Intestinal stromal tumors in a simian immunodeficiency virus-infected, simian retrovirus-2 negative rhesus macaque (Macaca mulatta).

Multifocal submucosal stromal tumors were diagnosed in a 5.5-year-old rhesus macaque (Macaca mulatta) experimentally infected with simian immunodeficiency virus, strain SIVsmE660, and CD4+ T cell depleted. The animal was negative for simian retroviruses, SRV-1, -2, and -5. Polymerase chain reaction analysis of DNA from tumor and spleen tissue revealed abundant, preferential presence of retroperitoneal fibromatosis herpesvirus, the macaque homologue of the Kaposi sarcoma-associated herpesvirus (human herpesvirus-8), in the tumors. This was corroborated by demonstration of viral latent nuclear antigen-1 in the nuclei of a majority of the spindeloid tumor cells. Low levels of an additional macaque herpesvirus, rhesus rhadinovirus, were also detected in the spleen and tumor tissues. The spindeloid cells labeled positively for vimentin and CD117 but were negative for CD31, CD68, desmin, and smooth muscle cell actin. Collectively, these findings suggest a relation to but not absolute identity with simian mesenchymoproliferative disorders (MPD) or typical gastrointestinal stromal tumors (GISTs).

An immunoblot assay for detection of immunoglobulin M antibody to human herpesvirus 6.

We identified the human herpesvirus 6 (HHV-6)-dominant immunoglobulin M (IgM)-reactive virion protein as being the same 101-kDa protein (101K) previously identified as the major IgG immunoreactive protein and a specific serologic marker of HHV-6 infection. An immunoblot assay (IB) to detect HHV-6-specific IgM antibodies against the 101K protein in human serum samples was developed. The assay was validated by using acute- and convalescent-phase serum collected from children under 2 years of age in which we previously detected IgG seroconversion to the HHV-6 101K protein. Of 32 serum pairs which previously demonstrated IgG seroconversion to the 101K protein, 29 had IgM reactivity to the same protein in the acute-phase sample and the remaining 3 had reactivity in the convalescent-phase sample. We also detected HHV-6 IgM activity in sera collected from individuals > or =4 years of age who were also IgM seropositive to measles or rubella. Results of cross-adsorption studies using measles virus-, rubella virus-, and HHV-6-infected cells as the adsorbing antigen indicated no cross-reactivity between measles or rubella IgM and HHV-6 IgM in human serum samples. The IgM IB detected HHV-6-specific IgM antibody to the 101K protein in 78% (63 of 81) of tested acute-phase serum collected from young children with an undifferentiated rash illness by using a single serum dilution.

Two distinct lineages of macaque gamma herpesviruses related to the Kaposi's sarcoma associated herpesvirus.

BACKGROUND: KSHV, Kaposi's sarcoma-associated herpesvirus, is a necessary cofactor for the development of Kaposi's sarcoma (KS). We have previously reported KSHV-related DNA sequences in retroperitoneal fibromatosis (RF) tissue from two species of macaque. The putative herpesvirus was called RFHV for RF-associated herpesvirus. These data suggested that KSHV is a human representative of a larger family of primate herpesviruses. OBJECTIVE: To identify and characterize other members of a putative family of KSHV-related herpesviruses in macaques in order to obtain information on the evolutionary history of KSHV infection in humans. STUDY DESIGN: Lymphoid tissue cells and blood leukocytes from rhesus-, cynomolgus- and pigtailed-macaques were tested for the presence of unknown herpesviruses using degenerate primer-driven PCR amplification. The sequences obtained were compared against known herpesvirus sequences. RESULTS: We have identified new herpesvirus DNA sequences in each of the three macaque species. Sequence comparisons indicate that these new viruses are most related to each other and form a separate phylogenetic lineage within the gamma herpesviruses. Screening of PBMC from Indonesian-origin quarantine animals suggests that these viruses (MGV, macaque gamma virus) are species-specific, and highly prevalent in the wild. They are readily cultured in vivo, and share a common tissue tropism with the previously identified RFHV. CONCLUSIONS: MGV and RFHV represent two independent introductions of an ancestral gamma herpesvirus into macaque precursors.

Activation in vivo of retroperitoneal fibromatosis-associated herpesvirus, a simian homologue of human herpesvirus-8.

Retroperitoneal fibromatosis-associated herpesvirus of rhesus macaques (RFHVMm) is a gammaherpesvirus closely related to human herpesvirus-8 (HHV-8), which is thought to be a necessary cofactor for the development of Kaposi's sarcoma (KS) in humans. Here, RFHVMm infection of rhesus macaques exposed to the D-type retrovirus simian retrovirus-2 (SRV-2) is described. Development of SRV-2 viraemia, infection with simian immunodeficiency virus or administration of cyclosporin A could result in persistent RFHVMm viraemia. From this, it is concluded that productive retrovirus infection or otherwise-induced immune suppression has the ability to activate this herpesvirus in vivo. Elevated levels of circulating interleukin-6, a cytokine that plays a central role in KS, were found in RFHVMm-viraemic animals. In viraemic animals, RFHVMm was found in tissues that are common sites for the development of AIDS-associated KS, especially the oral cavity. Together, these data suggest a common biology between RFHVMm infection of macaques and HHV-8 infection and pathogenesis in humans.

Identification of two homologs of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis of different macaque species.

Simian retroperitoneal fibromatosis (RF) is a vascular fibroproliferative neoplasm which has many morphological and histological similarities to human Kaposi's sarcoma (KS). Like epidemic KS in AIDS patients, RF is highly associated with an immunodeficiency syndrome (simian acquired immunodeficiency syndrome [SAIDS]) caused by a retrovirus infection. Recently, a new gammaherpesvirus, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), has been identified in KS tumors, suggesting that KS has a viral etiology. Our previous experimental transmission studies and epidemiological data suggest that RF also has an infectious etiology. In order to determine whether a similar virus is also associated with RF, we have assayed for the presence of an unknown herpesvirus using degenerate PCR primers targeting the highly conserved DNA polymerase genes of the herpesvirus family. Here we provide DNA sequence evidence for two new herpesviruses closely related to KSHV from RF tissues of two macaque species, Macaca nemestrina and Macaca mulatta. Our data suggest that KSHV and the putative macaque herpesviruses define a new group within the subfamily Gammaherpesvirinae whose members are implicated in the pathogenesis of KS and KS-like neoplasms in different primate species.

Increased enzyme-linked immunosorbent assay specificity with solubilized simian retrovirus 2-infected cell membranes.

Simian type-D retrovirus (SRV) infection is a health problem in captive and wild-caught macaques; it interferes with acquired immune deficiency syndrome-related research. Because the enzyme-linked immunosorbent assay (ELISA) with gradient-purified SRV-2 virus yields a high percentage of false-positive results, the assay was modified with membrane antigens from SRV-2-infected and uninfected A549 cells. The SRV-2 membrane antigen contains the major proteins detected in positive sera by Western blotting: env proteins gp70 and gp20 and gag proteins p27, p14, p12, and p10. The original purified virus ELISA had a specificity of 74% compared with Western immunoblot. The modified ELISA using the difference in optical density between infected and control cell membrane antigens resulted in a specificity of 100% when the same samples were tested.

The effect of combined rotavirus and Escherichia coli infections in rabbits.

In rabbits, experimentally induced rotavirus infection results in soft feces only; thus it is unlikely that it is the sole cause of the severe, often fatal diarrhea of weanling rabbits with which it is associated. To determine whether rotavirus acts synergistically with another pathogen, New Zealand White rabbits (10 to 38 weeks old) were inoculated with rotavirus (L:ALA:84) and/or Escherichia coli 015:H-(RDEC-1) via orogastric tube. A single dose of high-titer (10(6) fluorescent focus-forming units) rotavirus was used, whereas E. coli was administered in various doses (10(2) to 10(9) CFU) to determine the titer of E. coli that induced only mild diarrhea but, when combined with rotavirus, resulted in diarrheal disease. Doses of E. coli > 10(6) CFU resulted in infection in almost all rabbits 10 to 16 weeks old, as detected by fecal shedding, regardless of whether rotavirus was inoculated simultaneously. However, inoculation of > 10(6) CFU of E. coli, in conjunction with rotavirus, resulted in increased morbidity and mortality due to diarrheal disease compared with E. coli alone. Inoculation of rabbits 28 to 38 weeks old with similar doses of rotavirus and E. coli caused infection but failed to induce diarrhea, indicating that older rabbits were more resistant to the pathogenic effects of these two agents. A synergistic effect between rotavirus and E. coli occurred, causing more severe diarrheal disease in weanling rabbits than that resulting from either pathogen alone.

Use of polymerase chain reaction for diagnosis of type D simian retrovirus infection in macaque blood.

We developed a simple and specific polymerase chain reaction (PCR) method for the detection of type D simian retrovirus (SRV) infection (SRV-1, SRV-2, and SRV-3) using whole blood samples from macaques. Each pair of primers for the three serotypes of SRV was highly specific for its respective envelope proviral DNA and was sensitive enough to easily detect about five copies of the SRV-2 proviral genome. The PCR products were confirmed by Southern blot hybridization with digoxigenin-labeled internal oligonucleotide probes. For diagnostic purposes the three sets of primers were mixed together. The molecular weight of the PCR product for each of the three serotypes differed. Serotypes were confirmed by hybridization with a mixture of SRV-2 and SRV-1 and -3 internal probes. The PCR analysis of 39 whole blood samples correctly identified five SRV-1 and nine SRV-2 culture-positive samples. It also detected SRV-2 in two culture-negative blood samples from monkeys from which SRV had been previously isolated.

Prevalence of coronavirus antibodies in rabbits.

Antibodies to coronavirus were detected by an indirect fluorescent antibody test in rabbit sera from six rabbitries. The prevalence ranged from 3 to 40% in different rabbitries and most seropositive rabbits were more than 4 months old. A rabbitry with high prevalence of antibodies and high incidence of diarrhea could serve as a source of virus and aid in studying the natural history of coronavirus infection in rabbits.

Viral persistence of simian type D retrovirus (SRV-2/W) in naturally infected pigtailed macaques (Macaca nemestrina).

To elucidate sites of SRV-2/W persistence, tissue DNA from three groups of naturally infected Macaca nemestrina was analyzed for provirus: vertically transmitted, viremic, seronegative macaques; horizontally transmitted, viremic, seronegative macaques, and nonviremic seropositive macaques. In viremic animals infected vertically, provirus was found in many tissues, whereas in those infected horizontally, proviral DNA was limited. In V-Ab+ macaques, provirus was detected in bone marrow and/or ileocecal junction, confirming the presence of provirus in V-Ab+ animals.

Immune function in offspring of nonhuman primates (Macaca nemestrina) exposed weekly to 1.8 g/kg ethanol during pregnancy: preliminary observations.

A preliminary investigation of immune host response was conducted in a group of fetal alcohol-exposed nonhuman primates (Macaca nemestrina) who were part of a broader ongoing study of ethanol teratogenicity. The mothers of the offspring received weekly oral doses of ethanol (1.8 g/kg) for the first 3 or 6 or the entire 24 weeks of gestation. A control group received sucrose solution weekly throughout pregnancy. Four of the 18 ethanol-exposed animals (22%) died or were euthanized after infectious disease or failure to thrive during the first year of life; none of the seven control animals died. This imbalance in survival prompted the present review of immune function in the remaining offspring. Parameters assessed included: (1) white blood cell count (WBC), (2) peripheral blood leucocyte subsets (CD4+, CD8+, CD20+, and CD11c+), (3) T-cell proliferation after activation with phytohemagglutinin (PHA), staphylococcus enterotoxin B (SEB), and tetanus toxoid (TT), (4) phagocytic activity of monocytes, and (5) serum immunoglobulin levels and serum antibody titers after TT vaccination. Mean T-cell proliferation to TT was significantly decreased (p = 0.01) in all ethanol-exposed animals relative to controls, with near-significant decreases (p = 0.06) in response to SEB in the ethanol-exposed animals. Lymphocyte proliferation in response to PHA was not altered. Ethanol-exposed animals had significantly lower TT titers than controls after initial vaccination and booster. WBC, leukocyte subsets, serum immunoglobulins, and monocyte phagocytic activity were not significantly different from control values. These preliminary observations suggest that T-cell proliferation and antigen-specific memory responses may be altered in offspring exposed to weekly doses of ethanol in utero and warrant further evaluation for confirmation.

Method for detection of simian immunodeficiency virus neutralizing antibodies using a noncommercial antigen capture enzyme-linked immunosorbent assay.

A neutralization test (NT) using a noncommercial antigen capture enzyme-linked immunosorbent assay (ELISA) to detect simian immunodeficiency virus (SIV) growth in vitro was developed. The capture antibody was a mixture of purified macaque anti-SIV immunoglobulin G (IgG) and a monoclonal antibody to SIV p27. Captured antigens were detected by using purified macaque anti-SIV IgG conjugated to horseradish peroxidase. The NT reliably and sensitively detected differences when various amounts of SIV were used with positive and negative control macaque sera. Dilutions of sequential sera from a macaque (Macaca nemestrina) that had been experimentally infected with SIV were tested for neutralizing antibody with 300 50% tissue culture infective doses of SIV. In this macaque, neutralizing activity and anti-SIV IgG levels in serum (detected by ELISA) increased with time after SIV inoculation, and high IgG titers were required in serum before neutralization occurred in vitro. This simple NT, which detects the presence of SIV serum neutralizing antibodies at a low cost, will be useful for investigating the role of neutralizing antibodies in the SIV-infected macaque model for AIDS.

Maternal-fetal transmission of SIV in macaques: disseminated adenovirus infection in an offspring with congenital SIV infection.

To develop a nonhuman primate model for maternal-fetal transmission of HIV infection, we have inoculated pregnant Macaca nemestrina with uncloned SIVMne. Three animals inoculated during the third trimester delivered healthy infants. One of the three infants, a male born 31 days after the mother was inoculated with SIV, became virus-positive but failed to produce SIV-specific antibody and died with overt simian immunodeficiency and disseminated adenovirus (SV20) infection at age six and one-half months. SIV and adenovirus antigen could be demonstrated by immunohistochemical methods in multiple organ systems.

A novel subgroup of exogenous avian leukosis virus in chickens.

An avian leukosis virus with a wide host range belonging to a new subgroup for chickens was isolated from meat-type chicken lines. The virus, of which HPRS-103 strain is the prototype, was of low oncogenicity in chickens but appeared to behave like an exogenous leukosis virus. Neutralizing antibodies to the virus were found in three of five meat-type chicken lines, but not in seven layer lines. The virus and its Rous sarcoma virus pseudotype did not replicate in, or transform, mammalian cells.

Maternal transmission of type D simian retrovirus (SRV-2) in pigtailed macaques.

Pregnant macaques were used as a natural model for maternal-infant transmission of SRV-2 retrovirus. Fifty-one pregnant females were placed into one of four virus/antibody groups. Nonviremic mothers produced 100% virus-negative offspring at birth. In contrast, viremic mothers produced offspring which were 17% virus-negative and 83% virus-positive at birth. SRV-2 infection occurred principally in utero by the transplacental route. Infants born to viremic mothers exhibited low birth weight, prematurity, high perinatal death, and increased incidence of SAIDS.

Inoculation of Macaca fascicularis with simian immunodeficiency virus, SIVmne immunologic, serologic, and pathologic changes.

Previous studies had tested the susceptibility of two macaque species, Macaca nemestrina and M. mulatta, to infection with the primate lymphotropic lentivirus SIVmne. In this report we describe the results obtained after infecting eleven M. fascicularis with SIVmne. Six of the animals had previously been immunized with a recombinant vaccinia virus expressing the envelope gene of HIV-1. All eleven animals became seropositive. To date ten animals have died 43 to 155 weeks post infection of an AIDS-like disease.

Transmission of the simian immunodeficiency virus SIVmne in macaques and baboons.

A primate lymphotropic lentivirus was isolated on Hut 78 cells after cocultivation of a lymph node from a macaque that died with malignant lymphoma. In earlier studies SIV/Mne was inoculated into 17 macaques and two baboons. All of the macaques became viremic and seropositive. Fifteen of the macaques succumbed to a classic AIDS-like disease, whereas the baboons did not become viremic. The SIV/Mne virus has now been molecularly cloned and inoculated into Macaca nemestrina and baboons. A new transmission study has been initiated to test the effects of route and dosage on disease.

Pathogenicity of rotavirus in rabbits.

The role of rotavirus in diarrheal disease of rabbits was investigated, and a model for human rotavirus infection was established. Orogastric inoculation of 8- and 12-week-old New Zealand White rabbits with a rabbit strain of rotavirus (L:ALA:84) resulted in fecal shedding of virus for 6 to 8 days from 2 to 5 days after inoculation. Most rabbits exhibited diarrhea, coincident with the onset of viral shedding, which persisted for 2 to 4 days. Diarrhea was characterized by soft or fluid stools and fecal staining of the perineum. Inoculation of 3-week-old rabbits resulted in a briefer period of viral shedding and diarrhea of a milder nature. Histopathologic examination during the period of viral shedding revealed a mild, nonsuppurative enteritis. Inoculated rabbits exhibited antibodies in serum to rotavirus by enzyme-linked immunosorbent assay. Sham-inoculated or uninoculated rabbits maintained in the same cage or the same room with inoculated rabbits acquired rotavirus infection. The mild diarrheal disease which resulted with a rotavirus isolate from severe field cases suggests that cofactors were involved.

Antigenic and biological diversity of feline coronaviruses: feline infectious peritonitis and feline enteritis virus.

Antigenically related feline coronaviruses cause two distinct disease manifestations in infected cats. The diseases are feline infectious peritonitis (FIP), in which the virus is widely disseminated, and feline enteric coronavirus (FECV), a mild disease in which the virus is usually limited to the villi. These two viruses were found to differ in their growth in cell culture. FIPV grows to higher titer, forms larger plaques and switches off host cell protein synthesis more effectively than FECV. Cross neutralization studies showed antigenic differences between the strains. There also appeared to be a difference in the nucleoprotein molecular weight of the viruses causing these two different disease syndromes.

Rotavirus-associated diarrhea in a commercial rabbitry.

An epizootic of diarrheal disease occurred in a commercial specific-pathogen-free rabbitry, and was characterized by sudden onset, rapid spread, and high morbidity and mortality among sucklings. Affected rabbits rapidly became dehydrated and most died within two days of the onset of diarrhea. Eight of these rabbits were necropsied. Five had blunted and fused small intestinal villi with attenuated villous enterocytes. A rotavirus was isolated from four rabbits, and five survivors of affected litters had strong antibody responses to rotavirus.