Atherosclerosis induced by infection with Marek's disease herpesvirus in chickens.

BACKGROUND: This research was suggested after crystals that we observed in herpesvirus-infected cell cultures were identified as cholesterol. Other reports and the development of defined reagents led us to select the use of Marek's disease herpesvirus (MDV) infection of chickens to demonstrate a potential role of herpesviruses in the pathogenesis of atherosclerosis. Available for our use were a clone-purified strain of MDV of known virulence, genetically selected, specific pathogen-free chickens, and appropriate isolation facilities to design controlled experiments to fulfill Koch's postulates. METHODS AND RESULTS: Experiments were performed to test the roles of both MDV and dietary cholesterol in atherosclerosis. The birds were examined 7 months after MDV infection with and without cholesterol feeding for gross and microscopic arterial lesions. Atherosclerotic lesions were found only in infected normocholesterolemic or hypercholesterolemic birds. The character and distribution of these lesions closely resembled those found in the chronic human arterial disease. Atherosclerotic lesions were not found in uninfected birds even if the birds were hypercholesterolemic. CONCLUSIONS: Evidence was obtained from other experiments that after MDV infection, cholesterol and cholesteryl esters accumulated in cell cultures and in atherosclerotic lesions. These changes were associated with altered enzymatic activity of the cholesterol synthesis cycle. Immunization with turkey herpesvirus vaccine or SB-1 vaccine prevented atherosclerotic lesions.

Viruses: causal factors in atherosclerosis.

Herpesvirus infections may lead to atherosclerosis by altering arterial cell lipid metabolism. In a pathogen-free animal model, the virus-induced arterial disease closely resembled human atherosclerosis. These and other experimental findings may eventually lead to the control of human cardiovascular disease.

Virus-induced atherosclerosis. Herpesvirus infection alters aortic cholesterol metabolism and accumulation.

Infection of normocholesterolemic, specific-pathogen-free chickens with Marek's disease herpesvirus (MDV) has been shown histologically to lead to chronic atherosclerosis like that in humans. The development of herpesvirus-induced atherosclerosis in vivo and the presence of specific Marek's antigen within aortic cells suggested that MDV infection may modify lipid metabolism and lead to significant lipid accumulation. Experiments reported herein were designed to determine the types and quantity of lipid present in aortas from MDV-infected and uninfected chickens between 2 and 8 months of age following infection and assess one possible mechanism of lipid accumulation by evaluating the effect of MDV infection on aortic cholesterol and cholesteryl ester (CE) metabolism. Chromatographic-fluorometric analyses indicated that at 4 and 8 months of age after MDV inoculation, MDV-infected animals had a significant (P less than 0.05) two-fold to threefold increase in total aortic lipid accumulation characterized by significant increases in cholesterol, CE, triacylglycerol, and phospholipid as compared with aortas from uninfected animals. At 8 months of age, similar increases in aortic lipid accumulation were observed in MDV-infected animals as compared with those animals vaccinated with turkey herpesvirus and later challenged with MDV. CE synthetic activity was increased significantly by 50% at 4 months of age in the MDV-infected group as compared with the uninfected group, which could explain the initial increase in CE accumulation. By 8 months of age, the authors also observed a twofold increase in CE synthetic activity and a 30% and 80% reduction in lysosomal and cytoplasmic CE hydrolytic activities, respectively, in aortas of MDV-infected chickens as compared to controls. Moreover, infection with MDV blocked the activation of cytoplasmic CE hydrolytic activity by dibutyryl cyclic AMP or exogenous cyclic AMP-dependent protein kinase. Taken together, these results suggest that lipid accretion in aortas of MDV-infected chickens results, in part, from alterations in cholesterol/CE metabolism during early stages of the disease. These findings support the hypothesis that human atherosclerosis may result from specific herpesvirus infection which can alter lipid metabolism and lead to lipid accretion.

Altered cholesteryl ester cycle is associated with lipid accumulation in herpesvirus-infected arterial smooth muscle cells.

We describe herein the effects of Marek's disease herpesvirus (MDV) on cholesterol and cholesteryl ester metabolism in cultured chicken arterial smooth muscle cells. Infection of arterial smooth muscle cells from specific pathogen-free chickens with MDV, but not a virus control, herpesvirus of turkeys led to a 7-10-fold increase in the accumulation of free and esterified cholesterol and a 2-fold increase in phospholipids. The cellular lipid changes observed in the MDV-infected arterial smooth muscle cells resulted, in part, from the following: decreased low-density lipoprotein-cholesteryl ester hydrolysis due to decreased lysosomal (acid) cholesteryl ester hydrolytic activity; increased de novo synthesis of cholesterol; decreased excretion of free cholesterol; and, both increased cholesteryl ester synthetic activity and decreased cytoplasmic (neutral) cholesteryl ester hydrolytic activity which resulted in increased incorporation of oleic acid into cholesteryl ester. Other changes noted in the MDV-infected cells as compared to uninfected cells included a 2-fold increase in both total protein synthesis and lysosomal and microsomal marker enzyme activities. These alterations in lipid and protein metabolism in MDV-infected arterial smooth muscle cells may explain in part our in vivo findings that herpesvirus (MDV) infection of specific pathogen-free chickens fed a normocholesterolemic diet will induce arterial thickening and lipid accumulation resembling human atherosclerosis.

Herpesvirus-induced atherosclerosis in chickens.

Repeated experiments have established that infection with Marek's disease herpesvirus (MDV) leads to atherosclerosis in specific pathogen free (SPF) normocholesterolemic chickens. Neither normocholesterolemic nor hypercholesterolemic uninfected SPF chickens develop this disease. The MDV-induced arterial disease is remarkably similar to chronic human atherosclerosis. Cholesterol and saturated cholesteryl esters accumulated in cultured arterial smooth muscle cells (SMC) infected with MDV. Similar preliminary observations were made in vivo. These findings suggest that MDV-induced alteration of SMC lipid metabolism is of major importance in the pathogenesis of MDV-induced atherosclerosis. In addition, immunization with turkey herpesvirus, used commercially to prevent MDV-induced tumors in chickens, also protected against MDV-induced atherosclerosis. This animal model has introduced important new dimensions and tools in atherosclerosis research: a defined etiologic agent (MDV) that causes atherosclerosis in a defined animal of known genetic susceptibility to the etiologic agent. With these tools, important mechanisms in the pathogenesis of atherosclerosis may be established in a relatively short period of time. Further, this animal model should be considered important in other models of atherosclerosis research because herpesvirus infections are ubiquitous in these animals. Finally, because humans are widely and persistently infected with up to five herpesviruses, these studies may lead to the understanding and eventual control of human atherosclerosis.

Herpesvirus infection enhances cholesterol and cholesteryl ester accumulation in cultured arterial smooth muscle cells.

In our previous experiments, atherosclerosis similar to that in humans was reproducibly induced in both normocholesterolemic and hypercholesterolemic specific-pathogen-free (SPF) chickens by infection with Marek's disease herpesvirus (MDV). In contrast, uninfected chickens fed either relatively cholesterol-poor or cholesterol-supplemented diets did not develop this arterial disease. In experiments reported here, the hypothesis that infection of arterial smooth muscle cells (SMCs) with MDV would enhance lipid accumulation in these cells was tested. The number of MDV-infected SMCs with lipid stained with oil red O was assessed, and the lipid content of these cells was quantitated chemically by chromatographic and fluorometric analyses. These data were compared to those of uninfected control cells and, in the case of chemical analyses, were also compared to SMCs infected with a second avian herpesvirus, turkey herpesvirus (HVT). Results indicate the following: 1) The percentage of MDV-infected SMCs containing stainable lipid was significantly greater than the percentage of uninfected SMCs; 2) Increased total lipid accumulation was observed in MDV-infected SMC, particularly cholesterol (CH) and cholesteryl esters (CEs), as compared with uninfected or HVT-infected cells; 3) The types of CEs and nonesterified fatty acids (NEFA) accumulating in MDV-infected cells (particularly saturated types of CEs and NEFAs) were significantly different than those in uninfected or HVT-infected SMCs. These qualitative and quantitative differences in lipid content between infected and uninfected SMCs suggest that infection with MDV results in altered intracellular lipid metabolism. Results support the hypothesis that lipid accumulation in arteries of normocholesterolemic chickens may result from MDV infection acting at the cellular level to induce lipid accumulation that resembles that in human atheroarteriosclerosis.

Serological responses to the cell associated herpesvirus and the manx calicivirus of SPF male cats with herpesvirus-induced urolithiasis.

The feline cell associated herpesvirus (CAHV), but not the Manx calicivirus, was previously reported to induce urolithiasis in specific pathogen free (SPF) cats. Serum neutralization (SN) antibody studies, reported here, revealed that the experimental SPF cats did not have SN antibodies either against the CAHV or the Manx calicivirus in preinoculation serum samples. However, all cats inoculated with the CAHV (either alone or in combination with the Manx virus) developed SN antibodies against the herpesvirus. SN antibodies against the CAHV were detected 21 days post inoculation (PI) in 7 cats, 41 days PI in 4 cats, and 89 days PI in 1 cat. Cats inoculated with the Manx calicivirus alone, or in combination with the CAHV developed SN antibodies against the calicivirus in 7 to 21 days PI with that virus.

Atheroarteriosclerosis induced by infection with a herpesvirus.

Atheroarteriosclerosis closely resembling that in humans was induced in normocholesterolemic and hypercholesterolemic chickens by infection with Marek's disease herpesvirus (MDV). Four comparably sized groups of chickens were used. Each group was initially fed a diet relatively poor in cholesterol. Group I and II were inoculated intratracheally at 2 days of age with MDV. At 15 weeks, one group of virus-infected chickens (Group II) and one group of uninfected controls (Group IV) were fed a 2% cholesterol supplement for an additional 15 weeks. Group I, infected, and III, uninfected, were continued on a cholesterol-poor diet. All groups were killed at 30 weeks. Striking grossly visible atherosclerotic lesions were seen in large coronary arteries, aortas, and major aortic branches of both Groups I and II but not in those of Groups III and IV. Microscopically, arterial changes in infected animals were characterized by occlusive fibromuscular intimal thickening, which formed fibrous caps overlying areas of atheromatous change. This change closely resembled chronic atherosclerosis in humans. These results may be important to our understanding of human arteriosclerosis, since there is widespread and persistent infection of human populations with as many as five herpesviruses.

Virus-induced atherosclerosis.

Of four groups of chickens, two (groups I and II) were infected with MDV and two were not (groups III and IV). Groups I and III were fed diets low in lipid, and groups II and IV were fed cholesterol-supplemented diets. Striking grossly visible atherosclerotic lesions were seen in large coronary arteries, aortas, and major aortic branches of infected normocholesterolemic and hypercholesterolemic chickens (groups I and II). In contrast, grossly visible atherosclerotic lesions were not seen in uninfected normocholesterolemic chickens (group III), nor in uninfected hypercholesterolemic chickens (group IV). Microscopically, arterial changes in the infected animals were characterized by occlusive fibromuscular intimal thickening which formed fibrous caps overlying areas of atheromatous change. This change closely resembled chronic atherosclerosis in man. These results may have important bearing on our understanding of the etiology and pathogenesis of human arteriosclerosis since there is widespread and persistent infection of human populations with up to five different herpes-viruses.

Identification and characterization of a second feline herpesvirus.

A feline virus isolate previously reported as adenovirus-like has more recently been identified and characterized as a new feline herpesvirus. This herpesvirus, which appears to be cell associated, has some characteristics of the cytomegaloviruses and has been found to induce chemical crystal formations in infected cell cultures.

Virus-induced cholesterol crystals.

One of the crystal types induced in cell cultures by a new feline herpesvirus was identified as cholesterol by crystal structure, polarized light microscopy, and mass spectroscopy.

Intracellular and extracellular mineral crystal formation induced by viral infection of cell cultures.

Intracellular mineral crystals were observed in cell cultures infected with one or more feline viruses. One was a feline syncytium-forming virus, and the other was a new feline virus which produced intranuclear inclusions in cell cultures. Crystals were noted both in autogenous cell cultures from trypsinization of kidneys and urinary bladders of infected cats (three passages) and in subcultures of culture fluids from these, made in a stable feline kidney cell line. Free mineral crystals were also noted in the fluids of the cell cultures. In two bladder and two kidney infected autogenous cultures, structures resembling the urinary calculi observed in obstructed cats were seen. These consisted of mineral crystals in a matrix of cellular debris.