Viral arthritides.

Viral infections may manifest as acute or chronic arthritis. Joint involvement arises from either direct infection of the joint, through an immunological response directed towards the virus or autoimmunity. Epidemiological clues to the diagnosis include geographic location and exposure to vector-borne, blood-borne or sexually transmitted viruses. Although not always possible, it is important to diagnose the pathogenic virus, usually by serology, nucleic acid tests or rarely, viral culture. In general, viral arthritides are self-limiting and treatment is targeted at symptomatic relief. This article focuses on the causes, clinical features, diagnosis and treatment of viral arthritides.

[The temperature stability of bovine parvovirus]. / Zur Temperaturstabilität des bovinen Parvovirus.

The stability of the bovine Parvovirus, strain Haden against moist heat in the temperature range 75 to 90 degrees C was tested. It was found that the resistance depended largely on the medium (distilled water, water of standardized hardness (WSH), plasm) in which the viruses were suspended during heating. In WSH the resistance was highest. When heated in plasm, the viruses were inactivated in two phases. From the temperature dependence of the D-values, z-values of 5,6 for distilled water and 8,2 for WSH were found. It is recommended to use the Parvovirus for the evaluation of chemo-thermic and thermic disinfection procedures in view of virucidal effectiveness. The usual pasteurizing procedures fail to inactivate the Parvovirus. To eliminate Parvoviruses from blood products, other methods are, therefore, necessary.

A parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae.

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but lacks serological relationships with the other members of this genus.

Encapsidation of a recombinant LuIII parvovirus genome by H1 virus and the fibrotropic or lymphotropic strains of minute virus of mice.

We previously constructed a recombinant LuIII parvovirus genome lacking viral coding sequences and used it to generate luciferase-transducing virions, by cotransfection of cells with a helper plasmid expressing LuIII viral proteins. Here, we describe similar cotransfections using alternative, replication-defective helpers encoding the non-structural and capsid proteins of parvovirus H1, or of either the fibrotropic or lymphotropic parvovirus strain of minute virus of mice [MVM(p) or MVM(i)]. Each cotransfection generated transducing virus which directed luciferase expression after infection of HeLa cells. The transducing activity of virus produced using either LuIII or H1 helper plasmids could be specifically neutralized by antiserum raised against the corresponding infectious virus. When the recombinant LuIII parvovirus was pseudotyped with MVM(p) or MVM(i), the resulting virions efficiently expressed luciferase after infection in human or murine cells known to be permissive for both MVM strains. The MVM(p) pseudotyped virus also expressed this reporter efficiently when infected into the murine A9 fibroblast line. In contrast, the recombinant virus generated with an MVM(i) helper gave luciferase expression that was barely detectable after infection of A9 cells which are highly restrictive for MVM(i) productive infection. These results support the notion that the allotropic determinant of these MVM strains functions through their capsid proteins. Pseudotyping of recombinant parvovirus genomes should be useful in controlling their host range as vectors, and in studying mechanisms influencing the permissiveness of parvovirus infections.

Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells.

A putative parvovirus related to minute virus of mice (MVM), but distinct from MVM-prototype and MVM-immunosuppressive, was identified, using serologic techniques and Southern blot analysis, in maintenance cultures of established T cell clones. This putative viral agent resulted in a lytic infection of cloned L3 cytotoxic T cells but was unable to produce a productive infection in BHK.21 or EL-4(G) cells. Moreover, maintenance cultures of several distinct subsets of cloned T cells apparently contaminated with this putative viral agent contained poorly growing cells and erythrocyte aggregates. The aggregation of mouse erythrocytes appeared to be a reliable indicator of infection with this putative virus and may be related to the ability of this agent to agglutinate mouse erythrocytes. This putative virus also was found to inhibit the proliferative response of certain cloned T cells to IL-2 and Ag. Viremic mice and secondary MLC supernatant were identified as two potential sources of contamination and represent ways of propagating this agent in vitro. The finding that this agent interferes with the ability of T cell clones to thrive and, therefore has the potential to alter immune responses, emphasizes the importance of identifying and excluding parvoviral infections in cultures of murine T lymphocytes.

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts.

Characterization of the stage(s) in the virus replication cycle at which the host-cell specificity of the feline parvovirus subgroup is regulated in canine cells.

Feline panleukopenia virus (FPLV), mink enteritis virus (MEV), and canine parvovirus (CPV) are classified as a host-range variants. They show different host-range specificity in vivo and host-cell specificity in vitro. For instance, FPLV and MEV cannot grow or can grow only inefficiently in canine cell lines such as MDCK and the canine fibroma cell line A72. Here we have studied the mechanism(s) by which the different cell tropism is mediated in vitro. When FPLV or MEV was inoculated to A72 cells, viral DNA replicated slightly, few viral-antigen-positive cells were detected, and the culture fluid contained the threshold level of infectivity. On the other hand, when an infectious molecular clone of MEV (pMEV) was introduced into A72 cells, viral DNA replicated efficiently, and the culture fluid of pMEV-transfected cells contained much higher infectivities than that of MEV-infected cells. In spite of the restrictive growth in A72 cells, MEV could bind to A72 cells as efficiently as CPV. No detectable viral RNA was produced in MEV-infected A72 cells. In contrast, efficient viral transcription occurred in pMEV-transfected A72 cells. These results suggest that the restrictive infections of MEV and FPLV in A72 cells are not mediated by the attachment of the virus to the cells or by the events occurring after the viral transcription. It appears to be caused by the stage(s) in the virus replication cycle, which exists between a postadsorptional step required for virus penetration and the initiation of viral transcription.

The NS and capsid genes determine the host range of porcine parvovirus.

Porcine parvovirus is an autonomous parvovirus which normally infects pigs and multiplies in porcine cells in vitro. In this report, we describe the properties of a variant designated P2, which has extended its host range to include canine cells. The variant was able to produce cytopathic effects (CPE) in canine cells, unlike the prototype NADL-2 strain. The variant also produced higher viral antigen and infectivity titers in canine cells than the NADL-2 strain, whereas both strains produced CPE and similar titers in porcine cells. Generation of recombinant plasmids between the P2 variant DNA and an infectious clone of NADL-2, and analysis of the properties of the virus stocks produced from these recombinant plasmids, indicated that two changes were necessary for this extension in the host range. One change was located in the nonstructural protein coding region and the other in the capsid coding region.

Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle.

Circular double-stranded replication intermediates were identified in low-molecular-weight DNA of cells of the avian leukemia virus-induced lymphoblastoid cell line 1104-X-5 infected with chicken anemia virus (CAV). To characterize the genome of CAV, we cloned linearized CAV DNA into the vector pIC20H. Transfection of the circularized cloned insert into chicken cell lines caused a cytopathogenic effect, which was arrested when a chicken serum with neutralizing antibodies directed against CAV was added. Chickens inoculated at 1 day of age with CAV collected from cell lines transfected with cloned CAV DNA developed clinical signs of CAV. The 2,319-bp cloned CAV DNA contained all the genetic information needed for the complete replication cycle of CAV. The CAV DNA sequence has three partially overlapping major reading frames coding for putative peptides of 51.6, 24.0, and 13.6 kDa. The CAV genome probably contains only one promoter region and only one poly(A) addition signal. Southern blot analysis using oligomers derived from the CAV DNA sequence showed that infected cells contained double- and single-stranded CAV DNAs, whereas purified virus contained only the minus strand. It is the first time that the genome of one of the three known single-stranded circular DNA viruses has been completely analyzed.

Adaptation of the viral haemorrhagic disease virus of rabbits to the DJRK cell strain.

Liver emulsion of rabbits which had died of viral haemorrhagic disease (VHD) was inoculated onto DJRK cell culture. After two passages, specific cytopathic effect was observed. Immunofluorescence was found in the nucleus at the early stage of infection and later also in the cytoplasm. The virus propagated in cell culture at the fifth, tenth and sixteenth passages was found to cause typical VHD. Electron microscopy showed that there were numerous virions in the infected cells. The cultured virus, inactivated with formaldehyde, could elicit haemagglutination inhibition antibodies in the inoculated rabbits and protect them against the challenge of virulent VHD virus.

In vitro propagation of parvovirus B19 in primary foetal liver culture.

The culture of parvovirus B19 in foetal liver tissue has been described recently. We have established the technique in our laboratory and studied parameters affecting the yield of B19 virus. Replication of the virus was detected by radioimmunoassay for B19 antigen and dot blot hybridization assay of B19 DNA, and the virus was localized by immunofluorescence and thin section electron microscopy. B19 DNA and antigen production became detectable at day 2 and reached a maximum at day 5. Virus particles were seen mainly in cell nuclei, but some cytoplasmic membranes were lined with virus particles. The amount of virus produced depended on the age of the foetus and the cell culture and the concentration of erythropoietin and interleukin 3 in the culture medium.

Multiplication of attenuated and virulent porcine parvoviruses in colostrum-deprived, neonatal pigs.

Colostrum-deprived, neonatal, 2 days old pigs were inoculated with the attenuated HT-/SK or the virulent 90HS strain of porcine parvovirus (PPV) by the oral or subcutaneous route and sacrificed 2, 4 or 6 days after inoculation. Then, comparison was made on viral multiplication in pigs between the two strains. Pigs inoculated with the HT-/SK strain showed no detectable viremia or HI antibody responses against PPV within 6 days after inoculation. Only in pigs inoculated by the subcutaneous route, a small amount of virus was recovered from the spleen, liver, or mesenteric lymph nodes. These viruses were distinguished from the parental virulent 90HS strain, as examined for rct maker in vitro. When pigs were inoculated with the virulent 90HS strain, viremia appeared in all of them 1 day after inoculation and continued for up to the sacrificed day. Moreover, a considerable amount of virus was also detected from all tissues, including brain, lung, liver, spleen, pancreas, small intestine, and lymph node tissues, in all pigs tested. HI antibodies were first detected 6 days after inoculation.

Persistence of parvovirus H-1 DNA in human B- and T-lymphoma cells.

Persisting DNA of parvovirus H-1 could be demonstrated in cells of two human lymphoma cell lines, the Burkitt lymphoma cell line BL2 and the T-cell leukemia cell line Jurkat which survived infection with parvovirus H-1. Persistence of H-1 DNA rendered the cells resistant to a second H-1 infection. This resistance to H-1 superinfection persisted even after loss of H-1 DNA occurring after approximately 150-200 cell generations. Resistance to H-1 superinfection was accompanied by reduced uptake of infectious particles and by a block of H-1 DNA replication. This suggests that persistent H-1 infection leads to modifications of cellular functions involved in the permissivity for H-1.

Development of a vaccine preventing parvovirus-induced reproductive failure in pigs.

An inactivated porcine parvovirus (PPV) vaccine for the prevention of PPV-induced reproductive failure in pigs was developed, using virus grown in cell culture, inactivated with beta-propiolactone and adjuvanted with aluminum hydroxide. The vaccine was tested for safety by subcutaneous injection into pregnant gilts. There were no signs of abnormal reactions nor evidence of PPV infection in the gilts or their foetuses when they were sacrificed 6 weeks after vaccination. To demonstrate that the vaccine was immunogenic, pigs were immunised either once or twice with 4 weeks between doses. Resulting antibody titres (haemagglutination inhibition - HAI) ranged from less than 8 to 64 (geometric mean of 30) after one dose of vaccine, and from 128 to 512 (geometric mean 256) after two doses. To demonstrate that the vaccine was protective, antibody-negative gilts were vaccinated twice, with 4 weeks between doses, joined after the second dose, and were then infected with virulent PPV 40 to 50 days after joining. In litters from 10 vaccinated gilts, none of 93 foetuses showed evidence of PPV infection. In contrast, in litters from two unvaccinated gilts, all 13 foetuses showed evidence of PPV infection and 10 of these were mummified. The average number of live piglets per litter was 9.2 from vaccinated gilts and 1.5 from unvaccinated gilts. The vaccine was therefore considered to be effective in preventing PPV reproductive failure in susceptible gilts.

Susceptibility of human erythropoietic cells to B19 parvovirus in vitro increases with differentiation.

B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus-induced transient aplastic crisis.

General characteristics and viral susceptibility of a newborn pig kidney (NPK) continuous culture.

We have developed a fibroblastic-like continuous culture of newborn pig kidney (NPK). The current cell line was serially passaged 160 times and appeared to be well suited for production and assay of a number of viruses affecting pigs, such as pig parvovirus, pseudorabies and transmissible gastroenteritis. The cell line appeared aneuploid, with a modal chromosome number of 36 and induced tumors, classified as fibrosarcoma, in athymic mice.

Limitations to the expression of parvoviral nonstructural proteins may determine the extent of sensitization of EJ-ras-transformed rat cells to minute virus of mice.

The FR3T3 and NRK rat cell lines and their human EJ Ha-ras-1 oncogene-transformed derivatives, termed FREJ and NREJ, were compared for their susceptibility to the parvovirus MVMp. For a similar production of p21ras protein, FREJ clones are markedly sensitized to killing by MVMp, whereas the NREJ cells are not. Such a contrasting effect of ras transformation on the sensitivity of cells of different origins to MVMp can be traced back to their respective abilities to support the parvoviral life cycle. The FR3T3 line produces a substantial amount of viral DNA whose expression in the form of the nonstructural protein NS-1 is stimulated in its transformed derivatives. Conversely, NRK cells offer an early block to parvoviral DNA replication and expression that appears to persist in the ras-transformed clones. Thus, at least two intracellular restrictions can protect normal rat cells against MVMp infection, and transformation by ras relieves one of them at the level of parvoviral gene expression. A fair correlation was also found between the degree of sensitivity of the various lines to MVMp-induced killing and their capacity to synthesize the nonstructural viral proteins, suggesting a possible role of parvoviral nonstructural proteins in cytotoxicity.

Feline parvovirus propagates in cat bone marrow cultures and inhibits hematopoietic colony formation in vitro.

Feline parvovirus (FPV) causes leukopenia in naturally infected cats. We investigated the mechanism of hematopoietic depression by this virus in feline bone marrow cultured in vitro. In suspension cultures we demonstrated FPV propagation and replication using DNA molecular hybridization. Viral RNA and DNA were observed by in situ hybridization in about 10% of marrow cells at day 3. Granulocytes and their precursors were virtually absent from infected cultures after six days. Infected cells showed viral capsid protein predominantly in nuclei by immunofluorescence. In clonal assays, FPV most efficiently inhibited hematopoietic colony formation by myeloid progenitor cells (CFU-GM), but erythroid colony formation (BFU-E and CFU-E-derived) was also depressed in the presence of virus. Inhibition of colony formation could be abrogated by physical inactivation of the virus or preincubation with specific neutralizing antibodies. Recombinant human colony stimulating factors GM-CSF and G-CSF supported feline myelopoiesis in progenitor assays, and FPV completely inhibited factor dependent colony formation.

Characterization of a parvovirus isolated from a pig fetus.

A small hemagglutinating virus belonging to the Parvoviridae Family was isolated from a 70 day-old pig fetus in a breeding herd in which infertility, metritis and abortions were reported. The virus, named 85/193L, was isolated either in primary pig kidney (PK) cells or in a continuous cell line of minipig kidney origin (MPK), both cell cultures actively growing. It produced a typical cytopathic effect (CPE) starting from the 3rd passage and intranuclear inclusions surrounded by a halo were observed in stained preparations. The isolate was completely resistant to ether, chloroform and to pH 3; it was not inactivated after treatment at 56 degrees C for 4 h and at 70 degrees C for 2 h, while it was completely inhibited by the 80 degrees C/30 min temperature. It contained deoxyribonucleic acid (DNA). The highest infectious titer was reached at 96 h post infection. The infectivity and the hemagglutinating activity of the isolated strain were both inhibited by the reference immune serum against NADL-2 pig parvovirus. This further confirmed that the 85/193L isolate belongs to the parvovirus genus.

Inhibition by parvovirus H-1 of the formation of tumors in nude mice and colonies in vitro by transformed human mammary epithelial cells.

The formation of tumors in adult nude mice from transformed human mammary epithelial cells was drastically inhibited (greater than 80%) both after coinjection of tumoral cells and virus or after a single s.c. injection of parvovirus H-1 at the site of cell implantation prior to tumor formation. Moreover, when injected i.v. in animals bearing preformed tumors, H-1 virus was able to slow down and even in some cases to revert neoplastic growth. Thus, H-1 virus achieved the suppression of implanted tumors of human origin under conditions where the immune antitumor mechanisms of the recipient animals were dramatically impaired. Viral infection was not accompanied by detectable deleterious side effects. Imprints of H-1 virus DNA were found in one residual tumor. Normal human mammary epithelial cells were also compared with homologous transformed cells, either derived from tumors (three lines) or containing simian virus 40 (one line), for their susceptibility to the lytic replication of H-1 virus in vitro. Transformed cell lines were more sensitive to virus-induced killing than secondary cultures of normal cells. Moreover, the former had much greater abilities than the latter to amplify viral DNA and to express the viral nonstructural protein NS-1. Altogether, these results are compatible with the idea that the oncosuppressive activity exerted by H-1 virus may be mediated, at least in part, by virus replication in developing tumors.