Within-host genetic diversity of endemic and emerging parvoviruses of dogs and cats

Viral emergence can result from the adaptation of endemic pathogens to new or altered host environments, a process that is strongly influenced by the underlying sequence diversity. To determine the extent and structure of intrahost genetic diversity in a recently emerged single-stranded DNA virus, we analyzed viral population structures during natural infections of animals with canine parvovirus (CPV) or its ancestor, feline panleukopenia virus (FPV). We compared infections that occurred shortly after CPV emerged with more recent infections and examined the population structure of CPV after experimental cross-species transmission to cats. Infections with CPV and FPV showed limited genetic diversity regardless of the analyzed host tissue or year of isolation. Coinfections with genetically distinct viral strains were detected in some cases, and rearranged genomes were seen in both FPV and CPV. The sporadic presence of some sequences with multiple mutations suggested the occurrence of either particularly error-prone viral replication or coinfection by more distantly related strains. Finally, some potentially organ-specific host effects were seen during experimental cross-species transmission, with many of the mutations located in the nonstructural protein NS2. These included residues with evidence of positive selection at the population level, which is compatible with a role of this protein in host adaptation.     

Puma lentivirus is controlled in domestic cats after mucosal exposure in the absence of conventional indicators of immunity

A high percentage of free-ranging pumas (Felis concolor) are infected with feline lentiviruses (puma lentivirus, feline immunodeficiency virus Pco [FIV-Pco], referred to here as PLV) without evidence of disease. PLV establishes productive infection in domestic cats following parenteral exposure but, in contrast to domestic cat FIV, it does not cause T-cell dysregulation. Here we report that cats exposed to PLV oro-nasally became infected yet rapidly cleared peripheral blood mononuclear cell (PBMC) proviral load in the absence of a correlative specific immune response. Two groups of four specific-pathogen-free cats were exposed to PLV via the mucosal (oro-nasal) or parenteral (i.v.) route. All animals were PBMC culture positive and PCR positive within 3 weeks postinfection and seroconverted without exhibiting clinical disease; however, three or four oro-nasally infected animals cleared circulating proviral DNA within 3 months. Antibody titers reached higher levels in animals that remained persistently infected. PLV antigen-induced proliferation was slightly greater in mucosally inoculated animals, but no differences were noted in cytotoxic T-lymphocyte responses or cytokine profiles between groups. The distribution of virus was predominantly gastrointestinal as opposed to lymphoid in all animals in which virus was detected at necropsy. Possible mechanisms for viral clearance include differences in viral fitness required for crossing mucosal surfaces, a threshold dose requirement for persistence, or an undetected sterilizing host immune response. This is the first report of control of a productive feline or primate lentivirus infection in postnatally exposed, seropositive animals. Mechanisms underlying this observation will provide clues to containment of immunodeficiency disease and could prompt reexamination of vaccine-induced immunity against human immunodeficiency virus and other lentiviruses.     

Clearance of a productive lentivirus infection in calves experimentally inoculated with caprine arthritis-encephalitis virus

Lentiviruses have long been considered host-specific pathogens, but several recent observations demonstrated their capacity to conquer new hosts from different species, genera, and families. From these cross-species infections emerged new animal and human infectious diseases. The successful colonization and adaptation of a lentivirus to a nonnatural host depends on unspecific and specific host barriers. Some of those barriers exert a relative control of viral replication (i.e., cytotoxic T-lymphocyte response, viral inhibitory factors), but none of them was found able to totally clear the infection once the retrovirus is fully adapted in its host. In this study we examined the evolution of the host-lentivirus interactions occurring in an experimental animal model of cross-species infection in order to analyze the efficiency of those barriers in preventing the establishment of a persistent infection. Five newborn calves were inoculated with caprine arthritis-encephalitis virus (CAEV), and the evolution of infection was studied for more than 12 months. All the animals seroconverted in the first 0.75 to 1 month following the inoculation and remained seropositive for the remaining time of the experiment. Viral infection was productive during 4 months with isolation of replication competent virus from the blood cells and organs of the early euthanized animals. After 4 months of infection, neither replication-competent virus nor virus genome could be detected in blood cells or in the classical target organs, even after an experimental immunosuppression. No evidence of in vitro restriction of CAEV replication was observed in cells from tissues explanted from organs of these calves. These data provide the demonstration of a natural clearance of lentivirus infection following experimental inoculation of a nonnatural host, enabling perspectives of development of new potential vaccine strategies to fight against lentivirus infections.     

Sequence variations of Env signal peptide alleles in different clinical stages of HIV infection

The human immunodeficiency virus has been shown to increase its infectivity throughout the course of infection. This virus selection property has been associated with genome mutations and recombinations among virus variants, causing amino acid residue alterations in important viral proteins. In order to explore the contribution of Env signal peptide (Env-sp) to Env glycoprotein expression and its possible relationship to increased virus infectivity observed at late stages of infection, we characterized Env-sp sequences derived from twelve patients at “early” and “late” stages of HIV infection without antiretroviral therapy use. In spite of the remarkable overall similarity between both stages, we observed the deletion of a sequence of neutral and basic residues at the Env-sp amino terminus in virus from early stage specimens and the insertion of basic residues in the hydrophobic region on late-stage viral isolates. The Env-sp sequence alterations may have viral adaptive functions during HIV infection.     

Frequent transmission of immunodeficiency viruses among bobcats and pumas

With the exception of human immunodeficiency virus (HIV), which emerged in humans after cross-species transmissions of simian immunodeficiency viruses from nonhuman primates, immunodeficiency viruses of the family Lentiviridae represent species-specific viruses that rarely cross species barriers to infect new hosts. Among the Felidae, numerous immunodeficiency-like lentiviruses have been documented, but only a few cross-species transmissions have been recorded, and these have not been perpetuated in the recipient species. Lentivirus seroprevalence was determined for 79 bobcats (Lynx rufus) and 31 pumas (Puma concolor) from well-defined populations in Southern California. Partial genomic sequences were subsequently obtained from 18 and 12 seropositive bobcats and pumas, respectively. Genotypes were analyzed for phylogenic relatedness and genotypic composition among the study set and archived feline lentivirus sequences. This investigation of feline immunodeficiency virus infection in bobcats and pumas of Southern California provides evidence that cross-species infection has occurred frequently among these animals. The data suggest that transmission has occurred in multiple locations and are most consistent with the spread of the virus from bobcats to pumas. Although the ultimate causes remain unknown, these transmission events may occur as a result of puma predation on bobcats, a situation similar to that which fostered transmission of HIV to humans, and likely represent the emergence of a lentivirus with relaxed barriers to cross-species transmission. This unusual observation provides a valuable opportunity to evaluate the ecological, behavioral, and molecular conditions that favor repeated transmissions and persistence of lentivirus between species.     

Role of the nonspecific DNA-binding region and alpha helices within the core domain of retroviral integrase in selecting target DNA sites for integration

Retroviral integrase plays an important role in choosing host chromosomal sites for integration of the cDNA copy of the viral genome. The domain responsible for target site selection has been previously mapped to the central core of the protein (amino acid residues 49-238). Chimeric integrases between human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) were prepared to examine the involvement of a nonspecific DNA-binding region (residues 213-266) and certain alpha helices within the core domain in target site selection. Determination of the distribution and frequency of integration events of the chimeric integrases narrowed the target site-specifying motif to within residues 49-187 and showed that alpha 3 and alpha 4 helices (residues 123-166) were not involved in target site selection. Furthermore, the chimera with the alpha 2 helix (residues 118-121) of FIV identity displayed characteristic integration events from both HIV-1 and FIV integrases. The results indicate that the alpha 2 helix plays a role in target site preference as either part of a larger or multiple target site-specifying motif.     

A lion lentivirus related to feline immunodeficiency virus: epidemiologic and phylogenetic aspects.

Feline immunodeficiency virus (FIV) is a novel lentivirus that is genetically homologous and functionally analogous to the human AIDS viruses, human immunodeficiency virus types 1 and 2. FIV causes immunosuppression in domestic cats by destroying the CD4 T-lymphocyte subsets in infected hosts. A serological survey of over 400 free-ranging African and Asian lions (Panthera leo) for antibodies to FIV revealed endemic lentivirus prevalence with an incidence of seropositivity as high as 90%. A lion lentivirus (FIV-Ple) was isolated by infection of lion lymphocytes in vitro. Seroconversion was documented in two Serengeti lions, and discordance of mother-cub serological status argues against maternal transmission (in favor of horizontal spread) as a major route of infection among lions. A phylogenetic analysis of cloned FIV-Ple pol gene sequences from 27 lions from four African populations (from the Serengeti reserve, Ngorongoro Crater, Lake Manyara, and Kruger Park) revealed remarkably high intra- and interindividual genetic diversity at the sequence level. Three FIV-Ple phylogenetic clusters or clades were resolved with phenetic, parsimony, and likelihood analytical procedures. The three clades, which occurred not only together in the same population but throughout Africa, were as divergent from each other as were homologous pol sequences of lentivirus isolated from distinct feline species, i.e., puma and domestic cat. The FIV-Ple clades, however, were more closely related to each other than to other feline lentiviruses (monophyletic for lion species), suggesting that the ancestors of FIV-Ple evolved in allopatric (geographically isolated) lion populations that converged recently. To date, there is no clear evidence of FIV-Ple-associated pathology, raising the possibility of a historic genetic accommodation of the lion lentivirus and its host leading to a coevolved host-parasite symbiosis (or commensalism) in the population similar to that hypothesized for endemic simian immunodeficiency virus without pathology in free-ranging African monkey species.     

Coadaptation and immunodeficiency virus: lessons from the Felidae

The emergence of pathogenic viruses in new species offers an unusual opportunity to monitor the coadaptation of viruses and their hosts in a dynamic ongoing process of intense biological selection. Tracking lentivirus epidemics in man, monkeys and cats reveals genomic struggles at three levels: quasispecies divergence within an individual; coadaptation of virus and host genomes subsequent to disease outbreaks; and transmission, spread and pathogenesis in related host species. Aspects of each level are revealed by examining the genetic diversity of feline immunodeficiency virus in domestic and wild cat species. This approach has been facilitated by the recent genetic characterization of a novel lentivirus in lions.     

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.     

Decline in CD4+ cell numbers in cats with naturally acquired feline immunodeficiency virus infection.

T-cell subsets were studied by fluorescence-activated cell sorter analysis in 57 feline immunodeficiency virus (FIV)-seropositive cats with naturally acquired FIV infection to see whether CD4(+)-CD8+ alterations were comparable to those observed in human immunodeficiency virus-infected patients. CD4+ values were decreased and CD8+ values were increased. The CD4+/CD8+ ratio was reduced to 1.6, compared with 3.3 in 33 FIV-seronegative control cats. Variance analysis of data showed a significant influence of FIV seropositivity, sex, and spaying of female cats on CD4+ values. CD8+ values were significantly influenced by FIV seropositivity, age, and breed. These findings indicate a similarity between FIV and human immunodeficiency virus infections, as far as alterations of T-cell subsets are concerned.     

Roles of the auxiliary genes and AP-1 binding site in the long terminal repeat of feline immunodeficiency virus in the early stage of infection in cats.

To examine the roles of auxiliary genes and the AP-1 binding site in the long terminal repeat of feline immunodeficiency virus (FIV) in vivo, three mutant viruses, which are defective in the vif gene ([delta]vif), ORF-A gene (deltaORF-A), and AP-1 binding site (deltaAP-1), and wild-type virus as a positive control were separately inoculated into three specific-pathogen-free cats. These cats were assessed by measuring the number of proviral DNA copies in peripheral blood mononuclear cells (PBMCs), the CD4/CD8 ratio and antibody responses to FIV for 16 weeks and then examining histological changes at necropsy. Although viral DNAs were detected in PBMCs from all 12 cats to various degrees until 16 weeks postinoculation, no virus was recovered from PBMCs of cats infected with (delta)vif virus during the observation period. However, a very weak antibody response was induced in one cat infected with the (delta)vif virus. In contrast, despite the successful recovery of virus from both groups of cats infected with deltaORF-A and deltaAP-1 virus, antibody responses and decrease in the CD4/CD8 ratio in the groups were milder than those in cats infected with wild-type virus. Furthermore, the numbers of proviral DNA copies in PBMCs from the two groups were not able to reach the level in cats infected with wild-type virus during the observation period. From these results, we conclude that these mutant viruses are still infectious for cats but failed in efficient viral replication and suggest that these auxiliary genes and enhancer element are important or essential to full viral replication kinetics and presumably to full pathogenicity during the early stage of infection in vivo.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Serological survey of the Iriomote cat (Felis iriomotensis) in Japan

The Iriomote cat (Felis iriomotensis) was first discovered on Iriomote Island in the Yaeyama Islands of Japan in 1965. Ten male and 11 female adult cats were captured during the 6 yr period from 1983 to 1988. These were examined for evidence of viral and mycoplasmal infections. Neither Mycoplasma sp. nor Ureaplasma sp. were detected in swab samples of oropharyngeal and urogenital regions. A foamy virus was isolated from the oropharyngeal swab of a female cat examined in 1988. Feline leukemia virus was not detected in any of the cats. All cats were negative for serum antibodies to feline panleukopenia virus, feline herpesvirus, feline immunodeficiency virus and rotavirus. Eleven of 19 (58%), 14 of 17 (82%) and 6 of 17 cats (35%) had serum antibodies against feline calicivirus, coronavirus and feline syncytium forming virus, respectively.     

Retroviruses and sexual size dimorphism in domestic cats (Felis catus L.).

Hochberg and co-workers have predicted that an increase in host adult mortality due to parasites is balanced by an earlier age at first reproduction. In polygynous species we hypothesize that such a pattern would lead to diverging selection pressure on body size between sexes and increased sexual size dimorphism. In polygynous mammals, male body size is considered to be an important factor for reproductive success. Thus, under the pressure of a virulent infection, males should be selected for rapid growth and/or higher body size to be able to compete successfully as soon as possible with opponents. In contrast, under the same selection pressure, females should be selected for lighter adult body size or rapid growth to reach sexual maturity earlier. We investigated this hypothesis in the domestic cat Felis catus. Orange cats have greater body size dimorphism than non-orange cats. Orange females are lighter than non-orange females, and orange males are heavier than non-orange males. Here, we report the extent to which orange and non-orange individuals differ in infection prevelance for two retroviruses, feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV). FIV is thought to be transmitted almost exclusively through aggressive contacts between individuals, whereas FeLV transmission occurs mainly through social contacts. The pattern of infection of both diseases is consistent with the higher aggressiveness of orange cats. In both sexes, orange cats are significantly more infected by FIV, and tend to be less infected by FeLV than other cats. The pattern of infection is also consistent with an earlier age at first reproduction in orange than in non-orange cats, at least for females. These results suggest that microparasitism may have played an important role in the evolution of sexual size dimorphism of domestic cats.     

Epidemiology,genetic diversity,and evolution of endemic feline immunodeficiency virus in a population of wild cougars

Within the large body of research on retroviruses, the distribution and evolution of endemic retroviruses in natural host populations have so far received little attention. In this study, the epidemiology, genetic diversity, and molecular evolution of feline immunodeficiency virus specific to cougars (FIVpco) was examined using blood samples collected over several years from a free-ranging cougar population in the western United States. The virus prevalence was 58% in this population (n = 52) and increased significantly with host age. Based on phylogenetic analysis of fragments of envelope (env) and polymerase (pol) genes, two genetically distinct lineages of FIVpco were found to cooccur in the population but not in the same individuals. Within each of the virus lineages, geographically nearby isolates formed monophyletic clusters of closely related viruses. Sequence diversity for env within a host rarely exceeded 1%, and the evolution of this gene was dominated by purifying selection. For both pol and env, our data indicate mean rates of molecular evolution of 1 to 3% per 10 years. These results support the premise that FIVpco is well adapted to its cougar host and provide a basis for comparing lentivirus evolution in endemic and epidemic infections in natural hosts.     

Feline viruses in wildcats from Scotland

Few data are available on the prevalence of feline viruses in European wildcats (Felis silvestris). Previous surveys have indicated that wildcats may be infected with the common viruses of domestic cats, apart from feline immunodeficiency virus (FIV). In the present study, 50 wildcats trapped throughout Scotland (UK) between August 1992 and January 1997 were tested for evidence of viral infection. All were negative for FIV by several serological or virological methods. By contrast, 10% of the cats were positive for feline leukemia virus (FeLV) antigen and infectious virus was isolated from 13% of a smaller subset. Of the wildcats tested for respiratory viruses, 25% yielded feline calicivirus (FCV) and although no feline herpesvirus was isolated, 16% of the samples had neutralizing antibodies to this virus. Antibodies to feline coronavirus (FCoV) were found in 6% of samples. Feline foamy virus (FFV) was an incidental finding in 33% of samples tested. This study confirms that wildcats in Scotland are commonly infected with the major viruses of the domestic cat, except for FIV.     

Experimental infection of dogs with a feline endogenous retrovirus RD-114

Background  

The feline endogenous retrovirus RD114 is contained in the genome of cats. The virus may contaminate live canine vaccines based on cultured feline cells. The in vivo infectivity, acute and subacute pathogenicity, and viral proliferation of the RD114 virus were evaluated by experimental infection of dogs.     

Feline immunodeficiency virus tropism

Feline immunodeficiency virus (FIV) induces a disease similar to acquired immunodeficiency syndrome (AIDS) in cats, yet in contrast to human immunodeficiency virus (HIV), CD4 is not the viral receptor. We identified a primary receptor for FIV as CD134 (OX40), a T cell activation antigen and costimulatory molecule. CD134 expression promotes viral binding and renders cells permissive for viral entry, productive infection, and syncytium formation. Infection is CXCR4-dependent, analogous to infection with X4 strains of HIV. Thus, despite the evolutionary divergence of the feline and human lentiviruses, both viruses use receptors that target the virus to a subset of cells that are pivotal to the acquired immune response. Further, we applied the new method for FIV receptor to Ebola virus entry factors with some modifications, and identified receptor-type tyrosine kinases, Axl and Dtk (members of Tyro3 family). Distribution of the molecules matches well with the Ebola virus tropism.     

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.