Introduction of Rodent Parvoviruses Introduction of Rodent Parvoviruses Introduction of Rodent ParvovirusesDr. Cho-Hua WanSchool of Veterinary Medicine National Taiwan University Rodent parvoviruses are recognized as important infectious agents in laboratory rodents and cell lines. Three prototypic rodent parvoviruses were discovered in the late 1950s and early 1960s, including Kilham rat virus (KRV) and H-1 virus in rats and minute virus of mice (MVM) (19, 26, 43). In 1990s, indication of the existence of additional rodent parvoviruses, other than MVM, KRV and H-1, was initially recognized in several rodent colonies, based on detection of parvovirus antibodies by an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescent assay (IFA) and the absence of antibodies specific to MVM, KRV and H-1 hemagglutinin (1, 2, 7, 37, 41, 44). Since then, several rodent parvovirus species distinct from prototypic rodent parvoviruses have been isolated from infected cell lines and rodents and characterized, including mouse parvovirus (MPV), hamster parvovirus (HaPV), rat parvovirus (RPV), and rat minute virus (1, 2, 7, 31, 44). Molecular Analysis and Replication of Parvoviruses Autonomous parvoviruses are small (15-28 nm) nonenveloped DNA viruses of the family of Parvoviridae, which consist of a single-stranded genome about 5 kb in length. Replication of autonomous parvoviruses requires the presence of host cellular factors expressed during the S phase. The molecular biology of MVM has been well characterized and has been used as the model of other autonomous parvoviruses (17). Parvoviruses have palindrome sequences at both the 5' and 3' termini of the genome and these palindromes are involved in viral replication (17). Most rodent parvoviruses replicate with monomer and dimer DNA intermediates and encapsidate monomer single-stranded DNA, which is predominantly minus sense. Parvoviruses encode two non-structural proteins, NS-1 and NS-2, and three capsid proteins, VP-1, VP-2, and VP-3. NS proteins, involved in transcription and viral replication, are conserved among different parvoviruses, whereas VP proteins exhibit heterogeneity among different species of parvoviruses. Autonomous parvoviruses are generally host specific, and parvoviruses of laboratory rodents are grouped according to the results of hemagglutination inhibition (HAI) or serum neutralization assays. Regulation of transcription and translation in MVM has also been well studied. MVM produces three mRNA transcripts, R1, R2, and R3 that all terminate at a single polyadenylation site at genetic map unit (m.u.) 95 (14). R1 arises from the P4 promoter (m.u. 4) and encodes the NS-1 protein that is required for viral DNA replication and transactivation of the P38 promoter (m.u.38). R2 arises from the P4 promoter also, but two introns are spliced out of the primary transcript, a large intron (nucleotides 515 to1989) and a small intron in three different sizes due to alternative splicing between m.u. 44 to 46 (18, 36). Alternative splicing of two donor sites (nucleotide 2280 and 2316) and two acceptor sites (nucleotide 2377 and 2399) on R2 generates three isoforms of the NS-2 protein (major, minor, and rare NS-2 protein). The three isoforms of NS-2 protein are identical in the first 182 amino acids, but differ at their carboxyl termini. NS-2 protein is required for viral replication in a cell-type-specific manner, i.e., the requirement for NS-2 during replication of the prototypic MVM strain (MVMp) is more pronounced in murine A9 fibroblasts than human NB324K cells (36). R3 arises from the P38 promoter and encodes two structural viral proteins, the VP-1 and VP-2 proteins. VP-2 is the major capsid protein and its amino acid sequences are contained within VP-1. A third structural viral protein, VP-3, is produced by proteolytic processing of VP-2 at the trypsin-sensitive REVR motif (42). Clinical Signs of Rodent Parvoviral Infection Natural parvoviral infections in rodents are usually asymptomatic. Natural infections in mice are clinical silent and the most common evidence of infection is seroconversion or detection of viral DNA by PCR. However, experimental infection of neonatal BALB/c, SWR, SJL, CBA, and C3H with MVM immunosuppressive strain (MVMi) has caused lesions of internal hemorrhage and hepatic hematopoiesis (10). For rat parvoviruses, KRV can occasionally cause severe pathogenic and fatal infections in fetal and infant rats (16, 25). Experimental inoculations of KRV and H-1 can cause severe or fatal diseases with hemorrhage and necrosis in liver and central nervous system in infant rats (15, 30). No pathogenic evidence has been identified in natural (RMV and RPV) and experimental (RPV) infected rats (2, 44). HaPV has caused an outbreak of a severe epidemic disease in hamsters, with high mortality of suckling and weaning hamsters with malformed and missing incisors (22). Pathogenesis and Potential Impact of Rodent Parvoviruses Although natural infections caused by rodent parvoviruses are usually asymptomatic, parvovirus infections can interfere with both rodent-based research and in vitro studies using contaminated cells or sera from infected animals (11, 24). The effects on research are attributable to their predilection for mitotically active cells. Prototypic rodent parvoviruses have been reported to be frequent contaminants of cell lines and transplantable tumors (23). The major distortions of biological responses induced by rodent parvoviral infections are associated with neoplastic growth or immune dysfunctions. MVMi was shown to suppress lymphocyte proliferation in response to mitogens and to inhibit T cell-dependant B cell responses in vitro (9, 21, 34). MVMi also has immunosuppressive effects in infant mice and potentially interferes with hematopoietic system in newborn or severely immunocompromised mice (27). MPV infection of adult mice can cause persistent immune dysfunction even after seroconversion. MPV also can modulate immune responses differentially, depending on the source of T cells (33). MPV also can cause immunosuppression with reduction of both cytokine- and antigen-induced T cell proliferation in vitro. Modulation of immune responses by MPV raises concerns about the full potential for such effects on immunologic research using mice. KRV usually causes subclinical infections in rats; however, an outbreak infection with hemorrhage and necrosis of the brain, testes, and epididymides has been reported in young adult rats (16). KRV has been shown to modulate immune function through its tropism for T-cell lymphocytes (33). KRV infection can induce autoimmune diabetes in diabetes resistant rats (13). KRV is lymphotropic and can induce immune function distortions, including the interference with T cell response to transplantable neoplasms and the diminishment in proliferative and cytolytic responses to alloantigens in mixed lymphocyte cultures (11, 12, 20, 32, 39). H-1 infection is subclinical in rats and the experimental infection of H-1 virus can cause similar lesions as KRV infection in rats (35). While the effects of KRV and H-1 in biomedical research have been well studied, there is limited knowledge regarding the pathogenicity of RPV-1 and RMV-1. In rats naturally infected with RPV-1a or RMV-1, no specific gross or histopathologic abnormalities are observed (2, 44). RPV-1a appears nonpathogenic for experimentally infected infant rats (2); however, rats experimentally infected with RPV-1a exhibit persistent infection of lymphoid tissues and reduction in tumor size when compared with tumors in uninfected rats. In an in vitro infection study, RPV-1a exhibited cytopathic effects on a transformed human cell line (2). These findings suggest that RPV-1a may interfere with function of the immune system, affect neoplastic growth in infected rats, and cause cytopathic effects on contaminated cell cultures. Even less is known about RMV-1 pathogenicity because an in vitro culture system has not been established for RMV-1 and no experimental infections have been performed. In naturally infected rats, RMV-1 viruses were detected in lymphoid tissues by PCR amplification (44). This suggests that RMV-1 can infect lymphoid tissues and may have immunomodulation effects on hosts during infection, as other rodent parvoviruses do. Recognizing the potential deleterious effects of parvovirus infections on research studies, it is important to identify infected laboratory rats and contaminated cell lines and transplantable tumors.HaPV has caused an outbreak with high mortality of suckling and weaning hamsters. Typical lesions of this natural HaPV infection include necrosis and inflammation of the dental pulp with mononuclear leukocytic infiltration of dental lamina and osteoclasis of alveolar bone (22). Despite the absence of clinical disease, rodent parvoviruses can have significant deleterious effects on research due to their immunomodulatory effects both in vivo and in vitro. Therefore, identification of infected laboratory rodents and contaminated biological materials is critical to minimize the impact of rodent parvovirus infections on research. Diagnosis of Rodent Parvoviral Infection Several diagnostic systems have been established to detect infections caused by rodent parvoviruses. Since natural infections of parvoviruses are usually asymptomatic, diagnosis cannot be made on the basis of clinical signs of disease. Instead, laboratory diagnostic methods, such as PCR and serologic assays are routinely used to identify parvovirus-infected rodents and/or parvovirus-contaminated biological materials. Serologic evaluation for the presence of antiparvovirus antibodies has typically been used to diagnose parvovirus infections in rodent colonies. The most common methods used for the diagnosis rodent parvovirus infections include the enzyme-linked immunosorbent assay (ELISA), the indirect fluorescent-antibody assay (IFA), and the hemagglutination inhibition (HAI) assay, with the ELISA being preferred due to its high-throughput capability. The high degree of homology among the NS proteins of the rodent parvoviruses and the significant cross-reactivity of anti-NS antibodies against other rodent parvoviruses binding to the NS proteins of MVM lead to the idea of developing a generic serologic assay for the diagnosis of rodent parvoviruses. The recombinant ELISA with conserved recombinant MVM NS-1 protein (rNS1 ELISA) was developed and had been the most extensively used assay for rodent parvovirus diagnosis for a few years (38). However, recent serologic results suggest the rNS-1 ELISA may have limited sensitivity for detecting MPV, RPV and RMV infections due to low levels of NS-1 antibodies produced at certain stages of infection (8; Riley, personal communication). In the past few years, several high-throughput species-specific serologic assays (rVP2 ELISA) using the recombinant VP2 proteins of MVM, MPV, RPV, and RMV as antigens have been developed and been applied to rodent parvovirus diagnosis (3, 29, 28, 46; Shek, personal communication). Results of the rVP2 ELISA are confirmed by IFA or HAI assays, which detect virus-specific antibodies. Serologic assays are the most commonly used methods for diagnosis of parvovirus infections, but there are several disadvantages to serologic diagnostic assays. First, serologic assays cannot detect early infections. Second, serologic assays can be applied only to serum from immunocompetent animals, but not to the serum from immunodeficient animals. Finally, serologic methods cannot be used to evaluate contamination of biological specimens, such as cell lines. Molecular biology methods, such as the polymerase chain reaction (PCR) assays, have been developed and frequently applied to disease diagnosis in recent years. A universal rodent parvovirus PCR assay and species-specific PCR assays for are used routinely for diagnosis of different rodent parvoviruses (4-6, 45). The universal rodent parvovirus PCR amplifies a region conserved among different rodent parvoviruses and detects all known rodent parvoviruses. Parvovirus species-specific PCR assays amplify a region of the genome that is unique to each parvovirus species; these assays can distinguish the target virus from other parvoviruses and other virus families. The PCR assays can detect both acute and endemic infections and PCR tests are rapid, sensitive and specific in parvovirus diagnosis. Importantly, PCR assays have been used to screen for parvovirus contamination in biological specimens, such as cell cultures, culture medium, and even environmental swabs (40). While parvovirus PCR assays are very sensitive and specific, the expense of PCR precludes its use as a primary diagnostic method (4, 5, 45). Virus isolation and DNA sequencing of PCR fragments have also been used to detect and identify newly recognized rat parvovirus species in research settings (2, 44; Bauer, unpublished data). However, these methods are expensive, time consuming and labor intensive to perform. As such, these methods are not suitable for routine diagnostic use. Control and Prevention of Rodent Parvoviral Infection Parvovirus infections can interfere with both rodent-based research and in vitro studies using contaminated cells or sera from infected animals, so that how to control and prevent rodent parvovirus infections is an important issue for rodent research and breeding facilities. First is to control the possible contaminated sources into the parvovirus-free facilities. Parvovirus screening should be applied on all incoming animals and biological materials/cell lines before entering to the facilities. If it’s not possible, these animals should be handled as “suspected positive animals” and hold in a quarantine room until these animals are proved to be parvovirus-negative. Once an outbreak is happened, quarantine of sero-positive rooms, and extensive serologic testing on animals in each room, and identifying the possible contaminated sources (animals, tissues, cells, biological materials, cages, or carriers) by possible diagnostic assays should be performed as soon as possible. It is important to closely follow up the movement of potentially affected animals or contaminated materials to avoid the spread of infection within the facility or to regional facilities. Elimination (depopulation) of infected rodents should be considered if they are an immediate threat to experimental or breeding colonies and they can be replaced. 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