses. The results from assays using the baculovirus-expressed NP antigens showed good correlation with the results obtained using bacterially expressed NP antigen as well as complement fixation. Therefore, baculovirus-expressed NP antigens have the potential to be used to develop reproducible and routine assays for the serodiagnosis of influenza virus infections as an alternative to the complement fixation or haemagglutination inhibition tests.

The sequence of the P (phosphoprotein) gene of mumps virus has been determined. It has two open reading frames, the first of which probably encodes the NS1 (or V) protein of mumps virus. Expression of the P protein requires the insertion of two non-templated residues to link the two ORFs in a process analogous to that observed in the P/V gene of simian virus type 5 to which mumps virus is closely related. Strain differences in the accuracy of insertion of non-templated G residues in the P/V gene transcripts have been described.

Pulmonary A2 strain respiratory syncytial virus infection of BALB/c laboratory mice persisted for up to 7 days after initial infection with peak virus titres being recovered on day 4. Virus antigen within the lungs was found to be restricted essentially to the alveolar regions. Similarly, pulmonary histopathological changes remained confined to the peri-alveolar regions being consistent with mild pneumonia. Infection was found to elicit a pulmonary major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) response which was first detectable 6 days after infection and optimal 7 to 9 days after infection. This local CTL response was preceded by a rapid transient virus-specific lymphocyte transformation response which was detectable only 3 days after intranasal infection. In addition, infection induced rapid interferon production within the lungs which was accompanied by an equally rapid rise in pulmonary natural killer (NK) cell cytotoxic activity. Enhanced NK cell cytotoxicity could be detected after only 1 day post-infection and continued to rise to maximum levels on day 3. This response like the acute CTL response was found to be restricted to the lower respiratory tract. IgG was the first class of virus-specific immunoglobulin to be detected in the lungs of infected animals after experimental infection. However, IgG was not detected until day 10 post-infection, 5 days after the initial decline of virus shedding. Virus-specific IgA although detectable did not appear in the lung until day 24.

The nucleotide and amino acid sequences of the mRNA and predicted polypeptide of the integral membrane small hydrophobic (SH) protein of human respiratory syncytial virus strain 18537 (a prototype strain of antigenic subgroup B) were determined from cloned cDNA. At the nucleotide and amino acid levels there was 78% and 76% identity, respectively, with the previously described SH mRNA and protein of strain A2 (a prototype strain of subgroup A). Most of the amino acid substitutions occurred in the predicted ectodomain (50% identity). The pattern of posttranslational processing of the strain 18537 SH protein was very similar to that of strain A2, yielding a non- glycosylated form and two glycosylated forms. Analysis of released virions of strain A2 by immunoprecipita- tion with SH-specific antibodies suggested that the major non-glycosylated species and one of the glycosylated species are virion structural components.

Three human and six bovine parainfluenza virus type 3 (PIV3) strains were examined by the use of 60 monoclonal antibodies (MAbs). Fifty-three MAbs to the human C243 strain were directed against six, four, nine and seven epitopes of the haemagglutinin- neuraminidase (HN), fusion (F), nucleocapsid (N) and matrix proteins, respectively. Seven MAbs to the bovine strain were directed against three epitopes of the HN protein and three epitopes of the F protein. Each strain was characterized in ELISA and immunofluorescence tests with all MAbs and in a haemagglutination inhibition assay with the anti-HN MAbs. There were marked differences between human and bovine viruses, primarily in the HN protein where five epitopes differed. One epitope of the F and one of the N protein also differed. Bovine PIV3 was found to be a homogeneous subtype and distinct from human PIV3.

Variations in epitopes on structural proteins of four isolates of parainfluenza virus type 4 (PIV-4) and the M r of polypeptides in these isolates were determined by radioimmune precipitation assay with monoclonal antibodies to parainfluenza virus type 4A (PIV-4A) and type 4B (PIV-4B). Three isolates antigenically resembled the prototype PIV-4A and the sizes of their structural proteins were 72K (HN protein), 61K (F0 protein), 61K (NP protein) and 40K (M protein). However, one virus isolate showed marked antigenic differences from both the 4A and 4B prototype viruses, particularly with regard to properties of the HN and F proteins. In addition, both the NP and F0 proteins of this isolate had a slightly increased M r of 63K.

Cytoplasmic nucleocapsid (NC) isolated from Vero (V) cells infected in the logarithmic phase of growth with Onderstepoort canine distemper virus consists of light- NC (L-NC) and dense-NC (D-NC), encapsidating full- length genomic RNA, and defective-NC (Df-NC), encapsidating variably truncated RNAs. The 70K host cell protein constituent of L-NC and Df-NC was shown to be a member of the 70K heat-shock protein (70K hsp) family. Specifically, 72K hsp is associated with L- NC, and 72K and 73K hsp are associated with Df-NC. Variable L-NC production by three different Vero cell sublines was compared to cellular 70K hsp levels. VI41 supported the highest level of L-NC production and expressed high basal levels of 70K hsp in uninfected cells. These high basal levels correspond to a large distribution of log phase VI41s in the S phase of the cell cycle. V138-L and V138-H cells produced lower amounts of L-NC and exhibited similar low basal levels of 70K hsp expression, corresponding to low percentages of log phase cells in the S phase cell cycle compartment. Heat shock was effective in inducing L- NC expression in V138-H, which otherwise produced D-NC. Similar cell subline differences in L-NC production were obtained for eight different virus pools derived from the same plaque-purified parental stock. Enhanced biological activity was associated with L-NC based on correlation between L-NC production, viral titre, and plaque areas measured over infected cells.

In an earlier report, we described the construction of the genetically engineered pseudorabies virus strain 2.4N3A which does not express glycoprotein gl. Although this strain showed a strongly reduced virulence in 10-week-old seronegative pigs, it could still cause severe disease or death in 3-day-old piglets. To attenuate the strain further, we constructed mutants with a deletion in the viral thymidine kinase gene. One mutant strain, designated 783, has a deletion of 19 base pairs and was shown to be highly immunogenic and safe for vaccination of pigs against pseudorabies virus.

The pathological changes and distribution of virus antigen in mouse brains were studied following intracranial inoculation of 3 week old BALB/c mice with the herpes simplex virus (HSV) type 2 strain HG52 and its deletion variant JH2604. The variant JH2604 failed to produce necrotizing encephalitis compared to the parental HG52. The morphological changes induced in JH2604-infected brains consisted of localized perivascular cuffing by lymphocytes and infiltration by immune cells. Immunohistochemical studies using polyclonal anti-HSV serum showed that JH2604 antigens were localized at the site of inoculation with no evidence of neuronal involvement. Wild-type HSV- infected brains demonstrated a wide distribution of antigens both in neuronal and supporting cells. These data provide evidence that the non-neurovirulent phenotype of JH2604 is due to inability to replicate within neuronal cells of the central nervous system and pinpoints a precise role for the HG52 sequences contained within the 1488 bp subfragment of TRL/IRL deleted in JH2604.

The RNA of hepatitis delta virus (HDV) 1682 nucleotides long, has been cloned from a human serum isolate. Comparison with the three complete published sequences shows that a region of the HDV genome, between positions 620 and 1350, which contains sequences involved in replication and possibly pathogenicity, is highly conserved.

We have used the polymerase chain reaction (PCR) to provide a very sensitive and unequivocal test for diagnosis of foetal rubella virus infection. RNA extracted from biopsy specimens (chorionic villi), placenta or products of conception was reverse- transcribed using a rubella virus-specific oligonucleotide primer and the cDNA was amplified by PCR. The specificity of the amplified fragment was confirmed by Southern blotting. Detection of rubella virus infection in five out of 41 clinical specimens examined by this approach was shown to be entirely consistent with clinical history and other methods of laboratory diagnosis in current use. The sensitivity of the test and the unequivocal nature of the results obtained could be invaluable in providing prenatal counselling following rubella virus infection during pregnancy.