Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.     

Interferon response in normal and Aleutian disease virus-infected mink.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.     

The evolution and control of parvovirus host ranges

Host ranges of parvoviruses are complex, and depend on both the strain of virus and on the cell or animal being inoculated. Viruses similar to feline panleukopenia virus infect cats and cat cells in tissue culture, as well as a variety of other host animals and their cultured cells. Canine isolates infect dogs and cultured canine cells, but replication in cats depends on the type of virus. Feline and canine host ranges are determined primarily by a small number of sequence differences in the capsid protein. DNA sequences of viruses from cats, mink, raccoons and foxes could not be readily distinguished from each other. Viruses from dogs or raccoon dogs formed a distinct group, which was subdivided between the two antigenic types. Host ranges of other parvoviruses—minute virus of mice and porcine parvovirus—are also mediated primarily by sequences in the capsid protein gene, although differences in the non-structural protein genes of the minute virus of mice determine some host-range differences.     

Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.     

Humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus.

BACKGROUND AND OBJECTIVES: Two serotypes of autonomously replicating parvoviruses infect laboratory mice. Genome regions coding for the nonstructural proteins of minute virus of mice [MVM] and mouse parvovirus [MPV] are almost identical, whereas capsid-coding sequences are divergent. We addressed these questions: Does humoral immunity confer protection from acute infection after challenge with homotypic or heterotypic parvovirus, and if it confers protection against acute MPV infection, does it also protect against persistent MPV infection? METHODS: Infant mice without maternal antibody or antibody to MVM or MPV and young adult mice given normal mouse serum or antibody to MVM or MPV were challenged with homotypic or heterotypic virus. In situ hybridization with target tissues was the indicator of infection. RESULTS: Humoral immunity failed to confer protection against acute heterotypic parvovirus infection. In passive transfer studies, MPV DNA was observed occasionally in lymph nodes, intestine, or the spleen of MPV-challenged mice given homotypic antibody and kept for 6 or 28 days. Variable proportions of mice given MPV antibody and homotypic challenge had viral DNA in lymphoid tissues 56 days after virus inoculation. CONCLUSION: A mouse or colony that has sustained infection with MVM or MPV is probably fully susceptible to infection with the heterotypic virus.     

Structure of adeno-associated virus serotype 5

Adeno-associated virus serotype 5 (AAV5) requires sialic acid on host cells to bind and infect. Other parvoviruses, including Aleutian mink disease parvovirus (ADV), canine parvovirus (CPV), minute virus of mice, and bovine parvovirus, also bind sialic acid. Hence, structural homology may explain this functional homology. The amino acids required for CPV sialic acid binding map to a site at the icosahedral twofold axes of the capsid. In contrast to AAV5, AAV2 does not bind sialic acid, but rather binds heparan sulfate proteoglycans at its threefold axes of symmetry. To explore the structure-function relationships among parvoviruses with respect to cell receptor attachment, we determined the structure of AAV5 by cryo-electron microscopy (cryo-EM) and image reconstruction at a resolution of 16 A. Surface features common to some parvoviruses, namely depressions encircling the fivefold axes and protrusions at or surrounding the threefold axes, are preserved in the AAV5 capsid. However, even though there were some similarities, a comparison of the AAV5 structure with those of ADV and CPV failed to reveal a feature which could account for the sialic acid binding phenotype common to all three viruses. In contrast, the overall surface topologies of AAV5 and AAV2 are similar. A pseudo-atomic model generated for AAV5 based on the crystal structure of AAV2 and constrained by the AAV5 cryo-EM envelope revealed differences only in surface loop regions. Surprisingly, the surface topologies of AAV5 and AAV2 are remarkably similar to that of ADV despite only exhibiting approximately 20% identity in amino acid sequences. Thus, capsid surface features are shared among parvoviruses and may not be unique to their replication phenotypes, i.e., whether they require a helper or are autonomous. Furthermore, specific surface features alone do not explain the variability in carbohydrate requirements for host cell receptor interactions among parvoviruses.     

Transformation of human fibroblasts by ionizing radiation, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H-1 virus and minute virus of mice.

Morphologically altered and established human fibroblasts, obtained either by 60Co gamma irradiation, treatment with the carcinogen 4-nitroquinoline 1-oxide, or simian virus 40 (SV40) infection, were compared with their normal finite-life parental strains for susceptibility to the autonomous parvoviruses H-1 virus and the prototype strain of minute virus of mice (MVMp). All transformed cells suffered greater virus-induced killing than their untransformed progenitors. The cytotoxic effect of H-1 virus was more severe than that of MVMp. Moreover, the level of viral DNA replication was much (10- to 85-fold) enhanced in the transformants compared with their untransformed parent cells. Thus, in this system, cell transformation appears to correlate with an increase in both DNA amplification and cytotoxicity of the parvoviruses. However, the accumulation of parvovirus DNA in the transformants was not always accompanied by the production of infectious virus. Like in vitro-transformed fibroblasts, a fibrosarcoma-derived cell line was sensitive to the killing effect of both H-1 virus and MVMp and amplified viral DNA to high extents. The results indicate that oncogenic transformation can be included among cellular states which modulate permissiveness to parvoviruses under defined growth conditions.     

Parvovirus infection-induced cell death and cell cycle arrest

The cytopathic effects induced during parvovirus infection have been widely documented. Parvovirus infection-induced cell death is often directly associated with disease outcomes (e.g., anemia resulting from loss of erythroid progenitors during parvovirus B19 infection). Apoptosis is the major form of cell death induced by parvovirus infection. However, nonapoptotic cell death, namely necrosis, has also been reported during infection of the minute virus of mice, parvovirus H-1 and bovine parvovirus. Recent studies have revealed multiple mechanisms underlying the cell death during parvovirus infection. These mechanisms vary in different parvoviruses, although the large nonstructural protein (NS)1 and the small NS proteins (e.g., the 11 kDa of parvovirus B19), as well as replication of the viral genome, are responsible for causing infection-induced cell death. Cell cycle arrest is also common, and contributes to the cytopathic effects induced during parvovirus infection. While viral NS proteins have been indicated to induce cell cycle arrest, increasing evidence suggests that a cellular DNA damage response triggered by an invading single-stranded parvoviral genome is the major inducer of cell cycle arrest in parvovirus-infected cells. Apparently, in response to infection, cell death and cell cycle arrest of parvovirus-infected cells are beneficial to the viral cell lifecycle (e.g., viral DNA replication and virus egress). In this article, we will discuss recent advances in the understanding of the mechanisms underlying parvovirus infection-induced cell death and cell cycle arrest.     

Vesicular stomatitis virus (VSV) therapy of tumors

Vesicular stomatitis virus (VSV) is an essentially nonpathogenic negative-stranded RNA virus, the replication of which is extremely sensitive to the antiviral effects of interferon (IFN). We demonstrate here that VSV selectively induces the cytolysis of numerous transformed human cell lines in vitro, with all the morphological characteristics of apoptotic cell death. Importantly, VSV can also potently inhibit the growth of p53-null C6 glioblastoma tumors in vivo without infecting and replicating in normal tissue. With our previous findings demonstrating that primary cells containing the double-stranded RNA-activated protein kinase PKR and a functional IFN system are not permissive to VSV replication, these results suggest that signaling by IFN may be defective in many malignancies. Thus VSV might be useful in novel therapeutic strategies for targeting neoplastic disease.     

Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/ HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.     

LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells

Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.     

Investigation of the pathogenesis of transplacental transmission of Aleutian mink disease parvovirus in experimentally infected mink.

The transplacental transmission of Aleutian mink disease parvovirus (ADV) was studied in experimental infection of 1-year-old female non-Aleutian mink. The ADV-seronegative female mink were inoculated with ADV prior to mating or after the expected implantation of the embryos during pregnancy. A group of uninfected females served as a control group. Animals from each group were killed prior to or shortly after parturition. The in situ hybridization technique with radiolabeled strand-specific RNA probes was used to determine target cells of virus infection and virus replication. In both infected groups, ADV crossed the endotheliochorial placental barrier, although animals infected before mating already had high antibody titers against ADV at the time of implantation. The percentage of dead and resorbed fetuses was much higher in dams infected before mating. In the placentae of these mink, virus DNA and viral mRNA were detected in cells in the mesenchymal stroma of the placental labyrinth and hematoma but only occasionally in the cytotrophoblast of the placental hematoma. Placentae of animals infected during pregnancy showed in addition very high levels of virus and also viral replication in a large number of cytotrophoblast cells in the placental hematoma, which exhibited distinct inclusion bodies. In both groups, neither virus nor virus replication could be detected in maternal endothelial cells or fetal syncytiotrophoblast of the placental labyrinth. Fetuses were positive for virus and viral replication at high levels in a wide range of tissues. Possible routes of transplacental transmission of ADV and the role of trophoblast cells as targets for viral replication are discussed.     

Comparison of canine parvovirus with mink enteritis virus by restriction site mapping.

The genomes of canine parvovirus and mink enteritis virus were compared by restriction enzyme analysis of their replicative-form DNAs. Of 79 mapped sites, 68, or 86%, were found to be common for both types of DNA, indicating that canine parvovirus and mink enteritis virus are closely related viruses. Whether they evolved from a common precursor or whether canine parvovirus is derived from mink enteritis virus, however, cannot be deduced from our present data.     

Partial reversion of conditional transformation correlates with a decrease in the sensitivity of rat cells to killing by the parvovirus minute virus of mice but not in their capacity for virus production: effect of a temperature-sensitive v-src oncogene.

The cytolytic effect of the autonomous parvovirus minute virus of mice, prototype strain (MVMp), was studied in cultures of ts 339/NRK rat cells that display a temperature-sensitive transformed phenotype as a result of their transformation with a Rous sarcoma virus strain matured in the v-src oncogene. A shift from restrictive (39.5 degrees C) to permissive (34.5 degrees C) temperature was associated with a marked sensitization of these cells to killing by MVMp. In contrast, ts 339/NRK cell derivatives supertransformed with a wild-type src oncogene were sensitive to MVMp at both temperatures, suggesting that the expression of a functional oncogene product may determine, at least in part, the extent of the parvoviral cytopathic effect. Although ts 339/NRK cells were quite resistant to parvoviral attack at 39.5 degrees C, they were similarly proficient in MVMp uptake, viral DNA and protein synthesis, and infectious particle production at both permissive and restrictive temperatures. Consistently, electron microscopic examination of infected ts 339/NRK cultures incubated at 39.5 degrees C revealed the presence, in the majority of the cells, of numerous full and empty virions that were predominantly located in autophagic-type vacuoles. Thus, in this system, the reversion of transformed and MVMp-sensitive phenotypes appears to correlate with the setting up of a noncytocidal mode of parvovirus production. These results raise the possibility that the physiological state of host cells may affect their susceptibility to parvoviruses by modulating not only their capacity for virus replication but also cellular processes controlling the cytopathic effect of viral products.     

Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

Background  

Adeno-associated virus (AAV) type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates.     

The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage

The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.