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Hybrids between rubella virus and a latent virus of baby hamster kidney cell line BHK21: characterization of rubella virus and type 2 hybrid virus genomes and determination of their physical homology.

The biochemical nature of rubella virus and type 2 hybrid virus, which is a recombinant between rubella virus and a latent retrovirus of BHK21 cells, has been characterized. Type 2 hybrid virus carries DNA polymerase able to copy exogenous DNA. However, disrupted type 2 hybrid virions do not synthesize detectable amounts of DNA using the endogenous viral RNA or synthetic poly(rA)/oligo(dT) primed as a template. Thus, the type 2 hybrid virus DNA polymerase has no detectable reverse transcriptase activity. Rubella virus and type 2 hybrid virus RNA can serve as templates for avian myeloblastosis virus (AMV) reverse transcriptase, although they are inefficient. The addition of oligo(dT) to these viral RNA showed no significant stimulation of their template activity for AMV reverse transcriptase. The oligo(dT)-cellulose affinity column bound neither rubella virus nor type 2 hybrid virus RNA. This suggests that both RNA genomes have a very short poly(A) tail at their 3' end. Thus, complementary DNA (cDNA) synthesis by AMV reverse transcriptase using oligo(dT) primers showed no preferential reverse transcription from the genomic 3' terminus and produced only short cDNA fragments (about 200 nucleotides). We cross-hybridized these short cDNA fragments with their viral RNA, assuming that they are copies of random sites of the genome. These cDNA-RNA hybridization analyses of physical homology between type 2 hybrid virus and rubella virus genomes revealed that about 70% of the type 2 hybrid virus genome is derived from about an 85% portion of the rubella virus genome. These values indicate that the size of the type 2 hybrid virus genome is about 21% larger than that of the rubella virus genome. Co-sedimentation studies of these viral RNA by sucrose density gradient centrifugation confirmed that the molecular weight of type 2 hybrid virus RNA is 20% higher than that of rubella virus RNA. We propose a genomic structure of the type 2 hybrid virus taking into account both physical and biochemical data.

Animals↗
PubMed1975

Virus development in enucleate cells: echovirus, poliovirus, pseudorabies virus, reovirus, respiratory syncytial virus and Semliki Forest virus.

A group of RNA viruses, echovirus, poliovirus, reovirus, respiratory syncytial virus and Semliki Forest virus have been examined for ability to grow in enucleate African green monkey kidney (BSCi) cells. Semliki Forest virus produced an almost normal yield of virus but poliovirus, echovirus, reovirus and respiratory syncytial virus, although showing clear evidence of virus replication when compared with a nuclear DNA virus (pseudorabies virus) gave much lower yields than those from nucleate cells. Analysis of enucleate cells infected with echovirus and reovirus showed no evidence of a specific block in the synthesis of any virus-specified polypeptide. Infection with vesicular stomatitis virus at intervals after enucleation demonstrated a diminishing ability to support virus growth with increasing time. It is suggested that the yield of virus obtained from an enucleate cell is related to the length of the growth cycle of the virus, the reduced yield obtained with some viruses reflecting the declining ability of the enucleate cell to support virus growth.

Autoradiography↗
PubMed1992

Interactions between human immunodeficiency virus-1, hepatitis delta virus and hepatitis B virus infections in 260 chronic carriers of hepatitis B virus.

To evaluate the factors determining the severity of chronic hepatitis B virus infection and the interactions of human immunodeficiency virus and hepatitis delta virus infections, we retrospectively analyzed 260 patients, 146 of whom were followed for a mean of 31.4 +/- 1.8 mo. Human immunodeficiency virus, hepatitis B virus, and hepatitis delta virus status and aminotransferase activities, histological activity index, alcohol consumption and the prevalence of cirrhosis were investigated. The patients included 54 homosexuals, 19 parenteral drug abusers and 187 subjects with other or unidentified risk factors for exposure to hepatitis B virus. Thirty-five patients (13%) were positive for antibody to human immunodeficiency virus; 27 were homosexual and 8 were drug abusers. The mean aminotransferase activities, histological activity index and the prevalence of cirrhosis were similar in the human immunodeficiency virus-positive and human immunodeficiency virus-negative subgroups. Actuarial survival was significantly lower in the human immunodeficiency virus-negative subgroups. Actuarial survival was significantly lower in the human immunodeficiency virus-positive group than in the human immunodeficiency virus-negative subjects (p = 0.004); the cause of death was clearly related to liver failure in four of the five human immunodeficiency virus-positive patients and two of the six human immunodeficiency virus-negative subjects who died. To evaluate the factors determining the severity of liver disease, we compared homogeneous subgroups of subjects. Among the homosexual patients, the prevalence of HBeAg and hepatitis B virus DNA, aminotransferase activities and the histological activity index did not differ according to human immunodeficiency virus antibody status.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed1991

Glycoprotein C of herpes simplex virus type 1 is essential for the virus to evade antibody-independent complement-mediated virus inactivation and lysis of virus-infected cells.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) is a receptor for the complement component C3b. We have previously isolated HSV-1 gC- strains (TN1, TN2 and TN3) from a patient with recurrent keratitis at three different times. These are very rare isolates because gC was thought to be essential for the virus in vivo. To determine whether gC modifies the interaction of complement with cell-free virus or virus-infected cells, we constructed gC+ recombinant viruses in which the intact gC gene of strain KOS was inserted into the TN1 virus genome. TN1 virus was inactivated by complement and TN1 virus-infected cells were lysed by complement; however, gC+ recombinant viruses became resistant to these effects of complement. These results suggest a role for gC in protection of both the virion envelope and the infected cell surface against damage by complement. TN1 virus was inactivated by complement from rats (Wistar, WKA, F344 and SD), guinea-pigs (Hartley) and humans, but not by complement from mice (C3H, DDD and BALB/c), which indicates that mice seem to be inappropriate as an experimental model for the study of HSV infection in which complement factors need to be considered.

Animals↗
PubMed1992

Virus-specific antibodies to Epstein-Barr virus, varicella-zoster virus and rubella virus in renal transplant patients with cytomegalovirus infections.

Renal transplant patients with primary and recurrent cytomegalovirus (CMV) infection had higher antibody titres to Epstein-Barr virus viral capsid antigen (EBV-VCA-IgG) before and after transplantation than healthy blood donors. The geometric mean titres (GMT) of EBV-VCA-IgG were higher in renal transplant patients without CMV infection than in renal transplant patients with CMV infection. Four-fold or greater rises in EBV-VCA-IgG antibody were detected in six patients and a similar rise in antibody to EBV early antigen (EBV-EA-IgG) was detected in one other patient. IgM antibody to EBV-VCA (EBV-VCA-IgM) was detected in only three of these patients. EBV-EA-IgG was present in 39% patients and in 30% control subjects. IgG titres to varicella zoster virus (VZV-IgG) and rubella virus (rubella HI) were higher in patients without CMV infection compared to the patients with CMV infection. Raised titres were detected to VZV in five patients and to rubella virus in three patients. Reductions in antibody titre of four-fold or more were also detected in EBV-EA-IgG (one patient) and to rubella virus (one patient). Raised antibody titres to EBV, VZV, and rubella virus in renal transplant patients may indicate reactivation of these viruses without any symptoms.

Antibodies, Viral↗
PubMed1990

Incidence of antibodies against human immunodeficiency virus, human T-cell lymphotropic virus type 1, hepatitis B virus, hemorrhagic fever with renal syndrome virus and Chlamydia in Tonga and western Samoa.

Among the populations of Tonga and Western Samoa, serum antibodies against human immunodeficiency virus or hemorrhagic fever with renal syndrome virus were not detected (0/904 and 0/192). No serum samples were considered to be positive for antibody against human T-cell lymphotropic virus type 1 (0/527). Hepatitis B antigen and antibody were found in 4% (8/192) and 47% (90/192), respectively. Chlamydia trachomatis IgG and C. psittaci IgG antibodies were detected in 39% (75/192) and 47% (91/192), respectively. The possibilities of the spread of human immunodeficiency virus and hemorrhagic fever with renal syndrome virus on the islands when the viruses invade from abroad were discussed.

Adolescent↗
PubMed1975

Multiple sclerosis and parainfluenza 1 virus. History of the isolation of the virus and expression of phenotypic differences between the isolated virus and Sendai virus.

54 cultures were established from brain tissue obtained 2-3 hrs after death from 1 case of multiple sclerosis and 30 cultures from another case. Following fusion with indicator cells in the presence of lysolecithin, a parainfluenza type 1 virus (6/94 virus) was isolated from cultures representing one plaque area in the first case and one plaque area in the second case. A cell line chronically infected with the 6/94 virus has been maintained for more than 100 passages in vitro. A close relationship to the Sendai Hemagglutinating Virus of Japan (HVJ) is indicated from RNA-RNA hybridization and the patterns of electrophoretic mobilities of viral polypeptides. Conversely, differences in optima for growth-requirement temperatures, hemolytic activity and the capability to fuse mammalian cells, distinguishes 6/94 virus and HVJ as distinct phenotypic entities of a closely related genotype.

Adult↗
PubMed1992

Herpes simplex virus type 2 infection, syphilis, and hepatitis B virus infection in Haitian women with human immunodeficiency virus type 1 and human T lymphotropic virus type I infections. The Johns Hopkins University (JHU)/Centre pour le Developpement et la Santé (CDS) HIV Study Group.

Antibodies to herpes simplex virus type 2 (HSV-2), antibodies to hepatitis B virus (HBV) core antigen (anti-HBc), and VDRL antibodies (serologic evidence of syphilis) were evaluated in women known to be infected with human immunodeficiency virus type 1 (HIV-1) (n = 95) or human T lymphotropic virus type I (HTLV-I) (n = 45) and controls (n = 89). HIV-1-seropositive women were more likely than controls to have antibodies to HSV-2 (88% vs. 54%; P less than .001), anti-HBc (67% vs. 43%; P = .008), and VDRL antibodies (21% vs. 8%; P = .02). Similarly, HTLV-I-seropositive women were more likely than controls to have antibodies to HSV-2 (82% vs. 54%; P = .003) and anti-HBc (67% vs. 43%; P = .008). There was no evidence that HIV-1 or HTLV-I predisposed to chronic hepatitis B virus infection. The stronger associations between HIV-1 and HTLV-I with HSV-2 than the associations with syphilis or HBV are consistent with the hypothesis that recurrent disruptions of mucous membranes caused by HSV-2 infections predispose to sexual transmission of HIV-1 and HTLV-I.

Adolescent↗
PubMed1992

Evidence that pepper mottle virus and potato virus Y are distinct viruses: analyses of the coat protein and 3' untranslated sequence of a California isolate of pepper mottle virus.

Pepper mottle virus (PepMoV) is a member of the large and complex genus Potyvirus, and is classically distinguished from other members of the genus by differential host range and cytopathology as well as serology of the coat protein and cytoplasmic inclusion body proteins. Here we report the deduced amino acid sequence of the coat protein of a California potyvirus identified by a variety of classical methods as PepMoV (PepMoV C). Comparison of the 3' untranslated nucleic acid sequence and the deduced coat-protein amino acid sequence of the PepMoV C isolate with those of PVY and other potyviruses indicates that PepMoV C is sufficiently diverged to be considered a distinct virus species. Thus, comparative sequence analyses of the PepMoV C isolate support earlier serological and biological evidence that PepMoV and PVY are distinct viruses.

Amino Acid Sequence↗
PubMed1992

Use of single-gene reassortant viruses to study the role of avian influenza A virus genes in attenuation of wild-type human influenza A virus for squirrel monkeys and adult human volunteers.

The transfer of six internal RNA segments from the avian influenza A/Mallard/New York/6750/78 (H2N2) virus reproducibly attenuates human influenza A viruses for squirrel monkeys and adult humans. To identify the avian influenza A virus genes that specify the attenuation and host range restriction of avian-human (ah) influenza A reassortant viruses (referred to as ah reassortants), we isolated six single-gene reassortant viruses (SGRs), each having a single internal RNA segment of the influenza A/Mallard/New York/6750/78 virus and seven RNA segments from the human influenza A/Los Angeles/2/87 (H3N2) wild-type virus. To assess the level of attenuation, we compared each SGR with the A/Los Angeles/2/87 wild-type virus and a 6-2 gene ah reassortant (having six internal RNA segments from the avian influenza A virus parent and two genes encoding the hemagglutinin and neuraminidase glycoproteins from the wild-type human influenza A virus) for the ability to replicate in seronegative squirrel monkeys and adult human volunteers. In monkeys and humans, replication of the 6-2 gene ah reassortant was highly restricted. In humans, the NS, M, PB2, and PB1 SGRs each replicated significantly less efficiently (P less than 0.05) than the wild-type human influenza A virus parent, suggesting that each of these genes contributes to the attenuation phenotype. In monkeys, only the NP, PB2, and possibly the M genes contributed to the attenuation phenotype. These discordant observations, particularly with regard to the NP SGR, indicate that not all genetic determinants of attenuation of influenza A viruses for humans can be identified during studies of SGRs conducted with monkeys. The PB2 and M SGRs that were attenuated in humans each exhibited a new phenotype that was not observed for either parental virus. Thus, it was not possible to determine whether avian influenza virus PB2 or M gene itself or a specific constellation of avian and human influenza A virus specified restriction of virus replication in humans.

Adult↗
PubMed1975

Separation of sarcoma virus-specific and leukemia virus-specific genetic sequences of Moloney sarcoma virus.

We have studied the nucleic acid sequences in nonproducer cells transformed by Moloney sarcoma virus or Abelson leukemia virus (two types of replication-defective, RNA-containing, viruses isolated by passage of Moloney leukemia virus in BALB/c mice). DNA probes from the Moloney leukemia in virus detect RNA in both Abelson virus-transformed nonproducer cells and Moloney sarcoma virus-transformed nonproducer cells. A sarcoma-specific cDNA, prepared from the Moloney sarcoma virus, has extensive homology to RNA found in heterologous nonproducer cells transformed by Moloney sarcoma virus, has little homology to RNA in cells producing Moloney leukemia virus, and no detectable homology to RNA in nonproducer cells transformed by the Abelson virus. By analogy to earlier data on avian and mammalian sarcoma viruses, these results suggest that the Moloney sarcoma virus arose by recombination between a portion of the Moloney leukemia virus genome and additional sarcoma-specific information, and indicate that the expression of this information in not essential for Abelson virus-mediated fibroblast transformation.

Cell Line↗
PubMed1991

Homotypic and heterotypic protection against influenza virus infection in mice by recombinant vaccinia virus expressing the haemagglutinin or nucleoprotein of influenza virus.

Recombinant vaccinia virus expressing the influenza virus haemagglutinin (HA) or nucleoprotein (NP) genes from A/SW/Hong Kong/1/74 (H1N1) under the control of a hybrid promoter containing the P7.5 early promoter element and promoter of the gene encoding the major protein of cowpox virus A type inclusion body was constructed to investigate protective immunity against homologous and heterologous viruses in mice. These recombinant vaccinia viruses produced authentic influenza virus HA and NP in infected cells. The recombinant vaccinia virus-influenza virus HA conferred efficient subtype-specific protection although mice challenged with heterologous influenza viruses underwent initial infection. By contrast, immunization with the recombinant vaccinia-influenza virus NP limited virus multiplication in the lungs against challenge infection with all H1N1 and H3N2 influenza viruses examined, although less efficiently. These results will prompt the re-examination of the possibility of using the recombinant vaccinia virus-influenza virus NP as a cross-protective vaccine.

Animals↗
PubMed1992

Intrahepatic expression of pre-S1 and pre-S2 antigens in chronic hepatitis B virus infection in relation to hepatitis B virus replication and hepatitis delta virus superinfection.

Hepatocyte expression of pre-S1 and pre-S2 in relation to hepatitis B virus replication (hepatitis B virus-DNA in serum and HBcAg in the liver), histological activity and hepatitis delta virus superinfection was studied by indirect immunofluorescence on frozen sections of liver specimens from 68 patients with chronic hepatitis B virus infection. All 44 patients with chronic type B hepatitis had pre-S1 and pre-S2 display in the liver. The distribution of pre-S1 in the liver was membranous in one, mixed membranous and cytoplasmic in 12, and cytoplasmic in 31. The distribution of pre-S2 was membranous in one, mixed membranous and cytoplasmic in 26, and cytoplasmic in 17. Membranous expression of pre-S1 was significantly more prevalent in patients with active hepatitis B virus replication than in those without (13/28 v 0/16, p < 0.001), regardless of the histological activity, as was membranous expression of pre-S2 (27/28 v 0/16, p < 0.001). In contrast, a significantly higher extent of cytoplasmic expression of pre-S1 and pre-S2 was noted in patients without active hepatitis B virus replication than in those with. Of 24 patients with chronic type D hepatitis virus, eight had active hepatitis B virus replication, and the other 16 did not. The distribution and quantitative expression of pre-S1 and pre-S2 in the liver in these patients also correlated significantly with the status of hepatitis B virus replication and, moreover, showed little or no difference from those without hepatitis delta virus infection. In conclusion, all patients with chronic type B hepatitis had synthesis and display of pre-S1 and pre-S2 in the liver. The distribution and quantitative expression of pre-S1 and pre-S2, however, were closely related to the status of hepatitis B virus replication, but not to the histological activity. Hepatocyte expression of pre-S1 and pre-S2 in chronic type D hepatitis also correlated significantly with status of hepatitis B virus replication, and was not modulated by concurrent hepatitis delta virus infection.

Fluorescent Antibody Technique↗
PubMed1990

Sequential changes in the humoral immune response of pigs to pseudorabies virus after vaccination, exposure to virulent virus, and reactivation of latent virus.

Sequential changes in the humoral immune response of pigs to pseudorabies virus (PRV) after each of several exposures to the virus were evaluated by determining virus neutralization (VN) and radioimmunoprecipitation (RIP) activities of sera collected at selected intervals. Pigs were vaccinated intramuscularly with live attenuated virus (6 pigs), inactivated attenuated virus (6 pigs), or inactivated virulent virus (6 pigs). All pigs were challenged oronasally with virulent virus 3 weeks later and 12 (4 pigs of each vaccine group) were subsequently treated with dexamethasone in an attempt to reactivate latent virus. The relatively low serum titers of VN antibody that were raised by vaccination (titers ranged from 2 to 32) increased markedly (at least 16-fold) for all pigs after exposure to virulent virus. After dexamethasone treatment, the VN titers of 2 pigs increased 16-fold, whereas those of the other 10 dexamethasone-treated pigs and the 6 nontreated pigs either remained the same or increased only minimally (i.e., no more than 2-fold). The results of RIP using 35S-methionine-labeled viral proteins were initially similar to those of VN in that the low levels of serum RIP activity detected after vaccination increased markedly after subsequent exposure to virulent virus. In contrast to VN, however, most pigs (11 of 12) treated with dexamethasone had a clear increase in serum RIP activity. The increase was particularly striking for viral proteins of relatively low (less than 46K) molecular weight. Precipitating activity for 14C-glucosamine-labeled viral glycoproteins was not detected until after pigs were exposed to virulent virus. The increase in RIP activity detected after dexamethasone treatment was likely due to an additional antigenic stimulus associated with virus reactivation. However, virus was isolated from nasal swabs of only 4 of the 12 treated pigs. None of the results appeared to be affected appreciably by the type of vaccine used for initial immunization.

Animals↗
PubMed1975

Murine sarcoma virus related nucleic acid sequences in a non-transforming virus derived from an interspecies pseudotype sarcoma virus.

A number of sarcomagenic viruses recovered from hamster tumors induced originally by Moloney sarcoma virus or several of its mouse pseudotypes appear to be interspecies pseudotypes with structural proteins of hamster type-C viruses and nucleic acid sequences of both mouse and hamster specificity. A non-transforming virus (GLOH-minus) recovered from a sarcomagenic virus preparation obtained from a tumor induced by the Gross pseudotype of Moloney sarcoma virus was previously shown to retain mouse sequences. The ability to discriminate readily between Gross and Moloney viruses by molecular hybridization, especially with regard to thermal stability of inter-strain hybrid molecules, and the stability of Moloney virus sequences in a rat-mouse interspecies pseudotype, made it possible to specify that the mouse virus-specific sequences in GLOH-minus are derived from Moloney virus and not Gross. Thus, even though GLOH-minus is non-transforming in vitro or in vivo, it still retains sequences highly related to Moloney sarcoma virus. The lack of transforming ability is thus probably based on loss or change of specific sequences necessary for transformation at some point in the history of this virus.

AKR murine leukemia virus↗
PubMed1991

Vaccinia virus DNA ligase is nonessential for virus replication: recovery of plasmids from virus-infected cells.

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).

Animals↗