Divergent Simian Arteriviruses Cause Simian Hemorrhagic Fever of Differing Severities in Macaques.

UNLABELLED: Simian hemorrhagic fever (SHF) is a highly lethal disease in captive macaques. Three distinct arteriviruses are known etiological agents of past SHF epizootics, but only one, simian hemorrhagic fever virus (SHFV), has been isolated in cell culture. The natural reservoir(s) of the three viruses have yet to be identified, but African nonhuman primates are suspected. Eleven additional divergent simian arteriviruses have been detected recently in diverse and apparently healthy African cercopithecid monkeys. Here, we report the successful isolation in MARC-145 cell culture of one of these viruses, Kibale red colobus virus 1 (KRCV-1), from serum of a naturally infected red colobus (Procolobus [Piliocolobus] rufomitratus tephrosceles) sampled in Kibale National Park, Uganda. Intramuscular (i.m.) injection of KRCV-1 into four cynomolgus macaques (Macaca fascicularis) resulted in a self-limiting nonlethal disease characterized by depressive behavioral changes, disturbance in coagulation parameters, and liver enzyme elevations. In contrast, i.m. injection of SHFV resulted in typical lethal SHF characterized by mild fever, lethargy, lymphoid depletion, lymphoid and hepatocellular necrosis, low platelet counts, increased liver enzyme concentrations, coagulation abnormalities, and increasing viral loads. As hypothesized based on the genetic and presumed antigenic distance between KRCV-1 and SHFV, all four macaques that had survived KRCV-1 injection died of SHF after subsequent SHFV injection, indicating a lack of protective heterotypic immunity. Our data indicate that SHF is a disease of macaques that in all likelihood can be caused by a number of distinct simian arteriviruses, although with different severity depending on the specific arterivirus involved. Consequently, we recommend that current screening procedures for SHFV in primate-holding facilities be modified to detect all known simian arteriviruses. IMPORTANCE: Outbreaks of simian hemorrhagic fever (SHF) have devastated captive Asian macaque colonies in the past. SHF is caused by at least three viruses of the family Arteriviridae: simian hemorrhagic fever virus (SHFV), simian hemorrhagic encephalitis virus (SHEV), and Pebjah virus (PBJV). Nine additional distant relatives of these three viruses were recently discovered in apparently healthy African nonhuman primates. We hypothesized that all simian arteriviruses are potential causes of SHF. To test this hypothesis, we inoculated cynomolgus macaques with a highly divergent simian arterivirus (Kibale red colobus virus 1 [KRCV-1]) from a wild Ugandan red colobus. Despite being only distantly related to red colobuses, all of the macaques developed disease. In contrast to SHFV-infected animals, KRCV-1-infected animals survived after a mild disease presentation. Our study advances the understanding of an important primate disease. Furthermore, our data indicate a need to include the full diversity of simian arteriviruses in nonhuman primate SHF screening assays.

Equine viral arteritis.

Equine arteritis virus (EAV), the causative agent of equine viral arteritis (EVA), is a respiratory and reproductive disease that occurs throughout the world. EAV infection is highly species-specific and exclusively limited to members of the family Equidae, which includes horses, donkeys, mules, and zebras. EVA is an economically important disease and outbreaks could cause significant losses to the equine industry. The primary objective of this article is to summarize current understanding of EVA, specifically the disease, pathogenesis, epidemiology, host immune response, vaccination and treatment strategies, prevention and control measures, and future directions.

Semen quality of stallions challenged with the Kentucky 84 strain of equine arteritis virus.

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a respiratory and reproductive disease of equids. Some strains of EAV can cause fever, leukopenia, and dependent edema of the limbs, scrotum, and preputium in the acutely infected stallion. We hypothesized that fever and scrotal edema observed during the acute phase of the infection, but not the presence of EAV, have an adverse effect on semen quality. A group of seven stallions were intranasally inoculated with the Kentucky 84 (KY84) strain of EAV. Stallions were monitored for clinical signs of EVA until 42 days postinoculation (dpi). Semen was collected every other day for the first 15 days and 2 times a week up to 79 dpi. Additional samples were collected at 147, 149, and 151 dpi. Semen from each stallion was evaluated on the basis of motion characteristics, total number of spermatozoa, membrane integrity, and morphology. Virus infectivity titers were determined in RK-13 cells. Significant decreases in sperm quality were observed between 9 and 76 dpi. LOWESS (locally weighted scatterplot smoothing) curves for each horse were fit and integrated to quantify spermatozoa exposure to fever, virus, and edema over a period of 67 days before each ejaculation. Linear mixed models were then fit to isolate the effects of each factor on semen quality. Scrotal edema and fever were found to exert independent effects on all the semen quality parameters (P ≤ 0.002), whereas virus seems to exert little to no direct effect, as virus titers remained high long after semen quality returned to baseline.

Development and characterization of an infectious cDNA clone of the virulent Bucyrus strain of Equine arteritis virus.

Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant progeny virus derived from this clone caused severe disease in horses, characterized by pyrexia, oedema, leukopenia, high-titre viraemia and substantial nasal shedding of virus. The availability of infectious cDNA clones that produce recombinant viruses of different virulence to horses will facilitate characterization of the virulence determinants of EAV through reverse genetics.

Lactate dehydrogenase-elevating virus induces apoptosis in cultured macrophages and in spinal cords of C58 mice coincident with onset of murine amyotrophic lateral sclerosis.

Age-dependent poliomyelitis (ADPM) or murine amyotrophic lateral sclerosis (ALS) is a murine paralytic disease triggered in immunosuppressed genetically-susceptible mice by infection with the arterivirus lactate dehydrogenase-elevating virus (LDV). This disease provides an animal model for ALS, affecting anterior horn neurons and resulting in neuroparalysis 2-3 weeks after LDV infection. We have tested the hypothesis that spinal cord apoptosis is a feature of the LDV-induced murine ALS, since apoptosis is postulated to be a causal factor in human ALS. Gene microarray analyses of spinal cords from paralyzed animals revealed upregulation of several genes associated with apoptosis. Spinal cord apoptosis was investigated further by TUNEL and activated caspase-3 assays, and was observed to emerge concurrent with paralytic symptoms in both neuronal and non-neuronal cells. Caspase-3-dependent apoptosis was also triggered in cultured macrophages by neurovirulent LDV infection. Thus, virus-induced spinal cord apoptosis is a pre-mortem feature of ADPM, which affects both neuronal and support cells, and may contribute to the pathogenesis of this ALS-like disease.

Phylogenetic characterization of a highly attenuated strain of equine arteritis virus from the semen of a persistently infected standardbred stallion.

An avirulent, novel variant of equine arteritis virus (EAV; CA95G) was isolated from the semen of a persistently infected Standardbred stallion. The CA95G virus caused subclinical infection and seroconversion in susceptible horses, and virus was isolated only once from blood and nasal secretions collected from 6 experimentally infected horses. Sequence analysis of genes encoding the known EAV structural proteins shows that this highly attenuated strain of EAV is genetically similar to virulent field strains of EAV and, in particular, to a strain of EAV that was isolated during an outbreak of equine viral arteritis in western Canada in 1986. Not only is the carrier stallion the critical natural reservoir of EAV, but genetic diversity of the virus is generated in the course of persistent infection of carrier stallions. The subtle genetic changes that facilitate and maintain persistent EAV infection of the stallion's reproductive tract likely influence phenotypic properties of the virus such as virulence.

Comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure.

OBJECTIVE: To compare the virulence of selected strains of porcine reproductive and respiratory syndrome virus (PRRSV) relative to reproductive performance of pregnant gilts. DESIGN: 16 pregnant gilts (principals) were exposed oronasally to 4 strains (vaccine strain RespPRRS, field strains VR-2385, VR-2431, and NADC-8, 4 gilts/strain) of PRRSV on or about day 90 of gestation, 4 pregnant gilts (controls) were kept under similar conditions, except for exposure to PRRSV. Samples and specimens obtained from gilts, pigs (before ingestion of colostrum), and fetuses were tested for PRRSV and homologous antibody. ANIMALS: 20 pregnant gilts. PROCEDURE: The virulence of each strain of PRRSV was evaluated mainly on the clinical status of the corresponding litters at farrowing. RESULTS: Most gilts remained clinically normal throughout the study and farrowed normally at or near the expected farrowing time. All virus strains crossed the placenta of principal gilts to infect fetuses in utero. The number of late-term dead fetuses (which appeared to be the best measure of relative virulence) ranged from 0 for litters of control gilts and gilts exposed to strain RespPRRS, to 38 for gilts exposed to strain NADC-8. All principal gilts became viremic and developed antibody against PRRSV. All strains persisted in alveolar macrophages of at least some principal gilts for at least 7 weeks after exposure. CONCLUSION: Strains of PRRSV vary in virulence. CLINICAL RELEVANCE: The effects of PRRSV on reproductive performance are strain dependent and this should be considered in making a tentative diagnosis on the basis of clinical observations.

Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study.

Dual infections of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) followed by a second common respiratory virus, either porcine respiratory coronavirus (PRCV) or swine influenza virus (SIV), were studied. The aim was to determine if dual infections, as compared to single virus infections, result in enhanced clinical manifestations. It was also examined if PRRSV replication affects replication of PRCV or SIV in the respiratory tract. Groups of conventional 10 week old pigs were inoculated with PRRSV-only (3 pigs), PRCV-only (4 pigs) or SIV-only (4 pigs). Dual inoculations with PRRSV-PRCV (4 pigs) and PRRSV-SIV (3 groups of 4, 4 and 5 pigs) were performed at a 3 day interval. A group of uninoculated control pigs (8 pigs) was included. The infection with PRRSV-only induced a transient fever (40.2 degrees C) at 2 DPI, but no respiratory signs. The PRCV-only infection remained subclinical. The SIV-only infection resulted in a one day fever (40.1 degrees C) with moderate tachypnoea and dyspnoea. Mean weight gain in the virus-inoculated groups was retarded compared with the control group. The PRRSV-PRCV infection induced a 9 day lasting fever (peak 40.9 degrees C) with tachypnoea, dyspnoea and productive coughing. The PRRSV-SIV infection resulted in fever and respiratory signs in all 3 groups. Clinical signs, however, were more pronounced in group 1 than in groups 2 and 3. Pigs of group 1 showed fever during 10 days (peak 41.4 degrees C), tachypnoea, marked dyspnoea with abdominal breathing, and a productive cough. Pigs of groups 2 and 3 had fever for 5 and 3 days (peaks 40.6 and 40.3 degrees C) respectively and mild respiratory disorders. Mean weight gain during 14 DPI of the 2nd virus was 5.9 kg in the PRRSV-PRCV group and 4.0, 6.8 and 6.7 kg in PRRSV-SIV groups 1, 2 and 3 respectively. Mean weight gain during the corresponding period in the PRRSV-only group was 8.6 kg. It was concluded that dual infections with viruses causes more severe disease and growth retardation than single PRRSV infection. PRCV excretion curves were similar in single and dual virus inoculated groups. Excretion of SIV was delayed by 2 days in the dual inoculated pigs. Thus, replication of the second virus is not (PRCV) or only slightly (SIV) affected by a prior infection with PRRSV.

Comparative pathogenicity of nine US porcine reproductive and respiratory syndrome virus (PRRSV) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model.

One hundred forty-six 5-week- old cesarean-derived, colostrum-deprived (CDCD) pigs were inoculated intranasally with 1 of 9 US porcine reproductive and respiratory syndrome virus (PRRSV) isolates. Differences were found in severity of clinical respiratory disease, rectal temperatures (P < or = 0.001), gross lung lesions (P < or = 0.001), and microscopic lung lesions (P < or = 0.05). Gross lung lesions were generally most severe 10 days postinoculation and were distributed primarily in the cranial, middle, and accessory lobes and ventromedial portion of the caudal lung lobes. Mean gross lung lesion scores estimating the percentage of lung affected by pneumonia at 10 days postinoculation ranged from 16.7% +/- 2.8% (mean +/- SEM, n = 10) for isolate ISU-51 to 62.4% +/- 5.7% (n = 10) for isolate ISU-28. Microscopic lung lesions were characterized by hyperplastic and hypertrophied type 2 pneumocytes, septal infiltration by mononuclear cells, and accumulation of necrotic alveolar exudate. Lymph node follicular hyperplasia and focal necrosis was seen with all 9 isolates. This CDCD pig model was useful for demonstration of significant differences in pathogenicity among US PRRSV isolates. This difference in pathogenicity may help explain the variation of severity of clinical disease observed in field outbreaks of porcine reproductive and respiratory syndrome and should provide for meaningful comparison of PRRSV genotypes.

[Significance of PRRS virus infections for respiratory tract infections in swine--a literature review]. / Die Bedeutung des PRRS-Virus für Erkrankungen des Respirationstraktes beim Schwein--eine Literaturübersicht.

The paper summarises present knowledge concerning the possible role of a virus which has in the meantime been classified as belonging to the arteriviridae and which causes PRRS (Porcine Reproductive and Respiratory Syndrome), in the pathogenesis of respiratory diseases four years after first isolation of the virus. Although the fact that PRRSV-infection produces an immunity which protects swine from repeated bouts of PRRSV is common knowledge by now, reinfection with the virus might be possible. An immunosuppression, which has been suggested by many investigators, and which was thought to be the result of destruction of alveolar macrophages during virus replication, was not found as yet. The significance of the extent and the duration of the decrease in the number of alveolar macrophages, which belong to the unspecific immunity, for the total immune system of swine infected with PRRSV remains unclear. A general impairment of specific immunity through PRRSV-infection could not be shown. The present role of PRRS for the pathogenesis of respiratory disease is seen very differently. The significance of PRRSV as primary cause of an "influenza-like" illness which is principally followed by severe bacterial infection of the respiratory tract, is questioned by the results of studies which identify PRRSV-infection as a mainly subclinical disease. In these studies, clinical cases are the exemption. Attempts at experimental reproduction of a clinically manifest, respiratory disease was not successful as yet. The paper describes factors which might be responsible for these variable results. Control of PRRS is difficult in areas with a high density of the swine population, since the spreading of the virus with the wind seems to be important besides the recruitment of new, infected animals. A vaccine has not yet been registered in Germany. First experimental experiences with vaccination are available from Denmark and the USA.

Cytotoxic T cells are elicited during acute infection of mice with lactate dehydrogenase-elevating virus but disappear during the chronic phase of infection.

Lactate dehydrogenase-elevating virus (LDV) invariably establishes a life-long viremic infection in mice, which is maintained by replication of LDV in a renewable subpopulation of macrophages and escape from all host immune responses. We now demonstrate that cytotoxic T lymphocytes (CTLs) that specifically lyse LDV-infected macrophages and 3T3 cells producing the nucleocapsid protein of LDV were elicited in Swiss, B10.A, and (Swiss x B10.A)F1 mice. To detect target cell lysis, splenocytes needed to be expanded by a 5-day in vitro culture in the presence of recombinant interleukin 2 and syngeneic LDV protein-expressing cells. In vitro culture resulted in the specific expansion of CD8+ cells which mediated the lysis of target cells in a major histocompatibility complex class I-restricted manner. When CTLs were added to macrophage cultures at 1 h after infection with LDV, the lysis of the infected macrophages by the CTLs started about 5 h postinfection (p.i.) and, at an effector cell/target cell ratio of 25:1, resulted in the lysis of all LDV-infected macrophages in a culture by about 7 h p.i. However, lysis of the LDV replication in a culture was not rapid enough to significantly suppress the LDV yield in the culture. LDV replication in mice was also little affected by the presence of CTLs which were induced by immunization with 3T3 cells expressing the LDV nucleocapsid protein. Furthermore, all CTL precursor cells in infected mice had disappeared by 30 days p.i. Loss of CTL precursor cells in infected mice probably reflected high-dose clonal exhaustion, since LDV infection of a mouse results in massive production of LDV in all tissues of the mouse, but especially in lymphoidal tissues, and accumulation of LDV in newly formed germinal centers. Furthermore, slow LDV replication continues in the thymus and other lymphoidal organs.

Pathogenesis of in utero infection in porcine fetuses with porcine reproductive and respiratory syndrome virus.

Porcine fetuses were exposed in utero to porcine reproductive and respiratory syndrome virus (PRRSV) at stages of gestation ranging from 34 to 85 days and examined 17 to 31 days later to determine the effect of gestational age on fetal susceptibility. For each of the 8 litters tested during the study, all of the fetuses of 1 horn of the uterus were exposed to virus by intraamniotic injection; those of the other horn were exposed similarly to a sham inoculum that consisted of sterile cell culture medium. Viral infectivity titers associated with fetal tissues collected at necropsy indicated that, regardless of gestational age, the virus had replicated in fetuses exposed intraamniotically. In addition, virus had also spread and replicated in sham-inoculated littermates in 3 litters. On the basis of these findings it appears that there may be little or no temporal difference in fetal susceptibility to infection with PRRSV. If so, the lack of early fetal death as a commonly recognized feature of naturally occurring cases of PRRS may be due to a greater resistance of early gestational fetuses to the lethal effects of PRRSV, as suggested by this study, and/or a greater likelihood of transplacental infection during late gestation.

Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus.

Certain mouse strains, such as AKR and C58, which possess N-tropic, ecotropic murine leukemia virus (MuLV) proviruses and are homozygous at the Fv-1n locus are specifically susceptible to paralytic infection (age-dependent poliomyelitis [ADPM]) by lactate dehydrogenase-elevating virus (LDV). Our results provide an explanation for this genetic linkage and directly prove that ecotropic MuLV infection of spinal cord cells is responsible for rendering anterior horn neurons susceptible to cytocidal LDV infection, which is the cause of the paralytic disease. Northern (RNA) blot hybridization of total tissue RNA and in situ hybridization of tissue sections demonstrated that only mice harboring central nervous system (CNS) cells that expressed ecotropic MuLV were susceptible to ADPM. Our evidence indicates that the ecotropic MuLV RNA is transcribed in CNS cells from ecotropic MuLV proviruses that have been acquired by infection with exogenous ecotropic MuLV, probably during embryogenesis, the time when germ line proviruses in AKR and C58 mice first become activated. In young mice, MuLV RNA-containing cells were found exclusively in white-matter tracts and therefore were glial cells. An increase in the ADPM susceptibility of the mice with advancing age correlated with the presence of an increased number of ecotropic MuLV RNA-containing cells in the spinal cords which, in turn, correlated with an increase in the number of unmethylated proviruses in the DNA extracted from spinal cords. Studies with AKXD recombinant inbred strains showed that possession of a single replication-competent ecotropic MuLV provirus (emv-11) by Fv-1n/n mice was sufficient to result in ecotropic MuLV infection of CNS cells and ADPM susceptibility. In contrast, no ecotropic MuLV RNA-positive cells were present in the CNSs of mice carrying defective ecotropic MuLV proviruses (emv-3 or emv-13) or in which ecotropic MuLV replication was blocked by the Fv-1n/b or Fv-1b/b phenotype. Such mice were resistant to paralytic LDV infection. In utero infection of CE/J mice, which are devoid of any endogenous ecotropic MuLVs, with the infectious clone of emv-11 (AKR-623) resulted in the infection of CNS cells, and the mice became ADPM susceptible, whereas littermates that had not become infected with ecotropic MuLV remained ADPM resistant.

Relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt.

The relationship between stage of reproductive tract maturity and susceptibility to the experimental establishment of persistent infection with equine arteritis virus (EAV) was investigated in 21 prepubertal and 15 peripubertal colts. Five of six prepubertal colts inoculated intranasally remained infected in the reproductive tract from post-challenge day 28 to 93 and two of six from post-challenge day 120 to 180. No virus was detected in five of these animals killed on post-challenge day 210. Each of two peripubertal colts remained infected in the reproductive tract at post-challenge day 60 and one of nine was found to be persistently infected with EAV 15 months after challenge. These findings confirm that the virus can replicate in the reproductive tract of a significant proportion of colts for a variable period of time after clinical recovery in the absence of circulating concentrations of testosterone equivalent to those found in sexually mature stallions. Long-term persistent infection with EAV does not appear to occur in colts exposed to the virus before the onset of peripubertal development. We suggest that colts should be vaccinated at approximately 6 months of age, before peripubertal development but after the disappearance of maternally acquired antibodies.