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Although rhesus monkeys have been generally regarded as refractory to infection with poliomyelitis virus administered by the oral route, two of seven infant rhesus developed paralytic poliomyelitis when fed murine-adapted strains of virus. Preliminary intranasal treatment with zinc sulfate and negative serial sections of the olfactory bulbs of the positive animals ruled out the possibility that infection occurred by way of the olfactory pathway. Studies on the distribution of virus in the tissues of the infected animals yielded positive results in one animal only. In this instance, virus was widely distributed throughout the body being isolated from spinal cord, buccal mucosa, duodenal wall, colon contents, superficial lymph nodes, spleen, heart, and adrenals.

Cytomegalovirus (CMV) and Chlamydia pneumoniae (C. pneumoniae) antigens and DNA sequences have been demonstrated in atherosclerotic plaques by several investigators. Most significantly, CMV DNA was found both in atherosclerotic lesions as well as in uninvolved areas of aortas and carotid artery, whereas C. pneumoniae was mostly detected in advanced carotid atherosclerotic lesions.Atherosclerotic plaques removed from seventeen patients during carotid endarterectomy were analysed for the simultaneous presence of CMV and C. pneumoniae DNA sequences using polymerase chain reaction (PCR). Of the seventeen samples, nine (53%) were positive for CMV DNA sequences and seven (41%) contained C. pneumoniae DNA sequences. Four samples (24%) were positive for both CMV and C. pneumoniae DNA. CMV DNA or C. pneumoniae DNA was detected in 12 (71%) of 17 carotid plaques and 2 additional patients had high titers of antibodies to CMV. CMV DNA and C. pneumoniae DNA were found in the same tissue specimens in 4 (24%) patients.These results present evidence that CMV DNA and/or C. pneumoniae DNA can be detected in 71% of carotid atherosclerotic plaques and in some instances DNA of both agents in the same tissue. The possible pathogenetic role of these agents in the initiation or promotion of the development of atherosclerotic plaques deserves increased attention.

The biological properties of human cytomegalovirus are consistent with pathogenic involvement at several levels of the atherogenic process. Although the sites of latency of CMV have not been established, both smooth muscle cells and leukocytes are likely possibilities. The observations of viral antigens and nucleic acid sequences in arterial smooth muscle cells suggest that latent CMV infection of the arterial wall may be common in patients with atherosclerosis. The seroepidemiologic studies suggest that a periodically activated latent infection is present in patients with atherosclerosis. Important are the observations that in immunosuppressed heart transplant patients infected with CMV, atherosclerosis is prone to develop in the transplanted heart. This mainly circumstantial evidence of the involvement of CMV in human atherosclerosis provides an important basis for further investigation of the role of CMV in atherogenesis.

Oral poliovirus vaccine, which has almost prevented the appearance of new poliomyelitis cases in the world, has been stabilized by incorporating molar MgCl2 into the vaccine. With the stabilizer, the virus can be held for years at 4 degrees C, and for six weeks at 22-25 degrees C, with no loss in titre. The stabilized virus loses 0.5 log10 units in three days at 37 degrees C, and in three hours at 45 degrees C. The MgCl2-stabilized vaccine, after being held at 30 degrees C for 21 days, elicited an antibody response in humans equal to that of standard vaccine maintained in the frozen state and thawed just before administration. The goal of WHO is to have a vaccine that does not lose any potency when held at 45 degrees C for at least seven days. Several approaches have been explored, with the addition of heavy water (Crainic et al, this volume) plus MgCl2 being the best. When poliovaccine is formulated with heavy water and MgCl2, it is more thermostable than in ordinary water containing molar MgCl2. Reconstituted measles vaccine rapidly loses potency at room temperature. At 22-25 degrees C, there is usually a 50% loss in potency in one hour, and at higher temperatures the reconstituted vaccine loses potency even more rapidly, and cannot be used. When molar MgSO4 is added, measles vaccine is stabilized, losing only 0.3 log10 in 30 minutes at 50 degrees C.

To elucidate prognostic implications of recipient cytomegalovirus infection before heart transplantation, we prospectively followed the clinical outcome of 21 transplant recipients whose explanted hearts (myocardium and coronary arteries) were first examined for the presence of cytomegalovirus DNA with polymerase chain reaction. Subsequently, serial endomyocardial biopsy tissue samples obtained from the allograft during routine evaluation for rejection were analyzed by polymerase chain reaction for both an immediate early and late cytomegalovirus gene region of cytomegalovirus DNA. Humoral cytomegalovirus immunoglobulin G antibodies were also measured by radioimmunoassay. Both early and late antigens were present in 14 of 21 (67%) explants from patients with (12 of 15 explants) and without (2 of 6 explants) pretransplant cytomegalovirus antibodies. Although the presence of both early and late antigens was uncommon in allografts the first week after transplantation (5 of 20 allografts, 25%), their presence significantly increased at 1 month (14 of 21 allografts, 67%) and 2 to 3 months (13 of 17 allografts, 77%) regardless of pretransplantation cytomegalovirus antibody status. The presence of both early and late antigens in explant tissue strongly predicted allograft virus status during the follow-up periods. Of five patients in whom clinical cytomegalovirus disease subsequently developed, all had explants positive for both early and late antigens, and all allografts were positive for early and late antigens within 1 month after transplantation. These are the first prospective data to correlate pretransplantation serum antibodies and explant polymerase chain reaction status with the development of future allograft infections and overall clinical outcome.

Resolutions of the questions of how and where eradication of poliomyelitis can bc achieved will continue to be major objectivcs in the next few years. The World Health Organization Expanded Programme on Immunization was initiated with the aim of reducing morbidity and mortality rates from seven target diseases, including poliomyelitis, by providing immunization against them forcvery child in the world. The program depends heavily upon technical Cooperation with and among developing countries, particularly those in tropical regions. Results are already being seen: increasing numbers of countries now participate in the EPI program or are otherwise enhancing their polio vaccination efforts. Better and more complete surveillance programs have been implemented to detect and assess cases that occur, as global efforts increase to control polio.

The organizers of this meeting have assigned two tasks to me. The first is to describe the state of affairs in 1955 when several lots of Salk poliovaccine, which had been formalin-inactivated according to government regulation and had been released for use in children, were subsequently found to have retained a small amount of live virulent virus. The second task is to describe a series of experiments that we conducted 25 to 35 years ago on the influence of cations on the thermostability of viruses. Perhaps there are some lessons to be learned and some benefits for future research to be gained from this history.

The CD4 molecule was reconstituted into the bilayers of large liposomes. Fluorescence microscopy and electron microscopy showed that these liposomes interact with HIV-infected H9-HT cells, delivering their contents to the cell interior. Liposomes bearing CD4 did not interact in this way with noninfected H9-HT cells nor did liposomes without CD4 interact significantly with HIV-infected cells. From electron micrographs, it appeared that HIV binds to liposomes bearing CD4; no attachment of virions to liposomes without CD4 was observed.

A transmissible agent, the IM virus, antigenically related to the Japanese subacute myelo-optico-neuropathy virus, has been isolated from several human cerebrospinal fluids obtained from American patients with multiple sclerosis and other chronic diseases of the central nervous system. The isolates were propagated in human diploid fibroblast (MRC5) cells, and virus was released into the culture medium in the absence of overt cytolysis. Infection of MRC5 cells resulted in a subtle alteration in the normal growth pattern of the cells. In unstained cultures, the cell changes were so mild that it was necessary to carry out all virus assays under code to eliminate bias. Cells in late passages were more susceptible than vigorously growing cells in early passages. Analysis of the kinetics of replication revealed that newly synthesized progeny virus was first detected about 12 h postinfection, that maximal virus release occurred by 48 h postinfection, and that virus production was persistent throughout an 8-day period. Several inhibitors of DNA synthesis were effective in blocking viral replication, including cytosine arabinoside, iododeoxyuridine, and phosphonoacetic acid. A substantial decrease in infectivity was observed upon treatment of IM virus with ether, suggesting that a lipid-containing structure is essential for infectivity. Ultrafiltration studies approximated the size (diameter) of IM virus to be between 100 and 200 nm.

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.

A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 micron; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AlCl3 to 0.0005 M, collecting the virus on a 0.25-micron-pore Filerite disk (diameter, 25 mm) and and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 X 10(8) PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 X 10(7) to 4.5 X 10(7) PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools.

A synthetic peptide (SP1), corresponding to the amino acid residues 122 through 137 of the major polypeptide derived from hepatitis B surface antigen (HBsAg), subtype ayw, was analyzed for the presence of the major epitopes of HBsAg. Both a cyclic form, produced by introduction of an intrachain disulfide bond, and a linear form of the peptide were characterized. A panel of monoclonal antibodies with defined specificity for the cross-reactive group a antigenic determinant(s) and for the y and w subtype specificities was used for this analysis. The cyclic, but not the linear, form of SP1 reacted with five of 14 anti-a monoclonal antibodies, demonstrating that the cyclic peptide contains a conformation-dependent a epitope. Only one anti-a antibody was found to react with both cyclic and linear forms of SP1. Because SP1 failed to react with the remaining 8 anti-a monoclonal antibodies, it was concluded that the a antigenic reactivity associated with HBsAg contains an additional epitope(s) unrelated to that expressed on SP1. Both cyclic and linear SP1 reacted with three of three anti-y monoclonal antibodies, indicating that a sequential y epitope is also present on SP1; no w reactivity was detected. Analysis of the idiotypes associated with the monoclonal antibodies showed those that combined with cyclic SP1 also inhibited the binding of a common human anti-HBs (CHBs) idiotype with its rabbit anti-idiotype serum, whereas a monoclonal antibody that did not react with the cyclic SP1 epitope failed to inhibit the CHBs idiotype-anti-idiotype reaction. Thus, the conformational a epitope present on cyclic SP1 appears to contain the predominant epitope recognized by humans in response to a natural HBV infection.

Acquired immune deficiency syndrome (AIDS) in many aspects resembles formerly recognized immune deficiencies, but in its complexity and presumed viral etiology, it is a unique entity. Examples of overlapping features of AIDS and immune defects, infectious complications, and neoplasms are discussed. It is not known what are the exact underlying mechanisms that permit or prohibit infectious and neoplastic complications among the immunodeficiency syndromes compared, but both overlapping and differing features of these entities are significant enough to hold a clue for future understanding. AIDS resembles feline leukemia as this latter syndrome was described 10 years ago. The retrovirus causing feline leukemia is highly immunosuppressive; preleukemic and viremic cats often succumb to opportunistic infections. If patients with early AIDS harbor retroviruses related to the human T cell leukemia virus in their organs (lymph nodes, bone marrow), serotherapy with monoclonal antibodies directed to this virus may stop and reverse the severe immunosuppression, thus preventing the development of opportunistic infections.

Secondary sewage effluent containing about 3 X 10(4) plaque-forming units of polio virus type 1 (LSc) per ml was passed through columns 250 cm in length packed with calcareous sand from an area in the Salt River bed used for ground-water recharge of secondary sewage effluent. Viruses were not detected in 1-ml samples extracted from the columns below the 160-cm level. However, viruses were detected in 5 of 43 100-ml samples of the column drainage water. Most of the viruses were adsorbed in the top 5 cm of soil. Virus removal was not affected by the infiltration rate, which varied between 15 and 55 cm/day. Flooding a column continuosly for 27 days with the sewage water virus mixture did not saturate the top few centimeters of soil with viruses and did not seem to affect virus movement. Flooding with deionized water caused virus desorption from the soil and increased their movement through the columns. Adding CaCl2 to the deionized water prevented most of the virus desorption. Adding a pulse of deionized water followed by sewage water started a virus front moving through the columns, but the viruses were readsorbed and none was detected in outflow samples. Drying the soil for 1 day between applying the virus and flooding with deionized water greatly reduced desorption, and drying for 5 days prevented desorption. Large reductions (99.99% or more) of virus would be expected after passage of secondary sewage effluent through 250 cm of the calcareous sand similar to that used in our laboratory columns unless heavy rains fell within 1 day after the application of sewage stopped. Such virus movement could be minimized by the proper management of flooding and drying cycles.

A semiautomated instrument (Septi-Screen) and method are described that semiquantify bacteria colorimetrically in blood-inoculated cultures in 2 to 8 h. If the blood culture is positive, concentrates (of viable bacteria) are obtained free of blood cells. In pediatric cases and in septicemia attributed to indwelling catheters where bacteremia is often manifested by high bacterial counts, the total time required to obtain bacterial concentrates is only 2 to 3 h. In brief, at various intervals after inoculation of 10 ml of blood into 100 ml of culture fluid, 25-ml blood culture samples are passed through the Septi-Filter. Erythrocytes and leukocytes pass into the filtrate and are discarded, but bacteria are electrostatically retained on the filter surface. The entrapped bacteria are backflushed and recovered as a concentrate that is processed through a second filter (Septi-Count) for colorimetric quantitation. The bacteria are stained, and the filter fibers are decolorized. The color intensity of the filter surface is proportional to the number of bacteria in the concentrate processed. The total Septi-Screen processing time is 3 to 5 min.

Summary A number of recent reports concerning the inhibitory action of sulphated polysaccharides on d strains of poliovirus prompted a reinvestigation of the factors involved. It was found that polyglucose compounds in agar are not released in concentrations sufficient to inhibit viruses under the overlay conditions used for suppressing d strains of poliovirus. Furthermore, sulphated polyanions in agar cannot be the critical factor, since d strains of poliovirus are also suppressed under other solidifying agents (starch gel and methylcellulose) which are free of sulphated polyglucose. Purified agar free of sulphated polysaccharides, used with medium containing a low bicarbonate concentration attains the same high pH after equilibration as regular agar (containing polyanions) made up with high concentrations of bicarbonate, and it is this high pH which appears to explain the ready growth of d strains of poliovirus.

Discussion and summaryA tissue culture cell line of rabbit cornea (SIRC) has been discovered by Leerhoy(1) to show clear and readily detectable CPE when inoculated with rubella virus. This observation has been confirmed and extended. The SIRC cells have been shown to be as effective for primary isolation of rubella virus from throat swabs, rectal swabs, urine, cerebrospinal fluid, and lens tissue as the more widely used GMK cells. As previously noted for both GMK and primary human amnion cells(9), subcultivations are necessary with SIRC cells for maximum isolation of rubella virus. SIRC cells may be used for detecting rubella neutralizing antibody in human sera and titers obtained are comparable to those obtained in GMK. Serial passage of rubella virus in SIRC cells appears to yield higher virus titers than those obtained after serial passage in GMK.SIRC cells offer several advantages. They are a continuous cell line easily maintained in the laboratory. They produce direct CPE, eliminating the need for ch...

A retrospective serologic survey for antibodies to adeno-associated satellite viruses was conducted on serum specimens collected from military recruits over a 7-year period. The results showed that 14% to 23% of the subjects possessed antibody titers of 16 or greater to any of the four satellite serotypes. Monotypic and heterotypic type 4 antibody seroconversions, which have been seldom seen in human but often in simian sera, occurred more frequently in men who received respiratory virus vaccines prepared in monkey kidney tissue cultures. It is speculated that such responses may have occurred from infections due to activation of latent defective viruses in these preparations or to antigenic components in the vaccines themselves. No definite relationship between these satellite viruses and disease was observed although there was a suggestion of an association between seroconversion to type 1 satellite virus in patients with the diagnosis of pneumonia. Sera from rubella patients showed significantly higher preexisting antibody titer to satellite virus types 1, 2 and 3 than did those of subjects with other illnesses. Only a few of the rubella cases showed seroconversion to the type 1 satellite virus.

This paper describes the preparation and test procedures for a second batch of lyophilized LBM combination antiserum pools, A through H, used for identifying 42 enteroviruses. Each pool is selectively composed of 10 or 11 of 42 individual enterovirus equine sera so that it contains 500 antibody units of each serum component per 0.1 ml. The new pools have been constituted from equine monovalent antisera that were prepared during the period 1962-67 and then evaluated and standardized in a series of collaborative international studies. An essential aspect of preparing the new pools was ensuring that the individual sera had retained high antibody titres through the long period of storage. At the time of retesting, the original stocks of these monovalent sera had been stored frozen at -20 degrees C for periods ranging from 16 to 20 years.

Recent studies have provided a greater understanding of the movement of viruses in the environment by their attachment to solids. These studies have focused on solids-associated viruses present in wastewater discharged into the ocean and on viruses in sludge and wastewater that may be retained in soil following their land disposal. Such ocean or land disposal of wastewater and sludge may result in a discharge of one or more of 120 human enteric virus pathogens including those causing poliomyelitis, viral hepatitis A and acute gastroenteritis.Solids-associated viruses in effluents discharged into coastal waters accumulate in bottom sediments, which may contain 10 to 10 000 more virus per unit volume than the overlying seawater. Solids-associated viruses resuspended by water turbulence may be transported from polluted to distant non-polluted recreational or shellfish-growing water. Transmission of viruses causing hepatitis or gastroenteritis may result from contact by bathers or swimmers with these viruses in recreational waters, or from ingestion of raw or improperly cooked shellfish in which the solids-associated virus had been bioaccumulated.The land disposal of sludge and wastewater has a potential of causing infections in farm workers, contamination of crops, pollution of raw potable water sources or infiltration of ground water. Viruses retained on soils can be released by rain water and may contaminate ground water through lateral and vertical movements.

Hepatitis B virus surface antigen (HBsAg) mRNA has been enriched from a hepatoma cell line (PLC/PRF/5) by specific polysome immunoprecipitation and used for cDNA cloning. A HBsAg cDNA clone was identified by in situ hybridization with a cloned viral probe. It was characterized by restriction endonuclease mapping and DNA sequence analysis. Molecular hybridization of PLC/PRF/5 cellular DNA and RNA to [32P]-labeled HBsAg cDNA revealed the integration of at least six copies of the hepatitis B virus (HBV) DNA into the host genome and expression of three DNA species containing HBsAg-specific sequences. The possible role of HBV in the oncogenesis of primary hepatocellular carcinoma is discussed.