Rubella virus carrier cultures derived from congenitally infected infants.

Spontaneous rubella carrier cultures derived from tissues of infants with congenital rubella were studied in an attempt to elucidate a possible mechanism for viral persistence observed in these infants. Chronically infected cells were found to have a reduced growth rate and the cultures appeared to have a shortened life span. The rubella carrier state was not dependent on serum inhibitors or rubella antibodies. Virtually every cell in the carrier population was found to be producing virus. The carrier cultures could not be cured by rubella antibodies. The rubella-infected cells were resistant to superinfection with vesicular stomatitis virus and herpes simplex virus but were susceptible to infection with echovirus 11. The replication of vesicular stomatitis virus was apparently blocked at an intracellular site, for the virus readily adsorbed to the chronically infected cells and entered into an eclipse phase; however no infectious virus developed. No evidence of interferon production by these cells could be obtained. It is postulated that clones of rubella-infected cells in vivo, with properties similar to those in carrier cultures developed in vitro from tissues of in utero infected infants, might explain the observed viral persistence noted in congenital rubella.

Variation in the oncogenic potential of human adenoviruses carrying a defective SV40 genome (PARA).

The acquisition of the defective SV40 genome by a variety of human adenovirus serotypes by the process of transcapsidation has resulted in the addition of oncogenic potential for newborn hamsters to the previously nononcogenic adenovirus types 1, 2, 5, and 6. These serotypes have previously been grouped together by the high GC content of their DNA. Transcapsidation of the SV40 genome to weakly oncogenic adenovirus types 3, 14, 16, and 21 has failed to increase their oncogenic potential although the parent adenovirus type 7 carrying PARA is highly oncogenic. These serotypes belong to the group possessing a DNA of intermediate GC content. All the PARA-adenovirus populations, even those that were nononcogenic, were able to induce SV40 transplantation immunity and therefore carry the SV40 transplantation marker as well as the marker for synthesis of SV40 tumor or T antigen.

Adeno-associated satellite virus interference with the replication of its helper adenovirus.

Adeno-associated satellite virus type 4 interferes with the replication of its helper adenovirus. No interferon-like soluble substance could be detected in satellite-infected cultures and other DNA- and RNA-containing viruses were not inhibited by coinfection with satellite virus under conditions which reduced adenovirus yields by more than 90% in monkey cells. Altering the concentration of adenovirus in the presence of constant amounts of satellite resulted in a constant degree of interference over a wide range of adenovirus inocula and suggested that adenovirus concentration was not a significant factor in the observed interference. The interference with adenovirus replication was abolished by pretreating satellite preparations with specific antiserum, ultraviolet light or heating at 80 degrees C for 30 min. This suggested that infectious satellite virus mediated the interference. Satellite virus concentration was found to be a determinant of interference and studies indicated that the amount of interference with adenovirus was directly proportional to the concentration of satellite virus. 8 hr after adenovirus infection, the replication of adenovirus was no longer sensitive to satellite interference. This was true even though the satellite virus was enhanced as effectively as if the cells were infected simultaneously with both viruses. Interference with adenovirus infectivity was accompanied by reduced yields of complement-fixing antigen and of virus particles which suggested that satellite virus interfered with the formation and not the function of adenovirus products. When cells were infected either with adenovirus alone or with adenovirus plus satellite, the same proportion of cells plated as adenovirus infectious centers. However, the number of plaque-forming units of adenovirus formed per cell in the satellite-infected cultures was reduced by approximately 90%, the same magnitude of reduction noted in whole cultures coinfected with satellite and adenovirus. This suggested that all cells infected with the two viruses were producing a reduced quantity of adenovirus.

Abnormalities of in vitro lymphocyte responses during rubella virus infections.

In vitro rubella virus infections of lymphocytes from normal adult humans impaired their responsiveness to phytohemagglutinin (PHA) stimulations; a situation which seemed analogous to the PHA unresponsiveness of peripheral lymphocytes from babies with the congenital rubella syndrome. Such in vitro viral infection of normal cells also decreased the synthesis of normal nucleic acids and structural proteins, and abrogated the enhanced DNA synthesis induced by pokeweed and specific antigen stimulations. Furthermore, it was shown that live rubella virus, but not ultraviolet-irradiated virus, was necessary for the impaired mitogenic responses of normal leukocytes. These observations are interpreted to favor the view that the virus achieves its inhibitory effect on the action of mitogens by interference either directly or indirectly at an intracellular site. Such an action could reduce the functional potential of lymphocytes and impair their effectiveness as immunologically competent cells or as effectors in immunologic reactions.

Adenovirus endocarditis in mice.

Viral endocarditis developed in 24 percent of 50 newborn mice 6 to 8 days after intraperitoneal inoculation with murine adenovirus. Typical adenovirus intranuclear inclusions were seen in heart-valve lesions, and high titers of virus were recovered from heart tissue. Furthermore, adenovirions were directly visualized by electron microscopy within endothelial cells and fibroblasts of the heart valves.

Herpes simplex virus in mice: electron microscopy of neural spread.

Herpes simplex virus rapidly infected the trigeminal nerves of mice after intranasal inoculation. Centripetal neural spread was suggested by histologic evidence of encephalitis in the area of attachment of the trigeminal nerve. Furthermore, electron microscopy revealed virus replication primarily within Schwann cells of the trigeminal nerve, and neurons of the gasserian ganglion.

Antibody to hepatitis B virus induced by injecting antibodies to the idiotype.

Anti-idiotype reagents that recognize a common idiotype associated with antibody to hepatitis B surface antigen (anti-HBs) were used to induce anti-HBs in mice. The anti-idiotype-induced anti-HBs was found to recognize the group-specific a determinant of hepatitis B surface antigen and to express an interspecies idiotype. These findings suggest that anti-idiotypes may be useful as vaccines or vaccine primers.

Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions.

Based on physical and chemical determinations, the mnolecular weight of the type 4 adeno-satellite virus is 5.4 X 10(6) daltons, and the virion contains 1.4 X 10(6) daltons of DNA. Denaturation and renaturation studies indicate that the viral genome is a single-stranded DNA molecule and that each virion contains either a minus or a plus strand. Upon extraction, the minus and plus strands unite to form double-stranded DNA molecules with no obvious excess of unpaired strands.

Nonencapsidated infectious DNA of adeno-satellite virus in cells coinfected with herpesvirus.

Adeno-associated satellite viruses produce antigen detectable by immunofluorescence but not infectious virus in tissue culture cells coinfected with herpes simplex virus. Analysis of DNA extracts from these infected cells shows that large amounts of infectious satellite virus DNA are produced but not encapsidated in the system. This result indicates that satellite virus may be defective at the maturation step.

Herpesvirus type 2: association with carcinoma of the cervix.

By utilizing the kinetics of neutralization, it was found that an antibody response to type 1 (oral) and type 2 (genital) herpesvirus infection could be independently measured and correlated with type of virus isolated from the patient. The presence of antibodies to type 1 and type 2 herpesvirus was examined in patients with carcinoma of the cervix, in matched controls, random controls, and patients with other types of malignancy. Antibodies to type 2 were found with greater frequency in patients with carcinoma of the cervix (83 percent) than in the other groups tested (0 to 20 percent). The data suggest an association of genital herpesvirus and carcinoma of the cervix.

Replication of adenovirus type 7 in monkey cells: a new determinant and its transfer to adenovirus type 2.

A strain of human adenovirus type 7, adapted to replication in green-monkey kidney cells, requires the interaction of two particles to initiate plaque formation in the simian cells. One particle is a true adenovirion. The second, apparently defective, consists of a genome carrying amonkey-adapting component in an adenovirus capsid; this genome does not express known SV40 determinants. The addition of human adenovirus type 7 that is not adapted enhances the titer and changesconditions for plaque formation by the adapted virus to a one-particle requirement. Addition of nonadapted human adenovirus type 2 as helper virus results in the transfer of the monkey-adaptingcomponent from adenovirus type 7 to adenovirus type 2. The population containing the adenovirus 2 transcapsidant then has the ability to replicate in simian cells.

Immune response to hepatitis B surface antigen: enhancement by prior injection of antibodies to the idiotype.

Anti-idiotype reagents that recognize a common idiotype associated with the combining site of antibodies to hepatitis B surface antigen (anti-HBs) were used to manipulate the immune response to hepatitis B surface antigen in BALB/c mice. The injection of antibodies to the idiotype before antigenic stimulation resulted in an increase in the number of cells secreting immunoglobulin M antibodies to hepatitis B surface antigen. Anti-HBs-secreting cells were also induced by administration of antibodies to the idiotype without subsequent antigen exposure. These findings indicate that the immune response to hepatitis B surface antigen in mice is regulated through an idiotype-anti-idiotype network.

Effect of amantadine hydrochloride on the response of human lymphocytes to phytohemagglutinin.

Amantadine hydrochloride (Symmetrel), which is an antiviral agent marketed for the prevention of infection by influenza virus, inhibits the mitogenic response of human lymphocytes stimulated with phytohemagglutinin. The concentrations which effectively inhibited the response to phytohemagglutinin were similar to those which maximally inhibit virus replication. The drug inhibited the mitogenic response without affecting the ability of phytohemagglutinin to agglutinate leukocytes. The data suggest that phytohemagglutinin, amantadine, and certain lipid-containing RNA viruses take part in cell-membrane interactions of common nature.

Wastewater renovation and reuse: virus removal by soil filtration.

Secondary sewage effluent and renovated water from four wells at the Flushing Meadows Wastewater Renovation Project near Phoenix, Arizona, in operation since 1967, were assayed approximately every 2 months in 1974 for viruses during flooding periods. Viruses, regularly found in the secondary effluent, were not detected in any renovated water samples. Our results indicated that human viral pathogens do not move through soil into the groundwater, but are apparently absorbed and degraded by the soil and reduced in numbers by a factor of at least 10(4) (99.99 percent removal).

Viral inhibition of lymphocyte response to phytohemagglutinin.

The response to phytohemagglutinin by lymphocytes from eight of fourteen patients with congenital rubella was inhibited, whereas that of lymphocytes from patients with other diseases was not. The response of normal lymphocytes infected in vitro was also inhibited. The results suggest that early association of lymphocytes with virus inhibits the function of the cell and contributes to persistent carrying of virus in congenital rubella. This phenomenon may be a means of detecting viruses not now recognizable by routine methods of tissue culture.

Integrated state of subgenomic fragments of hepatitis B virus DNA in hepatocellular carcinoma from mainland China.

Hepatocellular carcinoma (HCC) samples from mainland China were examined for the presence and state of hepatitis B virus (HBV) DNA sequences. HBV DNA was detected by dot-blot hybridization in 13 of 17 cases of HCC from the Shanghai area and in three of six samples from Hangzhou. The HCC cases from Shanghai were then analyzed in more detail. Fifteen of the 17 patients had serologic evidence of past or present infection with HBV (with inadequate information available for the other two), and the 13 HCC samples positive for HBV DNA all came from serologically positive patients. Southern blot analysis showed that the HBV DNA sequences were always integrated in the HCC high-molecular-weight DNA; only one or two viral copies were present per tumor cell, and no common integration site was evident. Hybridization analyses using subgenomic probes of HBV DNA revealed that the tumors seldom retained an entire HBV genome. HBV S-region sequences were always present, X-region sequences were usually represented, and C-region sequences were rarely detectable in virus-positive tumors. A fragment within the HBV DNA X-region, between nucleotides 1441 and 1526, was found to hybridize nonspecifically with cellular DNA; reported sequence data indicated that this fragment would contain approximately 70% guanine + cytosine. Histologic sections were prepared from some of the frozen tissue specimens and stained by an indirect immunoperoxidase technique for hepatitis B surface antigen (HBsAg). Only 1 of 10 HBV DNA-positive samples contained HBsAg in the cytoplasm of tumor cells, although abundant HBsAg was present in adjacent normal cells in all 10 cases. There were no significant differences in histology between HCC that contained HBV DNA sequences and those that were virus negative. These data support the premise that HBV represents a major etiologic factor in the development of HCC in the Shanghai area of China, although the molecular basis of viral involvement remains obscure.

Experimental carcinoma of liver in macaque monkeys exposed to diethylnitrosamine and hepatitis B virus.

The effects of treatments with diethylnitrosamine (DENA) and hepatitis B virus (HBV) on macaque monkeys were investigated by virus serology and by light and electron microscopy. The experimental groups comprised 43 newborn or juvenile cynomolgus and rhesus monkeys of both sexes. HBV neither had a carcinogenic effect nor increased the oncogenic effect of DENA. However, HBV given to juvenile primates before treatment with DENA resulted in subsequent gross and microscopic alterations consistent with mild hepatitis and postnecrotic cirrhosis; multifocal liver carcinoma apparently developed within these cirrhotic nodules. The pathologic findings in the experimental animals were strikingly similar to those observed in liver cancer patients.

Smoking and infectious agents and risk of in situ cervical cancer in Sydney, Australia.

In a study of 116 in situ cervical cancer patients and 193 matched community controls in Sydney, Australia, smoking was found to be a major risk factor. Current smokers had a adjusted relative risk [RR] of 4.5 compared to nonsmokers [95% confidence interval (CI) 2.2-9.1] and exsmokers a RR of 1.3 [95% CI 0.6-3.0]. There was a stepwise dose-response relationship between risk and number of cigarettes smoked (30+ cigarettes/day, RR = 5.1, 95% CI 1.5-17.3); this dose-response relationship was more marked among current smokers. Years of cigarette smoking was not consistently related to risk. Exposures to herpes simplex virus type 2 and cytomegalovirus as measured by antibody prevalence were unrelated to risk (RR = 1.1 for both measures). However, cases appeared to have more exposure than controls to herpes simplex virus type 1 (RR = 2.3, 95% CI 1.1-4.4).

Studies on herpes simplex virus and cancer.

Virus-induced polypeptides of cells infected by herpes simplex virus (HSV) types 1 and 2 were investigated by analysis on polyacrylamide gels and by determination of their antigenicity. Some polypeptides, VP154 and VP134, had immunological reactivity common to both virus types, while others (VP175 and VP123) were type specific. Only the glycosylated polypeptides were able to induce neutralizing antibody. The expression of viral genetic information was studied in newborn mice infected with wild-type and ts mutant viruses; some mutants had become attenuated and had lost pathogenicity for newborn mice while others had not. From induction experiments in HSV=transformed hamster cells, it appears that detection of enhanced replication of ts mutants in human cancer cells would be an indication of resident HSV genetic information. Sera obtained from cancer patients were examined for antibodies to early proteins synthesized in HSV-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. Cervical cancer patients had sera with a higher reactivity to early nonstructural polypeptides than to breast cancer patients or to matched healthy women. In contrast to the results with early polypeptides, little difference was detectable between the matched sera in their reactivity with a major capsid polypeptide, which is synthesized late in the infectious cycle.

Optimal conditions for uptake of exogenous DNA by Chinese hamster lung cells deficient in hypoxanthine-guanine phosphoribosyltransferase.

Conditions were characterized for maximizing the uptake of exogenous mammalian cell DNA by hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster lung cells. Recipient cell cultures in an exponential growth phase were found to be more competent in taking up DNA than stationary cultures. Polyornithine enhanced the uptake of exogenous DNA more reproducibly and to a greater extent than did any of the other facilitators tested (DEAE-dextran, CaCl2, latex spheres, spermine, polylysine and polyarginine). Maximal DNA incorporation occurred when polyornithine and DNA were mixed together prior to inoculation. About 25-30% of the DNA inoculum became deoxyribonuclease-resistant in a typical experiment utilizing polyornithine as the facilitator. Both homologous and heterologous exogenous DNAs rapidly became associated with recipient cell nuclei: approximately 95% of the deoxyribonuclease-resistant donor DNA was nuclear-associated 15 min after inoculation.