Cytomegalovirus genome and the immediate-early antigen in cells of different layers of human aorta.

A number of data suggest that reactivation of cytomegalovirus (CMV) latent in arterial wall cells may contribute to atherogenesis; however, there is no direct evidence available. To address this issue, we have examined, using in situ hybridization or immunohistochemical staining, the frequency of occurrence of cells containing viral genome and of those expressing the IE 70 viral antigen in the endothelial layer and in deeper layers of human aortas with or without visible atherosclerotic lesions. Using endothelial cell cultures or tissue endothelial preparations, we found CMV-hybridizing endothelial cells in 6 of 8 grossly normal aortas and in 16 of 18 lesioned aortas. Antigen-positive endothelial cells were detected in 1 of 5 grossly normal vessels and in 6 of 7 lesioned vessels. Infected endothelial cells were abundant in areas adjacent to orifices of intercostal arteries of grossly normal aortas and in fatty spots of lesioned aortas, but no infected endothelial cells were observed in most plaques examined. In paraffin sections of grossly normal vessels, we detected CMV genome in cells adjacent to lumen and in cells randomly scattered through subendothelial intima and the media; however, no immunoreactive viral protein was found in the same tissue samples. In sections of lesioned vessels, clusters of CMV-hybridizing cells were found in the media in addition to infected cells randomly scattered through the intima and the media. In these samples of lesioned vessels, viral antigen was detected in cells adjacent to lumen and in cells clustered at the intima/media border. We found antigen-positive cells in grossly normal areas of lesioned aortas and in fatty lesions, but not in plaques of the same vessels. The data suggest that accumulation of the immediate-early CMV antigen in cells of endothelial layer and development of antigen-positive cell clusters in deeper layers of vascular wall accompany early atherogenic events in human aorta.

Frequency of coexistence of cytomegalovirus and Chlamydia pneumoniae in atherosclerotic plaques.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Cytomegalovirus infection and atherosclerosis.

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.

Cytomegalovirus infection, lipoprotein(a), and hypercoagulability: an atherogenic link?

A link between cytomegalovirus (CMV) infection and atherosclerosis has been suggested by experimental, clinical, and epidemiologic studies. We investigated the association between CMV antibody titers in serum collected in 1974 in 300 adult residents in Washington County, Md, and hemostatic parameters in plasma collected in 1987 through 1989, when these individuals participated in the baseline examination of the Atherosclerosis Risk in Communities Study. The cross-sectional association of CMV serum antibodies and hemostatic parameters was also explored in another set of Atherosclerosis Risk in Communities cases and controls. In the longitudinal analyses, CMV titers in 1974 were directly associated with 1987 through 1989 plasma levels of von Willebrand factor, factor VIII, and protein C and negatively associated with activated partial thromboplastin time. In the cross-sectional analyses, CMV titers were directly related to antithrombin III and fibrinogen levels. When the association between CMV antibodies and atherosclerosis was examined in stratified analyses, a significant association was restricted to individuals with high levels of lipoprotein(a) and fibrinogen. These results are compatible with previous evidence suggesting that CMV virus might have procoagulant properties. The possible synergism of CMV infection and resulting hypercoagulability with reduced fibrinolysis due to increased lipoprotein(a) levels deserves further investigation.

Cohort study of cytomegalovirus infection as a risk factor for carotid intimal-medial thickening, a measure of subclinical atherosclerosis.

BACKGROUND: Animal studies as well as clinical and cross-sectional epidemiological studies in humans have suggested a possible role of cytomegalovirus (CMV) and other herpesvirus infections in the development of cardiovascular disease. METHODS AND RESULTS: The present report is based on a case-control study nested within a historical cohort. The case group comprised 150 individuals with elevated carotid intimal-medial thickness (IMT) measured by B-mode ultrasound at the first two examinations of the Atherosclerosis Risk in Communities (ARIC) Study (1987 through 1992). The control group comprised 150 age- and sex-matched individuals with low IMT. Antibody titers for CMV and herpesvirus 1 and 2 were determined in sera obtained in 1974 as part of a community-wide survey conducted in Washington County, Maryland. Case subjects had higher mean CMV antibody titers in 1974 sera than control subjects, although the difference was not statistically significant when adjusted for other cardiovascular risk factors. There was evidence of a graded relation between the odds of intimal-medial thickening and the levels of CMV antibodies that remained significant after adjustment for the main cardiovascular risk factors (P = .013). The adjusted odds ratio for a high CMV antibody titer (a positive/negative value > or = 20) compared with a positive/negative value < 4 was 5.3 (95% confidence interval, 1.5 to 18.0). CONCLUSIONS: The results from this first population-based cohort study of CMV infection and carotid IMT are compatible with the hypothesis of a causal role of CMV in atherosclerosis.

Current status of poliovirus infections.

Two scientists who played leading roles in the conquest of poliomyelitis died recently. In 1954, Jonas Salk provided the first licensed polio vaccine, the formalin (and heat)-inactivated virus. Albert Sabin gave us the attenuated live virus vaccine, which was licensed in 1962. This paper takes the reader through the history of the disease, including its pathogenesis, epidemiology, vaccines, and future directions. The emphasis is on vaccines, for it seems that with proper vaccination the number of new cases is falling dramatically. It is hoped that by the year 2000, we will accomplish the goal of the World Health Organization of "a world without polio." Then, because there is no animal reservoir, we can seriously discuss when and how to eliminate the need for vaccination and ultimately destroy our stocks of poliovirus.

Prostitution, condom use, and invasive squamous cell cervical cancer in Thailand.

Cervical cancer is probably caused by a sexually transmitted agent. A case-control study was conducted in three hospitals in Thailand to investigate further the role of male sexual behavior, particularly regarding sexual contacts with prostitutes, in the development of this disease. Data were obtained from interviews with 225 married women with invasive squamous cell cervical carcinoma and 791 hospitalized controls, all of whom reported having only one sexual partner, and from interviews with their husbands. Risk of cervical cancer was strongly related to the women's husbands having visited prostitutes without using a condom when the husbands were less than 30 years old. A strong increasing trend in risk in relation to decreasing frequency of the husbands' condom use with prostitutes was observed, and a weaker increasing trend in risk with husbands' estimated lifetime total number of visits to prostitutes was found. The average latent period between the women's likely initial exposure to a sexually transmitted oncogenic agent and her diagnosis of invasive cervical cancer was about a quarter of a century. Regular use of condoms by customers of prostitutes could reduce the number of invasive cervical cancer cases in the general population of Thailand by at least one fourth.

PIP: Researchers compared data on 225 Thai married women with invasive squamous cell cervical cancer admitted to Siriraj and Chulalongkorn hospitals in Bangkok and at Maharaj Nakorn Chiang Mai Hospital during October 1979-September 1998 with data on 791 hospitalized Thai controls to examine the role of male sexual behavior and prostitution in the development of cervical cancer and the likely protective effect of condom use against cervical cancer. All the cases and controls claimed to have had only one sexual partner. Interviews were conducted with the husbands of all the cases and controls. There was a significant trend of increasing risk of cervical cancer as the frequency of husband's condom use with prostitutes declined (p = 0.004) (relative risk [RR] = 2.05 for rarely or never, 1.24 for sometimes, and 0.96 for always or frequently). The increasing risk of cervical cancer associated with little or no condom use with prostitutes was highest when the husbands were less than 30 years old (RR = 2.11 vs. 1.56-1.81 for age 20 or older). There was a weaker trend of increasing risk of cervical cancer with the husband's estimated lifetime total number of visits to prostitutes (p = 0.12). On average, the latent period between the wife's likely first exposure to a sexually transmitted oncogenic agent (i.e., date of their marriage) and her diagnosis of invasive cervical cancer was 24 years. These findings indicate the condom use with prostitutes would reduce the risk of cervical cancer.

My role in the discovery and classification of the enteroviruses.

The enteroviruses constitute one of the genera of the picornavirus family. The genus includes the polioviruses, the coxsackieviruses, and the echoviruses of humans, plus a number of enteroviruses of lower animals (e.g. monkeys, cattle, pigs, mice). Over 100 serotypes are recognized, of which the first to be discovered were the polioviruses. It was my good fortune to have been a scientist during the golden age of virology, when new techniques were being introduced into the field. These often led to the discovery of new viruses. This article details the isolation of the enteroviruses, their recognition as a separate genus of Picornaviridae, and my role in the process. Poliovirus, the most hazardous of the group, is almost gone from the world, but the other enteroviruses will be with us for some time. Several members of the Committee dealing with these agents--Enders,Sabin, Dalldorf, Syverton--have passed on, but the work of this Committee to which I was privileged to contribute will live long.

Thermostability of poliovirus and measles vaccines.

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.

Cytomegalovirus and atherosclerosis.

The investigations reviewed in this paper provide evidence that a member of the herpesvirus family can cause atherosclerosis in chickens. In vitro experiments, as well as studies of arteries from infected birds, suggest that a virus-induced alteration of cellular metabolism, which results in the accumulation of cholesterol and cholesteryl esters, may be the primary mechanism in development of viral atherosclerosis. In addition, the fact that the same avian virus induces a malignant lymphoma suggests that it may also have the potential to stimulate the proliferation of other cells, notably arterial smooth muscle cells. The evidence for involvement of one or more members of the herpesvirus family in human atherosclerosis is much more circumstantial. Cytomegalovirus (CMV) is prevalent, increasing with age, so that a majority of the human population becomes infected by adulthood. As with other herpesviruses, the infection with CMV is usually subclinical or latent. Although the sites of latency for CMV have not been established, both smooth muscle and leukocytes are likely possibilities. The observations of viral antigens and nucleic sequences, but not infectious virus, in arterial smooth muscle cells suggests that latent CMV infection of the arterial wall may be common in patients with atherosclerosis.

Cytomegalovirus and atherosclerosis.

Finding that an avian herpesvirus can cause atherosclerosis in chickens prompted studies of human herpesviruses in human atherosclerosis. Antigens and nucleic acid sequences of cytomegalovirus (CMV), a widespread member of the herpesvirus family, were found in arterial lesions in human atherosclerosis, but infectious virus has not been observed. In atherosclerosis patients, high levels of CMV antibodies are present, suggesting the presence of virus that had been activated from a latent state. Atherosclerosis also develops in immune-suppressed heart transplant patients infected with CMV. The properties of CMV are consistent with its involvement at several levels of the atherogenic process. If this concept is correct, immunization with a CMV vaccine should prevent CMV infection and atherosclerosis.

History and epidemiology of hepatitis A virus.

Hepatitis A was clearly recognized as an entity separate from other types of hepatitis during World War II, but only later did studies provide convincing evidence of the prevalence and transmission of hepatitis A virus (HAV). Disease incidence varies over time and geography, with wide differences from country to country and even within cities. Noted recently is a shift in prevalence in cases from childhood to adulthood. Incidence figures are unreliable. Epidemiology of the disease is best defined by measurement of anti-HAV antibodies. HAV is a very stable virus, frequently found in urban sewage. Infections occur early in life when sanitation is poor and living conditions crowded, but improvements in sanitation and hygiene have delayed infection, resulting in increasing numbers of adults susceptible to HAV. Transmission of HAV by blood is rare. High-risk persons include injection drug users, institutionalized persons and their caretakers, and those who travel from low-prevalence to high-prevalence countries.

Environmental virology: from detection of virus in sewage and water by isolation to identification by molecular biology--a trip of over 50 years.

Environmental virology began with efforts to detect poliovirus in sewage and water more than 50 years ago. Since that time, cell-culture methods useful for detection of enteroviruses have been replaced by molecular biology techniques for detection of pathogens (hepatitis A and E viruses, caliciviruses, rotaviruses, and astroviruses) that do not grow in cell culture or grow with great difficulty. Amplification of viral nucleic acid using the polymerase chain reaction (PCR) is the current preferred method. PCR or RT-PCR (to detect RNA viral genomes) is rapid, sensitive, specific, and quantitative. Method shortcomings include potential inhibition by substances in some environmental samples and an inability of test results to distinguish between infectious and noninfectious virus. Current questions involving use of PCR/RT-PCR tests for public health purposes include: What is the public health significance of a positive test, and should direct tests for viruses replace current public health-monitoring programs?

Cytomegalovirus DNA in arterial walls of patients with atherosclerosis.

The biological properties of cytomegalovirus (CMV) are consistent with a potential role in the pathogenesis of atherosclerosis. The evidence of such a role has so far been circumstantial, but CMV nucleic acid is beginning to be reported with increasing frequency in the arterial wall. Arterial specimens from 135 patients who underwent vascular surgery for symptomatic atherosclerotic vessel disease were analyzed by PCR for the presence of CMV nucleic acid. Samples were studied from the atheromatous plaque area and from uninvolved aortic tissues of patients undergoing surgery for vascular disease. One primer pair (LA) was used for detection of a late gene, and two other primer pairs (E1 and E2) were used for the immediate early gene region. Serum antibody to CMV was measured by radioimmunoassay. With the late gene primer, CMV nucleic acid was found in 76% of the tissue specimens tested, whereas the E2 gene primer complementary to the transforming mtr2 region was reactive in 90% of the arterial samples. There was no significant difference in the prevalence of CMV DNA in atherosclerotic plaque tissue and in uninvolved aortic tissue from the patients. A second early gene primer was not reactive with the tissue specimens, although it gave positive results with the positive control of infectious virus. Serum antibody to CMV was detected in 86% of the patients in whose tissue CMV DNA was demonstrated. CMV DNA was detected in a high proportion of atherosclerotic plaque tissues as well as in uninvolved aortic tissue of surgical patients, suggesting that latent CMV infection of the arterial wall may be a common occurrence in patients with atherosclerosis.

Poliomyelitis control in Israel, the West Bank and Gaza Strip: changing strategies with the goal of eradication in an endemic area.

Israel has faced the challenge presented by epidemic poliomyelitis by using different immunization strategies. In the 1950s, inactivated poliovirus vaccine (IPV) helped to reduce the total burden of the disease, but cases continued to occur. Introduction of oral poliovirus vaccine (OPV) in mid-1961 had a dramatic effect in controlling an extensive epidemic of poliomyelitis; however, poliovirus activity and cases continued during the 1970s, and at a low level in the 1980s. A localized outbreak of 15 cases of poliomyelitis in 1988 occurred in an area using enhanced potency IPV (eIPV) only. This led to a revision of poliomyelitis immunization policy. The successful poliomyelitis control in the West Bank and the Gaza Strip using both OPV and IPV since 1978 shows the advantages of a combined approach. This programme was therefore adopted in modified form in the whole of Israel, the West Bank and Gaza. Since late 1988, no cases of poliomyelitis have occurred in any of these three areas, indicating the success of the combined poliomyelitis immunization programme. These experiences may be helpful to other countries, especially those where there is a danger of importation of wild poliovirus, and to prevent vaccine-associated disease. The combined approach provides an additional immunization model in the international effort to eradicate poliomyelitis.

Cytomegalovirus/herpesvirus and carotid atherosclerosis: the ARIC Study.

Ubiquitous viruses such as members of the human herpes virus group, particularly cytomegalovirus (CMV), have been proposed to be clinically important agents in the initiation and progression of atherosclerosis. Antibodies to CMV and herpes simplex virus type 1 (HSV1) and type 2 (HSV2) were determined in 340 matched case-control pairs from the Atherosclerosis Risk in Communities (ARIC) Study. Cases were defined by B-mode ultrasonography as persons with thickened carotid artery walls consistent with early atherosclerosis but without a history of cardiovascular disease. Controls were defined as persons without thickened walls or history of cardiovascular disease. The case-control odds ratio for CMV antibodies was 1.55 (P = .03), for HSV 1.41 (P = .07), and for HSV2 0.91 (P = .63). When adjustment was made for potential confounders, the odds ratios were 1.36 for CMV (P = .24), 1.21 for HSV1 (P = .45), and 0.61 (P = .05) for HSV2. These results suggest a modest association between CMV and asymptomatic carotid wall thickening consistent with early atherosclerosis.

What are the implications of cardiac infection with cytomegalovirus before heart transplantation?

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.