Investigation of the Susceptibility of Human Cell Lines to Bovine Herpesvirus 4 Infection: Demonstration that Human Cells Can Support a Nonpermissive Persistent Infection Which Protects Them against Tumor Necrosis Factor Alpha-Induced Apoptosis

Abstract

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that has a worldwide distribution in the population of cattle. Many factors make human contamination by BoHV-4 likely to occur. In this study, we performed in vitro experiments to assess the risk and the consequences of human infection by BoHV-4. First, by using a recombinant BoHV-4 strain expressing enhanced green fluorescent protein under the control of the human cytomegalovirus immediate-early gene promoter, we tested 21 human cell lines for their sensitivity and their permissiveness to BoHV-4 infection. These experiments revealed that human cell lines from lymphoid and myeloid origins were resistant to infection, whereas epithelial cells, carcinoma cells, or adenocarcinoma cells isolated from various organs were sensitive but poorly permissive to BoHV-4 infection. Second, by using the HeLa cell line as a model of human cells sensitive but not permissive to BoHV-4 infection, we investigated the resistance of infected cells to apoptosis and the persistence of the infection through cellular divisions. The results obtained can be summarized as follows. (i) BoHV-4 nonpermissive infection of HeLa cells protects them against tumor necrosis factor alpha-induced apoptosis. (ii) BoHV-4 infection of HeLa cells persists in cell culture; however, the percentage of infected cells decreases with time due to erratic transmission of the viral genome through cell division. (iii)BoHV-4 infection has no effect on the rate of HeLa cell division. Altogether, these data suggest that BoHV-4 could infect humans. This study also stresses the importance of considering the insidious effects of nonpermissive infection when the biosafety of animal gammaherpesviruses for humans is being considered.Bovine herpesvirus 4 (BoHV-4) belongs to the Herpesviridae family, the Gammaherpesvirinae subfamily, and the Rhadinovirus genus (66). BoHV-4 has been isolated throughout the world from healthy cattle, as well as from cattle exhibiting a variety of diseases. The biological life cycle of BoHV-4 relies, as for all Herpesviridae, on the existence of two types of infection: lytic (or replicative) and latent infections. The former leads to the production of progeny virions and generally to the lysis of the infected cell. During a lytic infection, the expression of herpesvirus proteins is temporally regulated. The proteins are classified into three kinetic classes depending on the order of their synthesis during the infection (49). These proteins are expressed chronologically as immediate-early (IE), early (E), and late (L) proteins. IE proteins are expressed directly after release of the viral genome from the capsid into the nucleus. Although E protein expression occurs after synthesis of IE proteins, L protein expression depends on the expression of both IE and E proteins and viral DNA synthesis. Latent infection consists of a dormant state associated with the expression of a limited number of viral genes which might affect the biology of the infected cell. Various stimuli can turn the latent infect...

Bovine Herpesvirus 4 Induces Apoptosis of Human Carcinoma Cell Lines In vitro and In vivo

“…These effects were controlled on infected Madin-Darby bovine kidney cells by indirect immunofluorescent staining with antibodies raised against early and late proteins, as described previously (ref. 23; data not shown). The results presented in Fig.…”

“…Cell viability was assessed by Annexin V-FITC-propidium iodide staining as described earlier (23). The percentage of viable cells was defined as the percentage of cells excluding Annexin and propidium iodide stainings.…”

“…In vitro, BoHV-4 is able to replicate in primary cell cultures or cell lines from a broad spectrum of host species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, chickens, hamsters, rats, mice, and monkeys (12,(14)(15)(16)(17)(18)(19)(20)(21). Recently, several studies have shown that some human cell lines support BoHV-4 infection (15,(21)(22)(23).…”

“…The Vtest EGFP XhoI strain carries an enhanced green fluorescent protein (EGFP) expression cassette under the control of the human cytomegalovirus immediate-early promoter/enhancer inserted into a noncoding region of BoHV-4. BoHV-4 Vtest EGFP XhoI recombinant strain leads to EGFP expression in cells that are sensitive (supporting viral entry) and/or permissive (supporting viral replication) to BoHV-4 infection (23). Semipurified virus preparations prepared as described elsewhere (29) were used for cell inoculation.…”

The Art of Animal Cell Culture for Virus Isolation

“…BoHV-4 has been reported by others to be capable of infecting cells of various different species, including human cells [65]. In MDCK cells, cells infected with some strains of BoHV-4 produce vacuoles at the cell to cell junctions, and this is often mistaken for "cell stress" due to nutrient depletion.…”