Canine parvovirus: recent knowledge of the origin and development of a viral pathogen

Canine parvovirus (CPV) is a "new" virus that suddenly emerged in the mid 1970s. Antigenetically it is very similar to the long known feline panleukopenia virus (FPV). Soon after its appearance CPV was classified as a mutant of FPV. As with all "new" viruses, CPV continues to show active evolution, obvious by the appearance of new antigenic types. Interestingly, the new types, designated CPV-2a and CPV-2b, completely replaced the original type. This review summarizes the facts that are known about the emergence and evolution of CPV and discusses the relevance of the new antigenic types for the diagnosis of CPV and the vaccination against it.     

Two dominant neutralizing antigenic determinants of canine parvovirus are found on the threefold spike of the virus capsid

The 25-nm diameter parvovirus capsid is assembled from 60 copies of a sequence common to the overlapping VP1 and VP2 proteins. Here we examine the epitope specificity's of 28 monoclonal antibodies (MAb) prepared against canine parvovirus (CPV), feline panleukopenia virus (FPV), and raccoon-dog parvovirus or blue (Arctic) fox parvovirus. Comparing the reactivity of those MAb with various MAb-selected escape mutants, or with natural variants of CPV or mink enteritis virus (MEV) which differ at known sequences, showed that the binding of 20 of those MAb was strongly affected by variations of two regions on the threefold spike of the CPV capsid. One region was adjacent to the tip of the threefold spike, and the second was around VP2 residue 300, on the shoulder of that structure. MAb recognizing both antigenic sites efficiently neutralized the virus infectivity and inhibited hemagglutination. Mutations leading to natural antigenic variation have also been observed in both those sites in naturally variant strains of CPV or MEV, suggesting that they are important antigenic structures on these parvoviruses. The bindings of several MAb were not affected by the mutations at those antigenic sites, indicating that they recognized other, and perhaps conserved, structures.     

Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid

We analyzed a region of the capsid of canine parvovirus (CPV) which determines the ability of the virus to infect canine cells. This region is distinct from those previously shown to determine the canine host range differences between CPV and feline panleukopenia virus. It lies on a ridge of the threefold spike of the capsid and is comprised of five interacting loops from three capsid protein monomers. We analyzed 12 mutants of CPV which contained amino acid changes in two adjacent loops exposed on the surface of this region. Nine mutants infected and grew in feline cells but were restricted in replication in one or the other of two canine cell lines tested. Three other mutants whose genomes contain mutations which affect one probable interchain bond were nonviable and could not be propagated in either canine or feline cells, although the VP1 and VP2 proteins from those mutants produced empty capsids when expressed from a plasmid vector. Although wild-type and mutant capsids bound to canine and feline cells in similar amounts, infection or viral DNA replication was greatly reduced after inoculation of canine cells with most of the mutants. The viral genomes of two host range-restricted mutants and two nonviable mutants replicated to wild-type levels in both feline and canine cells upon transfection with plasmid clones. The capsids of wild-type CPV and two mutants were similar in susceptibility to heat inactivation, but one of those mutants and one other were more stable against urea denaturation. Most mutations in this structural region altered the ability of monoclonal antibodies to recognize epitopes within a major neutralizing antigenic site, and that site could be subdivided into a number of distinct epitopes. These results argue that a specific structure of this region is required for CPV to retain its canine host range.     

Differences in the evolutionary pattern of feline panleukopenia virus and canine parvovirus

Canine parvovirus (CPV) suddenly appeared in the late 1970s after which it showed continuous antigenic changes. Virological and molecular genetic analyses mainly focused on feline panleukopenia virus (FPLV) were conducted in this study because FPLV is the suspected ancestor of CPV; the way in which FPLV evolves may help to explain the emergence of CPV. Analysis of escape mutants against FPLV-specific monoclonal antibody showed that viruses possessing CPV-like properties were not easily detected in FPLV virus stocks. Phylogenetic analysis revealed that the nonstructural protein 1 (NS1) and capsid protein 2 (VP2) genes of FPLV changed with time. A similar tendency, however, was not observed in the FPLV VP2 proteins. In contrast, the topology of the phylogenetic tree of VP2 proteins of CPV basically concurred with that of the VP2 genes. Analysis of the ratio of nonsynonymous and synonymous substitutions revealed that synonymous substitutions exceeded nonsynonymous substitutions in both the NS1 and VP2 genes of FPLV, even when the analysis focused on specific regions in the VP2 gene that are known to be located on the capsid surface. Comparison of the CPV VP2 genes revealed that nonsynonymous substitution was found to dominate over synonymous substitution in one specific region in the VP2 gene. These results suggested that FPLV has changed mainly by random genetic drift. In contrast, after the appearance of CPV, changes in the CPV VP2 gene appear to be partly selected by certain positive selection forces. CPV and FPLV are known to be closely related viruses genetically and biologically, but the evolutionary mechanisms of the two viruses appeared to be different.     

Neutralizing antibodies protect against lethal flavivirus challenge but allow for the development of active humoral immunity to a nonstructural virus protein

Antibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion.     

Food animal and poultry retroviruses and human health

In summary, studies reported to date have largely failed to demonstrate human infection with animal and poultry retroviruses or an association between human diseases and these viruses. A number of studies, most of them serologic, have attempted to demonstrate human infection with these viruses. The lack of antibodies in apparently exposed groups of persons suggests an absence of infection. However, another possible explanation is that humans may be immunologically unresponsive to infection with these viruses. Although attempts to infect normal human cells in vitro with many of these viruses have not been reported, BLV and BIV appear to grow poorly or not at all. On the other hand, ALSV subgroup D infect and transform human cells in vitro. However, the production of infectious virus in vitro has been low or nonexistent. This may explain the absence of antibodies in human populations. Furthermore, many of the methods used to detect infection, either directly or indirectly, have either low sensitivity or problems with specificity. Several epidemiologic studies have tried to show a relationship between human and animal leukemia or lymphoma. In many of these studies the actual exposure to retroviruses is unknown and exposure to animals may merely represent exposure to other risk factors that are more important but were either not considered or are undefined; alternatively, a common exposure may be responsible for malignancy in humans and animals with no interspecies relationship. Based on the reported studies, these viruses appear unlikely to be responsible for any significant occurrence of human disease, particularly lymphoid malignancies. Although a definitive statement of no risk to human health is probably unwarranted, the evidence to date indicates that the risk is low and perhaps nonexistent. Thus, no specific public health recommendations regarding retrovirus-infected animals or poultry are warranted at this time.     

Congenital infection of pigs with ruminant-type pestiviruses

Congenital infections of pigs were induced with two ruminant-type pestiviruses isolated from pigs. One of the viruses was bovine viral diarrhoea virus-like and the other border disease virus-like. Both produced symptoms similar to those observed with low virulence strains of classical swine fever virus. A striking effect of persistent virus infection in post-natal life was stunting in viraemic animals. It was also shown that a congenitally infected pig shed virus for 2.5 years and in sufficient quantity to infect other pigs, even by indirect contact. Unlike ruminants, congenitally infected pigs sometimes had persistent viraemia but eventually eliminated the virus. Clearance of virus from the blood was related to the appearance of neutralizing antibodies. However, clearance from the tissues sometimes took as much as 5 months longer than from the blood.     

Kinetics of replication of a partially attenuated virus and of the challenge virus during a three-year intersubtype feline immunodeficiency virus superinfection experiment in cats

The effects of preinfecting cats with a partially attenuated feline immunodeficiency virus (FIV) on subsequent infection with a fully virulent FIV belonging to a different subtype were investigated. Eight specific-pathogen-free cats were preinfected with graded doses of a long-term in vitro-cultured cell-free preparation of FIV Petaluma (FIV-P, subtype A). FIV-P established a low-grade or a silent infection in the inoculated animals. Seven months later, the eight preinfected cats and two uninfected cats were challenged with in vivo-grown FIV-M2 (subtype B) and periodically monitored for immunological and virological status. FIV-P-preinfected cats were not protected from acute infection by FIV-M2, and the sustained replication of this virus was accompanied by a reduction of FIV-P viral loads in the peripheral blood mononuclear cells and plasma. However, from 2 years postchallenge (p.c.) until 3 years p.c., when the experiment was terminated, preinfected cats exhibited reduced total viral burdens, and some also exhibited a diminished decline of circulating CD4(+) T lymphocytes relative to control cats infected with FIV-M2 alone. Interestingly, most of the virus detected in challenged cats at late times p.c. was of FIV-P origin, indicating that the preinfecting, attenuated virus had become largely predominant. By the end of follow-up, two challenged cats had no FIV-M2 detectable in the tissues examined. The possible mechanisms underlying the interplay between the two viral populations are discussed.     

Activation of caspases in pig kidney cells infected with wild-type and CrmA/SPI-2 mutants of cowpox and rabbitpox viruses

The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1beta-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384-391, 1996). In our study, LLC-PK1 cells infected with CPV delta crmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPV delta SPI-2 resulted in cleavage activity against PARP and lamin A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys(biotinyl)-Asp-2,6-dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected with CPV delta crmA, wt RPV, or RPV delta SPI-2 but not wt CPV. Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP-cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.     

Ruminant pestiviruses

The ruminant pestiviruses, bovine virus diarrhoea virus (BVDV) and border disease virus (BDV) are highly successful and important pathogens which infect ruminant species worldwide. Although the serological relationships among ruminant pestiviruses require further clarification, there is growing evidence for two antigenic groups, one of which predominates in cattle and one in sheep. The success of pestiviruses stems from the ability of the non-cytopathic (NCP) biotype of the virus to cross the placenta and establish a persistent infection (PI) in the developing foetus. This biotype should be regarded as the 'normal' biotype with the cytopathic (CP) biotype being an abnormal virus that is usually isolated only from PI animals dying from mucosal disease. Recent molecular evidence points to CP viruses arising from their NCP counterparts by recombination events that include the insertion of host RNA and/or the duplication of viral RNA sequences. However, the biological mechanism through which CP viruses kill cells remains unknown. Virtually all CP and NCP viruses cause only mild, transient clinical symptoms in healthy adult animals and stimulate a protective immune response. Despite the urgent requirement for a safe, effective vaccine, there is still no commercial vaccine that has been shown to immunize dams so that foetal infection is prevented. In the absence of an effective vaccine, reliable diagnostic techniques are essential to implement effective control measures. There is now a range of monoclonal antibody-based enzyme-linked immunosorbent assays for identifying PI or convalescent animals. These tests are specific, rapid, sensitive and reliable but may themselves become redundant as they are superceded by ever-increasing molecular biology-based techniques.     

A review of alphavirus replication in neurons

Alphaviruses are important causes of mosquito-borne viral encephalitis. The prototype alphavirus, Sindbis virus, causes encephalomyelitis in mice. The primary target cell for nervous system infection is the neuron. Thus, Sindbis virus infection of mice provides a model system for studying virus-neuron interactions. The outcome of infection is dependent on the maturity of the targeted neurons and on the strain of Sindbis virus used for infection. Most Sindbis virus strains can induce programmed cell death or apoptosis in cultured lines of mammalian cells and in immature postmitotic neurons both in vitro and in vivo. As neurons mature they become increasingly resistant to Sindbis virus-induced apoptosis presumably due to increased expression with differentiation of cellular antiapoptotic proteins. Therefore, in the absence of an effective immune response, these relatively avirulent strains of Sindbis virus establish persistent nonfatal infection in mature neurons. More virulent strains of Sindbis virus can overcome this intrinsic resistance of mature neurons to apoptosis and cause neuronal death. Amino acid changes in the virion glycoproteins are the main determinants of neurovirulence and knowledge of the effects of specific changes allows the investigator to design Sindbis viruses of specified neurovirulence for animals of different ages.     

Influenza viruses select ordered lipid domains during budding from the plasma membrane

During the budding of enveloped viruses from the plasma membrane, the lipids are not randomly incorporated into the envelope, but virions seem to have a lipid composition different from the host membrane. Here, we have analyzed lipid assemblies in three different viruses: fowl plague virus (FPV) from the influenza virus family, vesicular stomatitis virus (VSV), and Semliki Forest virus (SFV). Analysis of detergent extractability of proteins, cholesterol, phosphoglycerolipids, and sphingomyelin in virions showed that FPV contains high amounts of detergent-insoluble complexes, whereas such complexes are largely absent from VSV or SFV. Cholesterol depletion from the viral envelope by methyl-beta-cyclodextrin results in increased solubility of sphingomyelin and of the glycoproteins in the FPV envelope. This biochemical behavior suggests that so-called raft-lipid domains are selectively incorporated into the influenza virus envelope. The "fluidity" of the FPV envelope, as measured by the fluorescence polarization of diphenylhexatriene, was significantly lower than compared with VSV or SFV. Furthermore, influenza virus hemagglutinin incorporated into the envelope of recombinant VSV was largely detergent-soluble, indicating the depletion of raft-lipid assemblies from this membrane. The results provide a model for lipid selectivity during virus budding and support the view of lipid rafts as cholesterol-dependent, ordered domains in biological membranes.     

Molecularly cloned feline immunodeficiency virus NCSU1 JSY3 induces immunodeficiency in specific-pathogen-free cats

A full-length feline immunodeficiency virus NCSU1 (FIV-NCSU1) genome (JSY3) was cloned directly from FIV-NCSU1-infected feline CD4+ lymphocyte (FCD4E) genomic DNA and identified by PCR amplification with 5' long terminal repeat, gag, env, and 3' long terminal repeat primer sets. Supernatant from FCD4E cells cocultured with JSY3-transfected Crandell feline kidney (CrFK) cells was used as an inoculum. Cell-free JSY3 virus was cytopathogenic for FCD4E lymphocytes but did not infect CrFK cells in vitro. To determine in vivo infectivity and pathogenesis, six young adult specific-pathogen-free cats were inoculated with cell-free JSY3 virus. Provirus was detected at 2 weeks postinfection (p.i.) and was still detectable at 25 weeks p.i. as determined by gag region PCR-Southern blot analysis of peripheral blood mononuclear cell lysates. Infectious virus was recovered from peripheral blood mononuclear cells at 6 and 25 weeks p.i., and an antibody response to FIV was detected by 4 weeks. In the acute phase of infection, JSY3 provirus was found only in the CD4+ lymphocyte subset; however, by 14 weeks p.i., the greatest provirus burden was detected in B lymphocytes. All six cats were panlymphopenic at 2 weeks p.i., CD4+/CD8+ ratios were inverted by 6 weeks p.i., and five of the six cats developed lymphadenopathy by 10 weeks p.i. To determine if the JSY3 molecular clone caused immunodeficiency similar to that of the parental wild-type FIV-NCSU1, the cats were challenged with the low-virulence ME49 strain of Toxoplasma gondii at 29 weeks p.i. Five of six cats developed clinical signs consistent with generalized toxoplasmosis, and three of six cats developed acute respiratory distress and required euthanasia. Histopathologic examination of the severely affected cats revealed generalized inflammatory reactions and the presence of T. gondii tachyzoites in multiple tissues. None of the six age- and sex-matched specific-pathogen-free cats inoculated with only T. gondii developed clinical disease. Our results suggest that the pathogenesis of the molecularly cloned NCSU1 JSY3 is similar to that of wild-type FIV-NCSU1.     

Human immunodeficiency virus inhibits multilineage hematopoiesis in vivo

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4(+) lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+ cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: possible role in G protein-coupled signal transduction

Although present in many copies in the mouse genome, xenotropic murine leukemia viruses cannot infect cells from laboratory mice because of the lack of a functional cell surface receptor required for virus entry. In contrast, cells from many nonmurine species, including human cells, are fully permissive. Using an expression library approach, we isolated a cDNA from HeLa cell RNA that conferred susceptibility to xenotropic envelope protein binding and virus infection when expressed in nonpermissive cells. The deduced product is a 696-aa multiple-membrane spanning molecule, is widely expressed in human tissues, and shares homology with nematode, fly, and plant proteins of unknown function as well as with the yeast SYG1 protein, which has been shown to interact with a G protein. This molecule also acts as a receptor for polytropic murine leukemia viruses, consistent with observed interference between xenotropic and polytropic viruses in some cell types. This xenotropic and polytropic retrovirus receptor (XPR1) is the fourth identified molecule having multiple membrane spanning domains among mammalian type C oncoretrovirus receptors and may play a role in G protein-coupled signal transduction, as do the chemokine receptors required for HIV entry.     

Replication of influenza virus in organ cultures of human and simian urogenital tissues and human foetal tissues

A survey of human adult tissues in organ cultures showed that influenza viruses (A/Moscow/1019/65 (h2n2) or a recombinant virus virulent for man (PR/8-A/England/939/69 Clone 7a(H3N2)) could infect uterus, bladder and conjunctiva but not oesophagus under the conditions employed; simian bladder and uterus were also susceptible. These results were similar to those already described for corresponding ferret tissues. Organ cultures of human foetal nasal mucosa, trachea, oesophagus, small and large intestine, and bladder consistently supported replication over 4 days or more with high virus yields. Lung, conjunctiva and umbilical cord were less consistently susceptible and gave lower yields. Placenta and kidney cultures allowed replication of virus in one of 8 and one of 4 experiments respectively, the yields being low and of short duration. Organ cultures of neural tissue (meninges and brain), lymphopoietic tissue (spleen, liver and thymus) and amnion did not support significant viral replication. The results are discussed in relation to possible infection of the foetus in utero with influenza virus.