Expression of the simian varicella virus glycoprotein E.

Simian varicella virus (SVV) is closely related to human varicella-zoster virus (VZV) and induces a varicella-like disease in nonhuman primates. The SVV genome encodes a glycoprotein E (gE) which is homologous to the gE of VZV and other alphaherpesviruses. The SVV gE was expressed in Escherichia coli and rabbits were immunized with the recombinant gE fusion proteins to generate polyclonal gE antiserum. Immunofluorescence and immunoprecipitation analyses demonstrated that the SVV gE is expressed on the surface and within SVV-infected cells. The gE is also expressed on SVV virions as indicated by serum neutralization assay. The mature SVV gE is glycosylated and is similar in size ( approximately 100 kd) to the mature VZV gE. Immunohistochemical analysis detected gE within skin vesicles and lung tissue of SVV-infected monkeys. Analysis of the humoral immune response to gE in an SVV-infected monkey determined that anti-gE antibody is induced as early as day 9 postinfection and persists at high titer for longer than 4 months. The simian varicella model offers an opportunity to investigate the role of gE in viral pathogenesis and immunity and to evaluate its potential as a varicella vaccine.

Outbreak of Orthoreovirus-induced meningoencephalomyelitis in baboons.

BACKGROUND AND PURPOSE: Spontaneous viral encephalitis is rare in the baboon; yet, during a 13-month period (1993-1994), eight juvenile baboons (Papio cynocephalus spp.) developed acute, progressive nonsuppurative meningoencephalomyelitis caused by an unknown agent. Clinical signs of disease included disorientation and truncal ataxia that rapidly progressed to hemiparesis or paraparesis. Clinicopathologic findings were not remarkable and appreciable gross lesions were not seen at necropsy. Microscopic examination revealed CNS lesions that were characterized by lymphoplasmacytic perivascular cuffing, microglial nodules, demyelination, axonal degeneration, vacuolization, and hemorrhage. Subsequently, a novel syncytium-inducing mammalian orthoreovirus was isolated from the brain tissue of five baboons with clinical signs of infection. METHODS: To confirm the etiologic role of the orthoreovirus, two juvenile baboons were inoculated with the virus, then were monitored for 6 weeks. RESULTS: Lesions similar to those seen in spontaneous cases were found in the CNS, and orthoreovirus was isolated from the brain of both animals. CONCLUSION: Analysis of the outbreak indicated juvenile baboons were most susceptible to disease and the virus had a possible incubation time of 46 to 66 days, but did not indicate a source of the virus or mode of transmission.

Genetically engineered Mengo virus vaccination of multiple captive wildlife species.

Encephalomyocarditis virus (EMCV), has caused the deaths of many species of animals in zoological parks and research institutions. The Audubon Park Zoo, (New Orleans, Louisiana, USA) attempted vaccination of several species with a killed EMCV vaccine with mixed results. This paper reports an attempt at vaccination against EMCV using a genetically engineered, live attenuated Mengo virus (vMC0) at the Audubon Park Zoo and Miami Metro Zoo, (Miami, Florida, USA) from December 1996 to June 1997. Several species of animals were vaccinated with vMC0, which is serologically indistinguishable from the field strain of EMCV. Serum samples were taken at the time of vaccination and again 21 days later, then submitted for serum neutralization titers against EMCV. The vaccinate species included red capped mangebey (Cercocebus torquatus), colobus (Colobus guereza), angolan colobus (Colobus angolensis), ruffed lemur (Lemur variegatus ruber and Lemur variegatus variegatus), back lemur (Lemur macaco), ring-tailed lemur (Lemur catta), siamang (Hylobates syndactylus), diana guenon (Cercopithicus diana), spider monkey (Ateles geoffroyi), common marmoset (Callithrix jacchus), talapoin monkey (Cercopithecus talapoin), Brazilian tapir (Tapirus terrestris), Baird's tapir (Tapirus bairdii), Malayan tapir (Tapirus indicus), dromedary camel (Camelus dromedarius), bactrian camel (Camelus bactrianus), gerenuk (Litocranius walleri), guanaco (Lama glama guanicoe), black duiker (Cephalophus niger), Vietnamese potbellied pig (Sus scrofa), babirusa (Babyrousa babyrussa), collard peccary (Tayass tajacu), and African crested porcupine (Hystrix africaeaustralis). The vaccine response was variable, with high virus neutralizing antibody titer responses in some primate species and mixed to poor responses for other species. No ill effects were seen with vaccination.

Rapid diagnosis of simian varicella using the polymerase chain reaction.

Simian varicella virus (SVV) causes sporadic epizootics of a varicella-like disease in nonhuman primates. Rapid diagnosis of simian varicella is critical in controlling epizootics. A polymerase chain reaction (PCR)-based diagnostic assay for detection of SVV DNA in cell culture and clinical samples from SVV-infected monkeys was developed. The assay is rapid, specific, and highly sensitive. The SVV DNA is readily detected in skin rash specimens and in peripheral blood lymphocytes of infected monkeys during the early stages of clinical varicella. In addition to providing an important diagnostic tool, the SVV PCR assay is also useful for investigating the epidemiology and pathogenesis of simian varicella.

Experimental simian varicella virus infection of St. Kitts vervet monkeys.

Experimental simian varicella virus (SVV) infection of St. Kitts vervet monkeys was evaluated as an animal model to investigate human varicella-zoster virus (VZV) infections. During the incubation period, viremia disseminated infectious virus throughout the body via infected peripheral blood lymphocytes (PBLs). A vesicular skin rash in the inguinal area, and on the abdomen, extremities, and face appeared on day 7-10 postinfection. Necrosis and hemorrhage in lung and liver tissues from acutely infected monkeys were evident upon histologic analysis. Recovery from simian varicella was accompanied by a rise in the serum neutralizing antibody response to the virus. SVV latency was established in trigeminal ganglia of monkeys which resolved the acute infection. This study indicates that experimental SVV infection of St. Kitts vervets is a useful animal model to investigate SVV and VZV pathogenesis and to evaluate potential antiviral agents and vaccines.

Intranasal Sendai virus vaccine protects African green monkeys from infection with human parainfluenza virus-type one.

Human parainfluenza virus-type I (hPIV-1) infections are a common cause of "group" and hospitalizations among young children. Here we address the possibility of using the xenotropic Sendai virus [a mouse parainfluenza virus (PIV)] as a vaccine for hPIV-1. Sendai virus was administered to six African green monkeys (Cercopithecus aethiops) by the intranasal (i.n.) route. A long lasting virus-specific antibody response was elicited, both in the serum and nasal cavity. Sendai virus caused no apparent clinical symptoms in the primates, but live virus was detected in the nasal cavity for several days after inoculation. No virus was detected after a second dose of Sendai virus was administered on day 126 after the initial priming. Animals were challenged with hPIV-1 i.n. on day 154. All six vaccinated animals were fully protected from infection while six of six control animals were infected with hPIV-1. The antibody responses induced by Sendai virus immunizations proved to be greater than those induced by hPIV-1. These results demonstrate that unmanipulated Sendai virus is an effective vaccine against hPIV-1 in a primate model and may constitute a practical vaccine for human use.

Further observations on coronavirus infection of primate CNS.

Previously, we demonstrated that intracerebral (IC) inoculation of a murine coronavirus, MHV-JHM, into two species of primates can result in acute encephalomyelitis (Murray et al., 1992a). Infectious virus isolated from acutely infected animals, designated JHM-OMp1, was inoculated IC into a second group of monkeys. In this report we describe observations on the acutely infected animals and those surviving the acute infection were sacrificed at later times post-infection. Results from dual in situ hybridization/immunohistochemistry screening of tissues show that astrocytes are target cells in white matter lesions during acute infection. In animals sacrificed 150 days post-infection, areas of demyelinated gliotic lesions, prominent in the spinal cord, were seen throughout the neuraxis. No virus products were detected in these late-infection lesions.

Temporal association of interferon-alpha and p27 core antigen levels in sera of simian immunodeficiency virus infected monkeys.

We report the temporal association of interferon (IFN) and p27 core antigen production during experimental simian immunodeficiency virus Delta B670 (SIV) infection in rhesus monkeys. Peak serum IFN-alpha levels (10(2.8-5.0)U/ml) occurred 10 days post infection (p.i.) and peak p27 levels (3.1-34.4 ng/ml) occurred 10-14 days p.i. Acid-stable IFN-alpha (10(1.6-2.5)U/ml) was detected 3-5 days before p27 in sera from three monkeys and was detected with p27 (0.06-3.06 ng/ml) in four monkeys during the primary infection. Serum IFN-alpha and p27 levels became undetectable 24-40 days p.i. Two monkeys remained asymptomatic for SIV after the primary p27 antigenaemia, three monkeys had recrudescent (3-4 months p.i.) acid stable interferonaemias (10(1-2.5)U/ml) with p27 antigenaemias (0.06-2.7 ng/ml) that persisted until death, and two monkeys had acute SIV infections (died < or = 7 months p.i.) with persistent acid-stable interferonaemia (10(1.6-2.5)U/ml) and p27 antigenaemia (6-9 ng/ml). Our results indicate that the detection of acid-stable IFN-alpha in serum is closely associated with detection of p27 (P = 0.0001) and suggest that detection of acid-stable IFN-alpha and p27 core antigen is indicative of active SIV infection.

Protection of non-murine mammals against encephalomyocarditis virus using a genetically engineered Mengo virus.

Genetically engineered Mengo viruses with artificial deletions in the 5' noncoding poly(C) tracts are highly attenuated for pathogenicity when introduced as live vaccines into the natural murine host. Inoculation produces lifelong protective immunity without disease or viral persistence. This report extends the vaccination studies to non-murine hosts, including baboons, macaques and domestic pigs, all of which are susceptible to severe cardiovirus epizootics. All animals of these species that were inoculated with vMC24, an engineered strain of Mengo, seroconverted. When the immunized animals were challenged, they were protected against lethal doses of encephalomyocarditis virus (EMCV) derived from currently circulating epizootic strains. In baboons, the neutralizing antibody titers induced by vMC24 were significantly higher than from an inactivated EMCV vaccine. Moreover, terminal histopathology on baboons (inoculated intramuscularly), macaques (inoculated intracerebrally), and pigs (inoculated intramuscularly) showed few, if any, gross lesions characteristic of EMCV-like disease, in the vMC24 vaccinates. We suggest that genetically engineered, short poly(C) Mengo viruses may be universally potent attenuated vaccines for many types of animals and can possibly provide safe, efficacious protection against all cardioviruses of the EMCV serotype.

Simian varicella virus antibody response in experimental infection of African green monkeys.

The humoral immune response to simian varicella virus (SVV) was investigated following primary and secondary experimental infection of African green monkeys. Neutralization and immunoprecipitation assays were used to determine antibody titers to SVV throughout the course of infection. The immune response to specific viral polypeptides was analyzed by immunoprecipitation analysis. The results demonstrate that the simian varicella model offers a useful approach to investigate immune mechanisms in human varicella zoster virus (VZV) infections.

Oral bioavailability of the antiretroviral agent 9-(2-phosphonylmethoxyethyl)adenine (PMEA) from three formulations of the prodrug bis(pivaloyloxymethyl)-PMEA in fasted male cynomolgus monkeys.

The bioavailability of PMEA from three oral formulations of the prodrug bis(POM)-PMEA has been evaluated in fasted male cynomolgus monkeys. The formulations examined included a hydroxypropyl-beta-cyclodextrin (HPBCD) complex, a PEG based cosolvent solution, and an aqeous suspension. Oral formulations containing 3H-bis(POM)-PMEA were compared to intravenous 3H-PMEA at 10.9 mg-eq/kg in a crossover study in four monkeys, with a 7 day washout period. No intact bis(POM)-PMEA or monoester were detected in plasma. Bioavailabilities of PMEA from the prodrug were 24.7 +/- 6.5%, 27.3 +/- 12.3% and 22.2 +/- 15.6% for the HPBCD complex, PEG solution and aqueous suspension, respectively. The oral bioavailability of PMEA from bis(POM)-PMEA was not limited by dissolution rate of the prodrug. Data for the PEG cosolvent solution and suspension indicate that the prodrug could potentially be formulated as a soft gelatin capsule or a tablet.

Effects of U-75875, a peptidomimetic inhibitor of retroviral proteases, on simian immunodeficiency virus infection in rhesus monkeys.

U-75875 inhibits human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus (SIV) proteases and blocks Gag-Pol protein processing and viral maturation and replication in vitro. Rhesus monkeys were treated with vehicle alone or with formulated U-75875 at doses of 7 or 20 mg/kg of body weight per day for 26 days by continuous intravenous infusion beginning 6 h prior to intravenous inoculation with 10 monkey 50% infectious doses of SIV Delta B670, and the monkeys were monitored until death. The effects of treatment on the level of SIV p26 antigenemia, the infectious virus titer in serum, and the level of proviral DNA in blood mononuclear cells evaluated by PCR were assessed. SIV infection of the controls resulted in an initial viral antigenemia that began 5 to 10 days postinoculation (p.i.), reached peak values on days 10 to 14 p.i., and lasted for more than 15 days. Proviral DNA was detectable in peripheral blood mononuclear cells by 7 to 11 days p.i., reached the mean peak level by 11 days p.i., and remained at high levels through day 24 p.i. Infectious virus was detected in serum from all of the infected controls by 24 days p.i. Treatment with U-75875 for 26 days resulted in a dose-related delay in the day of the peak level of antigenemia (P = 0.034). The level of proviral DNA in peripheral blood mononuclear cells at 11 days p.i. was significantly decreased in a dose-related fashion in the treated monkeys ( P

Entry of coronavirus into primate CNS following peripheral infection.

A previous report demonstrated that intracerebrally inoculated coronavirus produced CNS disease in two species of primates (Murray RS, Cai G-Y, Hoel K, et al., Virol 1992; 188: 274-84). We were therefore interested in testing the potential of coronaviruses to infect primate CNS tissue following peripheral inoculation. Four Owl monkeys (Aotus trivirgatus) were inoculated intranasally and ocularly and four were inoculated intravenously with coronavirus JHM OMp1 (Murray RS, Cai G-Y, Hoel K, et al., Virol 1992; 188: 274-84). Two intranasally and two intravenously inoculated animals received a second intravenous inoculum at 153 days post-infection. The animals were sacrificed 16, 35, 194, and 215 days post-infection. Tissue sections from brain and spinal cord were screened for viral products by in sity hybridization and immunostaining. Virus RNA and/or antigen was detected in the brains of all animals and the distribution corresponded to areas of inflammation and edema. Viral products were predominantly found in blood vessels and perivascular regions, suggesting hematogenous spread with entry into the central nervous system through endothelium.

Pharmacokinetics and antiviral activity of a novel isonucleoside, BMS-181165, against simian varicella virus infection in African green monkeys.

A novel nucleoside analog BMS-181165 with potent activity against varicella-zoster virus was tested for efficacy in a simian varicella virus infection in African green monkeys. BMS-181165 was effective in preventing the development of a rash, decreasing the development of viremia and preventing death in infected monkeys when administered orally at 4, 16 or 64 mg/kg/day. The compound is well orally absorbed in monkeys, between 44 to 50% oral bioavailability, and may prove of value in therapy of varicella-zoster infections in humans.

Effects of initiation of 3'-azido,3'-deoxythymidine (zidovudine) treatment at different times after infection of rhesus monkeys with simian immunodeficiency virus.

The effects of initiating treatment with 3'-azido-3'-deoxythymidine (zidovudine) at different times after inoculation of simian immunodeficiency virus (SIV) were investigated in rhesus monkeys. Zidovudine treatments of 100 mg/kg/day (25 mg/kg, subcutaneously every 6 h) were initiated 1, 8, 24, or 72 h after intravenous inoculation of 10 ID50 of SIV. Treatments continued for 28 days, and results were compared with those of saline-treated controls. Serum infectious virus titers 14 days after inoculation (AI) significantly decreased after treatment initiation 1, 8, or 24 h AI. Titers were correlated with the time treatment was initiated. Treatments initiated 1-72 h AI prevented the establishment of persistent SIV antigenemia; greater effects were observed with earlier initiation of treatment. Treatments initiated 1-8 h AI resulted in decreased levels of viral antigenemia 14 days AI and delayed decreases in CD4+CD29+ blood lymphocytes. Earlier treatment initiation resulted in delayed recurrence of antigenemia, with a tendency for longer survival. Early initiation of treatment may be important for limiting initial viral replication and dissemination in cases of known exposure.

Perfluorooctylbromide (PFOB) as a vitreous substitute in non-human primates.

We evaluated the toxicity of perfluorooctylbromide in the primate eye as a short-term postoperative vitreous substitute. Four eyes of 4 African green monkeys underwent complete vitrectomy and vitreous replacement with 1.5-2.0 ml of PFOB. One additional animal received BSS as a control vitreous substitute in one eye. Animals were examined twice weekly for clarity and consistency of the vitreous replacement substance. Anterior segment and lenses remained clear in all eyes, although in the immediate postoperative period one eye became inflamed and had a culture-negative vitritis. The other eyes showed a minimal anticipated postoperative vitreous inflammation. Emulsification of the PFOB began within 3 days of injection and progressed up to 3 weeks, precluding fundus examination and fluorescein angiography after 2 weeks. Eyes were enucleated and light microscopy performed at 2 days, 10 days, 33 days, and 45 days. No toxic effects to the retinal cells were detectable by histological examination, but perivasculitis of retinal vessels was noted at 45 days. Indirect examination was normal up to 10 days; thereafter, the fundus view was obscured by the emulsified PFOB. Because of cellular migration into the vitreous cavity and retinal perivasculitis, observed histologically, PFOB seems most suitable for intraoperative rather than postoperative use.

Efficacy of (-)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine in African green monkeys infected with simian varicella virus.

(-)-9-[4-Hydroxy-2-(hydroxymethyl)butyl]guanine was evaluated for its efficacy in African green monkeys infected with simian varicella virus. Treatment by intramuscular injection was initiated 48 h after virus inoculation and was continued for 10 days; the treatment showed therapeutic effects on rash and viremia at dosages down to 1 mg/kg of body weight per day.

Pharmacokinetics and antiviral activity of recombinant human interferon-beta ser17 in African green monkeys.

The pharmacokinetics and antiviral activity of recombinant human interferon-beta ser17 (Betaseron) were evaluated in African green monkeys. In one study, animals infected with simian varicella virus were administered Betaseron intravenously (i.v.), intramuscularly (i.m.), or subcutaneously (s.c.) at doses of 1 x 10(6) or 1 x 10(7) IU/kg twice daily for 10 days. In another study, infected animals received Betaseron s.c. at doses of 1 x 10(6) IU/kg twice daily, 2 x 10(6) IU/kg once daily, 4 x 10(6) IU/kg every other day, or 6 x 10(6) IU/kg every 3 days for 10 days. Following i.v. administration, mean clearance, steady-state volume of distribution, and terminal half-life values for Betaseron were 0.36 +/- 0.08 liters/hr.kg, 0.65 +/- 0.09 liters/kg, and 1.9 +/- 0.43 h, respectively. Although bioavailability following i.m. and s.c. administration was only 30-50%, antiviral activity, as measured by reduction in viremia and appearance of skin rash, was comparable for i.v., i.m., and s.c. administration of 1 x 10(6) IU/kg of Betaseron twice daily. With increasing dose (1 x 10(6) IU/kg to 1 x 10(7) IU/kg), both the area under the serum concentration-time curve (AUC) and antiviral activity of Betaseron tended to increase. When comparing various s.c. dosing regimens, there was significant accumulation of Betaseron in serum with repeated twice-daily dosing. However, no accumulation of Betaseron in serum was observed if the dosing interval was less frequent than once daily. Antiviral activity was greatest with twice-daily or once-daily s.c. administrations of Betaseron.

6-Dimethylamino-9-(beta-D-arabinofuranosyl)-9H-purine: pharmacokinetics and antiviral activity in simian varicella virus-infected monkeys.

6-Dimethylamino-9-(beta-D-arabinofuranosyl)-9H-purine (ara-DMAP) effectively prevented the development of rash and appreciably reduced viremia in simian varicella virus-infected monkeys. Doses of 100 and 50 mg/kg/day, administered orally, were highly effective. The lowest dose of 20 mg/kg/day was much less effective in preventing moderate viremia. However, the 20 mg/kg/day did prevent the development of rash in two of three monkeys. All three doses of ara-DMAP reduced liver infection as reflected by lower aspartate aminotransferase values in the sera of the African green monkeys. Orally administered ara-DMAP was rapidly absorbed. However, significant variation among individual monkeys in the AUC values, peak plasma levels, and plasma half-lives were observed.

Coronavirus JHM OMP1 pathogenesis in owl monkey CNS and coronavirus infection of owl monkey CNS via peripheral routes.

Two separate studies are described in this report. First, 5 Owl monkeys were inoculated intracerebrally (IC) with coronavirus JHM OMP1; this virus isolate was cultured from the brain of an animal inoculated with uncloned MHV JHM. Two of the animals became neurological impaired and were sacrificed; these animals had developed severe encephalomyelitis as previously described. Two of the remaining 3 healthy animals were inoculated IC again at 90 days post-inoculation (DPI) and all 3 were sacrificed approximately 5 months after the first virus inoculation. Despite the lack of detectable infectious virus, viral RNA and antigen, all 3 animals had significant white matter inflammation and areas of demyelination in the spinal cord. In the second study 4 Owl monkeys were inoculated intranasally (IN) and ocularly and 4 inoculated intravenously (i.v.) with JHM OMP1. The animals were sacrificed between 16 and 215 DPI with 2 IN and 2 i.v. animals receiving a second i.v. inoculum at 152 DPI. Viral RNA and/or antigen was detected in the brains of all animals and the distribution corresponded to areas of inflammation and edema. One of the animals that received the second inoculum developed neurological impairment and subsequent analysis of tissues showed viral antigen in both brain and spinal cord. Viral products were predominantly found in blood vessels suggesting hematogenous spread with entry into the central nervous system (CNS) through endothelium.