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C57BL/6J (B6) mice are commonly affected by ulcerative dermatitis (UD), a disease of unknown etiology with poor response to treatment. To study the possible role of diet in UD, we compared skin changes in B6 female mice fed a high-fat diet with those of mice fed a control diet. In addition, skin samples from mice with no, mild, moderate, and severe clinical signs of UD were examined by light and transmission electron microscopy (TEM). Mice fed a high-fat diet for 2 mo had more skin mast cell degranulation than did mice fed the control diet for the same period. Regardless of diet, older mice had more skin mast cells and more of these cells were degranulating as compared with younger mice. Microscopic changes in very early lesions were characterized by an increase in dermal mast cells and degranulation with focal areas of epidermal hyperplasia with or without hyperkeratosis. As the condition progressed, a mixed but predominantly neutrophilic inflammatory cell infiltrate appeared in the dermis, with or without epidermal erosion and scab formation. TEM showed that dermal mast cell membranes had disrupted and released of large number of electron-dense granules, whereas degranulated mast cells were filled with isolated and coalescing empty spaces due to fusion of granule membranes. Ulceration appeared to occur very quickly, probably as result of intense scratching due to the pruritogenic properties of the histamine released from mast cell granules. This study showed a direct correlation between dietary fat and skin mast cell degranulation in female B6 mice. In addition, the number of skin mast cells and degranulation rates was higher in older mice. Treatments directed at preventing mast cell degranulation may result in better outcomes when applied early in UD cases. As noted previously in studies using caloric restriction, lower fat content in rodent diets may help prevent UD.

BACKGROUND: Owl monkeys are commonly used in biomedical research which is affected by the high incidence of cardiomyopathy in this species. Occasionally, owl monkeys with no clinical signs of heart disease are found dead and at necropsy show no, or very mild, cardiomyopathy. A possible explanation for sudden death is acute myocardial infarction, however, early myocardial changes may be difficult to assess by conventional stains and light microscopy. METHODS: Complement component C9 immunohistochemistry was performed in paraffin-embedded heart tissue samples from owl monkeys who died suddenly, or were euthanized due to sickness, to determine if these animals suffered from acute myocardial infarcts. RESULTS AND CONCLUSION: C9 deposits were found in the myocardium of 19 out of 20 (95%) animals. The findings in this study suggest owl monkeys suffer from acute myocardial infarcts and complement component C9 immunohistochemistry may be a useful diagnostic tool.

An increased incidence of dilated cardiomyopathy and atrial thrombosis was noted in a breeding colony of BALB/c mice deficient in IL4 receptor α. The condition affected mice of both sexes and of various ages, and extensive testing (microbiology, serology, histopathology) failed to ascertain the cause. Transmission electron microscopy of heart samples showed structural defects in the myocardial intercalated disks, characterized by unorganized and heavily convoluted arrangement with lower density and less prominent desmosomes and adherens junctions, widening of the intercellular space, myofibrillar lysis adjacent to intercalated disks, occasional sarcomere lysis with marked myofiber degeneration, vacuolation, accumulation of cell debris, and myelin figures. The intercalated disk contains cell adhesion molecules that form cell junctions, allowing contraction coupling of cardiomyocytes and the electrical and mechanical connection between cardiac fibers. Thus, defects at this level result in poor myocardial contraction, intracardiac blood stagnation, and consequently cardiac dilation with clinical signs of heart failure. The background strain or, potentially, the Cre–loxP-mediated recombination system used to create these mice may have contributed to the elevated incidence of cardiomyopathy and atrial thrombosis in this colony. Due to the backcrossing breeding scheme used, we cannot discount the emergence and colonywide dissemination of a spontaneous mutation that affects the intercalated disk. This report underscores the importance of carefully monitoring genetically modified mice colonies for unexpected phenotypes that may result from spontaneous or unintended mutations or enhanced strain background pathology.

Eosinophilic aortitis is a rare condition in animals and humans, and it has been occasionally reported associated with parasitic migration and with a poorly understood complex group of autoimmune vasculitides. Here, we describe a case of eosinophilic aortitis with thoracic aortic aneurysm and rupture in a captive-born owl monkey and discuss the differential diagnoses.

Background Klebsiella pneumoniae can be a serious pathogen in non-human primates, particularly Neotropical monkeys. Methods During a K. pneumoniae outbreak in an owl monkey research colony, 13 K. pneumoniae isolates were DNA fingerprinted by automated repetitive extragenic palindromic-polymerase chain reaction and the profiles compared to isolates obtained from other non-human primate species during the same time period and isolates from previous outbreaks. Results Eleven different types of K. pneumoniae were circulating in the owl monkey colony at the time of the outbreak. When comparing owl monkey isolates relatedness to previous colony outbreak isolates and squirrel monkey and capuchin monkey isolates, all were different. Conclusions These results agree with recent reports where K. pneumoniae nosocomial isolates in hospital settings can have high genetic diversity, and multiple strains can be circulating simultaneously. This potential genetic diversity should be considered when designing strategies for controlling K. pneumoniae outbreaks in captive non-human primate colonies.

Background An adult male owl monkey (Aotus nancymae) underwent a splenectomy. When the spleen was removed, a small, nodular mass slightly bulging over the splenic surface was noted. Methods The mass was examined by light and transmission electron microscopy and by immunohistochemistry. Results On light microscopy, the mass was well-circumscribed, non-encapsulated, and composed of haphazardly arranged smooth muscle bundles admixed with numerous small capillary-like structures containing blood. Immunohistochemical (IHC) staining revealed the tumor was strongly positive for smooth muscle actin yielding vascular smooth muscle bundles, and for Factor VIII, staining endothelial cells within the smooth muscle bundles. Transmission electron microscopy (TEM) showed a large portion of the cells to be atypical appearing smooth muscle and a few cells had structures resembling Weibel–Palade bodies indicating endothelial cells. Conclusions Based on cell morphology, by light and TEM, and IHC a final diagnosis of splenic angioleiomyoma was made. This is, to our knowledge, the first report of an angioleiomyoma in a non-human primate.

Recent reports from Japan implicated wild Sika deer (Cervus nippon) in the zoonotic transmission of hepatitis E to humans. Seroprevalence studies were performed to determine if imported feral populations of Sika deer in Maryland and Virginia posed a similar risk of transmitting hepatitis E virus (HEV). Hunters collected blood on filter paper discs from freshly killed deer. The discs were desiccated and delivered to a collection point. The dried filters were weighed to estimate the amount of blood absorbed and were eluted and collected in one tube via a novel extraction system. The procedure was quantified and validated with negative and positive serum and blood samples obtained from domestic Sika deer before and after immunization with HEV recombinant capsid protein, respectively. None of the 155 tested samples contained antibody to HEV, suggesting that Sika deer in these populations, unlike those in Japan, do not pose a significant zoonotic threat for hepatitis E. However, the new method developed for collecting and eluting the samples should prove useful for field studies of many other pathogens.

Summary Background The outbreak of severe acute respiratory syndrome (SARS) in 2002 was caused by a previously unknown coronavirus—SARS coronavirus (SARS-CoV). We have developed an experimental SARS vaccine for direct immunisation of the respiratory tract, the major site of SARS-coronavirus transmission and disease. Methods We expressed the complete SARS coronavirus envelope spike (S) protein from a recombinant attenuated parainfluenza virus (BHPIV3) that is being developed as a live attenuated, intranasal paediatric vaccine against human parainfluenza virus type 3 (HPIV3). We immunised eight African green monkeys, four with a single dose of BHPIV3/SARS-S and four with a control, BHPIV3/Ctrl, administered via the respiratory tract. A SARS-coronavirus challenge was given to all monkeys 28 days after immunisation. Findings Immunisation of animals with BHPIV3/SARS-S induced the production of SARS-coronavirus-neutralising serum antibodies, indicating that a systemic immune response resulted from mucosal immunisation. After challenge with SARS coronavirus, all monkeys in the control group shed SARS coronavirus, with shedding lasting 5–8 days. No viral shedding occurred in the group immunised with BHPIV3/SARS-S. Interpretation A vectored mucosal vaccine expressing the SARS-coronavirus S protein alone may be highly effective in a single-dose format for the prevention of SARS.

Antigenic chimeric viruses in which the structural genes of dengue virus type 4 (DEN4) have been replaced with those derived from dengue virus type 2 (DEN2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. Specifically, the capsid, membrane precursor, and envelope (CME) or the membrane precursor and envelope (ME) gene regions of DEN2 were substituted for the corresponding genes of wild-type rDEN4 or vaccine candidate rDEN4delta30 which contains a 30 nucleotide deletion in the 3' untranslated region. The two DEN2/4 chimeric viruses lacking the delta 30 mutation were highly attenuated in tumor-bearing SCID-HuH-7 mice, mosquitoes, and rhesus monkeys, indicating chimerization with either the CME or ME regions lead to attenuation. In mosquitoes and SCID-HuH-7 mice, addition of the delta 30 mutation to the chimeric viruses resulted in comparable or only slightly increased levels of attenuation. In rhesus monkeys, addition of the delta 30 mutation rendered the CME chimeric virus non-infectious, indicating that the attenuation resulting from chimerization and the delta 30 mutation were additive for these animals. In contrast, the attenuation in rhesus monkeys of ME chimeric virus was not significantly modified by the addition of the delta 30 mutation. The satisfactory level of attenuation and immunogenicity achieved by the ME containing DEN2/4delta 30 chimeric virus, as well as its very low infectivity for mosquitoes, make it a vaccine candidate suitable for evaluation in phase I clinical trials.

The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100- to 1,000-fold restricted in replication in the respiratory tracts of nonhuman primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children, yet it is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild-type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and that the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA, and the levels of viral replication in vitro and in the respiratory tract of rhesus monkeys were determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tracts of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host range restriction of replication of BPIV3 for primates are polygenic, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher levels of HPIV3 hemagglutination-inhibiting serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild-type HPIV3. Furthermore, host range determinants could be combined with attenuating point mutations to achieve an increased level of attenuation. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccine candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3.

Human parainfluenza virus type 1 (HPIV1), a major cause of croup in infants and young children, accounts for 6% of hospitalizations for pediatric respiratory tract disease. The antigenically related Sendai virus, referred to here as murine PIV1 (MPIV1), is being considered for use as a live-attenuated vaccine to protect against HPIV1 (J. L. Hurwitz, K. F. Soike, M. Y., Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, 1997, Vaccine 15(5), 533–540) and also as a recombinant vaccine vector expressing antigens to protect against viral disease in humans. However, in the 1950s MPIV1 was reported to have been isolated from humans, suggesting that zoonotic transmission might have occurred. It is therefore important to examine the ability of MPIV1 to replicate in nonhuman primates, i.e., surrogate hosts for humans. In the present study the level of replication of MPIV1 and HPIV1 was compared in African green monkeys and chimpanzees. Surprisingly, MPIV1 replicated as efficiently as HPIV1 in the upper and lower respiratory tract of African green monkeys at doses of 104 and 106 and replicated only slightly less efficiently at both sites in chimpanzees. African green monkeys immunized with MPIV1 were highly resistant to subsequent challenge with HPIV1 even though MPIV1 did not induce a detectable HPIV1-neutralizing antibody response. The high level of replication of MPIV1 observed in the upper and lower respiratory tract of these primates suggests that MPIV1 likely would require significant attenuation before it could be given to humans as a vaccine against HPIV1 or as a vaccine vector. Its ability to efficiently replicate in nonhuman primates suggests that MPIV1 lacks a significant host range restriction in primates and could theoretically cause zoonotic disease in humans.

Mutant recombinant respiratory syncytial viruses (RSV) which cannot express the NS1 and M2-2 proteins, designated rA2ΔNS1 and rA2ΔM2-2, respectively, were evaluated as live-attenuated RSV vaccines. The rA2ΔNS1 virus contains a large deletion that should have the advantageous property of genetic stability during replication in vitro and in vivo. In vitro, rA2ΔNS1 replicated approximately 10-fold less well than wild-type recombinant RSV (rA2), while rA2ΔM2-2 had delayed growth kinetics but reached a final titer similar to that of rA2. Each virus was administered to the respiratory tracts of RSV-seronegative chimpanzees to assess replication, immunogenicity, and protective efficacy. The rA2ΔNS1 and rA2ΔM2-2 viruses were 2,200- to 55,000-fold restricted in replication in the upper and lower respiratory tracts but induced a level of RSV-neutralizing antibody in serum that was only slightly reduced compared to the level induced by wild-type RSV. The replication of wild-type RSV in immunized chimpanzees after challenge was reduced more than 10,000-fold at each site. Importantly, rA2ΔNS1 and rA2ΔM2-2 were 10-fold more restricted in replication in the upper respiratory tract than was the cpts 248/404 virus, a vaccine candidate that retained mild reactogenicity in the upper respiratory tracts of 1-month-old infants. Thus, either rA2ΔNS1 or rA2ΔM2-2 might be appropriately attenuated for this age group, which is the major target population for an RSV vaccine. In addition, these results show that neither NS1 nor M2-2 is essential for RSV replication in vivo, although each is important for efficient replication.

The shipping fever (SF) and Kansas (Ka) strains of bovine parainfluenza virus type 3 (BPIV3) are restricted in their replication in rhesus monkeys 100- to 1,000-fold compared to human parainfluenza virus type 3 (HPIV3), and the Ka strain also was shown to be attenuated in humans. To initiate an investigation of the genetic basis of the attenuation of BPIV3 in primates, we produced viable chimeric HPIV3 recombinants containing the nucleoprotein (N) open reading frame (ORF) from either BPIV3 Ka or SF in place of the HPIV3 N ORF. These chimeric recombinants were designated cKa-N and cSF-N, respectively. Remarkably, cKa-N and cSF-N grew to titers comparable to those of their HPIV3 and BPIV3 parents in LLC-MK2 monkey kidney and Madin-Darby bovine kidney cells. Thus, the heterologous nature of the N protein did not impede replication in vitro. However, cKa-N and cSF-N were each restricted in replication in rhesus monkeys to a similar extent as Ka and SF, respectively. This identified the BPIV3 N protein as a determinant of the host range restriction of BPIV3 in primates. These chimeras thus combine the antigenic determinants of HPIV3 with the host range restriction and attenuation phenotype of BPIV3. Despite their restricted replication in rhesus monkeys, the chimeric viruses induced a level of resistance to HPIV3 challenge in these animals which was indistinguishable from that conferred by immunization with HPIV3. The infectivity, attenuation, and immunogenicity of these BPIV3/HPIV3 chimeras suggest that the modified Jennerian approach described in the present report represents a novel method to design vaccines to protect against HPIV3-induced disease in humans.

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) appear to have originated by cross-species transmission of simian immunodeficiency virus (SIV) from asymptomatically infected African primates. Few of the SIVs characterized to date efficiently infect human primary lymphocytes. Interesting, two of the three identified to infect such cultures (SIVsm and SIVcpz) have appeared in human populations as genetically related HIVs. In the present study, we characterized a novel SIV isolate from an East African monkey of the Cercopithecus genus, the l’hoest monkey ( C. l’hoesti ), which we designated SIVlhoest. This SIV isolate efficiently infected both human and macaque lymphocytes and resulted in a persistent infection of macaques, characterized by high primary virus load and a progressive decline in circulating CD4 lymphocytes, consistent with progression to AIDS. Phylogenetic analyses showed that SIVlhoest is genetically distinct from other previously characterized primate lentiviruses but clusters in the same major lineage as SIV from mandrills (SIVmnd), a West African primate species. Given the geographic distance between the ranges of l’hoest monkeys and mandrills, this may indicate that SIVmnd arose through cross-species transmission from close relatives of l’hoest monkeys that are sympatric with mandrills. These observations lend support to the hypothesis that the primate lentiviruses originated and coevolved within monkeys of the Cercopithecus genus. Regarded in this light, lentivirus infections of primates not belonging to the Cercopithecus genus may have resulted from cross-species transmission in the not-too-distant past.

Decline in blood CD4+ lymphocytes during primary symptomatic infections with HIV is usually attributed to viral killing, and has not been considered in terms of altered lymphocyte migration and sequestration. We therefore sought to examine whether CD4+ cell loss from blood of macaques undergoing an acute primary SIV infection might be due to increased synthesis of cytokines, known to profoundly affect lymphocyte trafficking, rather than to direct lymphocyte destruction by virus. The findings indicate that rapid lymphocyte depletion following acute infection is not selective for CD4+ cells, correlates precisely with increased plasma IFN-gamma and tumor necrosis factor-alpha levels, and is reversible. CD4/CD8 ratios in lymph nodes with high viral burdens remain relatively unchanged despite lymphocyte loss from blood. Levels of cytokine mRNA measured in lymphoid organs reflect neither cytokine plasma levels nor their potential to induce sequestration. These results support a model of cytokine-induced lymphocyte extravasation to account for the acute HIV/SIV-induced CD4+ cell lymphopenia and raise questions regarding the extent to which altered lymphocyte migration plays a role in the gradual CD4+ cell depletion throughout infection.

Summary A 2-year-old, captive-born, male moustached tamarin was subjected to necropsy examination after a fatal head trauma. A solitary, circumscribed, subpleural mass (0.6 cm diameter) was found in the right caudal lung lobe. The mass was diagnosed as a mucinous cystadenoma. Histochemical and immunohistochemical tests were performed to further characterize the tumour. Surfactant proteins A, B, C and D were not found in the neoplastic cells, suggesting that the tumour arose from a non-surfactant-producing alveolar lining cell. Pulmonary mucinous cystadenomas are uncommon benign tumours in man and have not been reported previously in animals.

Optimal immunization of neonates against disease caused by respiratory syncytial virus (RSV) probably will require multiple doses of a vaccine containing viruses of both subgroups A and B. Live subgroup B RSV mutants were generated containing multiple attenuating mutations, ts (temperature-sensitive) and non-ts (host range), that were introduced by prolonged passage in cell culture or by chemical mutagenesis. The cold-passaged (cp)-52 mutant was restricted in replication compared to wild type virus in rodents and nonhuman primates. In addition, the attenuation phenotype of cp-52 was stable after prolonged replication in immunosuppressed rodents. One or two ts mutations were then introduced into the cp-52 mutant to generate additional candidate vaccine strains that were more attenuated in vivo than the cp-52 parental virus. Tests in humans are being done to determine if one or more of the RSV B-1 mutants exhibit a satisfactory balance between attenuation and immunogenicity.

A cold-passaged (cp) temperature-sensitive (ts) RSV mutant, designated RSV cpts-530, which possesses host-range mutations acquired during 52 passages at low temperature in bovine tissue culture and one or more ts mutations induced by chemical mutagenesis (shut-off temperature 39 degrees C) was found previously to be tenfold restricted in its replication in mice as compared to wild-type virus and stable genetically in nude mice. In the current study, we introduced additional attenuating mutations, such as small-plaque (sp) or ts mutations, into cpts-530 by chemical mutagenesis with 5-fluorouracil, with the intent of obtaining derivatives of cpts-530 that were more attenuated in mice or chimpanzees and that were more stable genetically following replication in vivo. Fourteen mutants of RSV cpts-530 which had acquired an additional ts mutation were identified and found to be more restricted in replication in BALB/c mice than the cpts-530 parental strain. One mutant, designated cpts-530/1009 (shut-off temperature 36 degrees C), was 30 times more restricted in replication in the nasal turbinates of mice and threefold more restricted in the nasopharynx of seronegative chimpanzees than its cpts-530 parent. Like its parent, this mutant was highly restricted (30,000-fold) in replication in the lower respiratory tract of chimpanzees even following direct intratracheal inoculation. The cpts-530 and cpts-530/1009 mutants exhibited a high level of stability of the ts phenotype during replication in chimpanzees.(ABSTRACT TRUNCATED AT 250 WORDS)

In the interval between the publication of the seventh and eighth editions of the Guide for the Care and Use of Laboratory Animals (Guide), much has changed with regard to the regulation and funding of highly pathogenic biologic agents and toxins (Select Agents). Funding of research involving highly pathogenic agents has increased dramatically during this time, thus increasing the demand for facilities capable of supporting this work. The eighth edition of the Guide briefly mentions Select Agents and provides a limited set of references. Here we provide some background information regarding the relevant laws and regulations, as well as an overview of the programmatic requirements pertaining to the use of Select Agents, with a focus on use in animals.

Simian immunodeficiency virus (SIV) exists within tissues of infected macaques as a mixture of diverse genotypes. The goal of this study was to investigate the biologic significance of this variation in terms of cellular tropism and pathogenicity. PCR was used to amplify and clone 3'-half genomes from the spleen of an immunodeficiency SIV-infected pig-tailed macaque (Macaca nemestrina). Eight infectious clones were generated by ligation of respective 3' clones into a related SIVsm 5' clone, and virus stocks were generated by transient transfection. Four of these viruses were infectious for macaque peripheral blood mononuclear cells (PBMC) or monocyte-derived macrophages (MDM). Three viruses with distinct tropism for macaque PBMC or MDM were tested for in vivo infectivity and pathogenicity. The ability of these three viruses to infect PBMC and macrophages correlated with differences in infectivity and pathogenicity. Thus, a virus that was infectious for both PBMC and MDM was highly infectious for macaques and induced AIDS in half of the inoculated animals. In contrast, virus that was less infectious for PBMC and not infectious for MDM induced only transient viremia. Finally, a virus that was not infectious for either primary cell type did not infect macaques. Chimeric clones exchanging portions of the envelope gene of the 62A and smH4 molecular clones and a series of point mutants were used to map the determinant of tropism to a 60-amino-acid region of gp120 encompassing the V3 analog of SIV. Naturally occurring mutations within this region were critical for determining tropism and, as a result, pathogenicity of these SIVsm clones.

Homologous SIVsm stocks were generated by passage of a macaque isolate of SIVsm (SIVsm/E660) in human CEM x 174 cells or macaque peripheral blood mononuclear cells. Macaques were immunized with whole inactivated SIV vaccine consisting of virus generated by transfection of CEM x 174 cells with the SIVsmH4 clone and were challenged with either cell-free stock. Only vaccinees challenged with virus generated in human cells were protected from infection. This confirms the species-specificity of whole inactivated vaccine-mediated protection.

Pulmonary thromboembolism associated with pancreatic endocrine neoplasia is extremely uncommon in man and animals. Post-mortem examination of an adult owl monkey (Aotus nancymae) revealed extensive pulmonary arterial thromboembolism and a well-demarcated mass attached to the pancreas. Microscopically, the mass consisted of areas of interstitial fibrosis with loss of acini and islets and replacement by nests and sheets of polygonal cells with amphophilic cytoplasm, an eccentric round nucleus with stippled chromatin and, in some cells, with a single prominent eccentric nucleolus. Clusters of these cells were noted within vessels and adjacent lymph nodes. The cells did not express S100 or insulin, but were labelled strongly with SP-1/chromogranin. Rare individual cells expressed glucagon and somatostatin. A few cells in pulmonary thrombi/emboli and the adjacent lymph node also expressed SP-1/chromogranin. Based on cell morphology, location and immunohistochemistry the tumour was classified as pancreatic endocrine (islet cell) carcinoma with metastasis to regional lymph nodes and lung.

Spontaneous intracardiac thrombosis is rarely reported in animals, particularly nonhuman primates. The finding of 2 cases of intracardiac thrombi in mustached tamarins (Saguinus mystax) that died as a consequence of congestive heart failure prompted us to do a retrospective study to determine the frequency of this condition. Clinical records, necropsy reports, and tissues from 60 mustached tamarins that died or were euthanized between 1996 and 2009 were reviewed. Of the 60 monkeys whose cases were reviewed, 10 (16.6%) had intracardiac thrombi, and 4 (6.6%) had dissecting aortic aneurysms. Of the 10 animals with intracardiac thrombosis, 3 had left ventricular involvement alone; 4 monkeys had thrombi only in the right ventricle, and the remaining 3 animals exhibited thrombi in both ventricles. Myocardial fibrosis and chronic renal disease were common findings in affected animals. The causes of the intracardiac thrombosis in the tamarins in the present study are not known, but the clinical signs and gross and microscopic lesions suggest that congestive heart failure secondary to cardiomyopathy is the primary contributor. In addition, the cause of the aortic dissecting aneurysms in the tamarins in this study is not known. Further studies are required to determine whether factors including aortic curvature, genetic background, or hypertension—alone or in combination—play a role. To our knowledge, the current retrospective study is the first report of intracardiac thrombosis and aortic aneurysms in mustached tamarins.

A variant of simian immunodeficiency virus (SIVSMM/PBj), isolated from a chronically infected pig-tailed macaque has been shown in previous studies to produce acutely fatal disease uniformly in pig-tailed macaques and in some rhesus macaques. The present study extends investigation of SIVSMM/PBj pathogenesis in rhesus and cynomolgus monkeys. Cynomolgus and rhesus macaques were found to be uniformly susceptible to infection, but as previously reported, the rhesus were found to not be uniform in their response during the acute disease. Homogenized tissues from a rhesus that died acutely from SIVSMM/PBj were passaged to 6 rhesus monkeys in an attempt to increase lethality. Five of 6 rhesus monkeys receiving intravenous inoculation of either spleen (10(3) TCID50) or lymph node (10(5) TCID50) homogenate developed acute disease; 4 died (days 8-10), 1 recovered, and one rhesus remained asymptomatic. Three of 3 cynomolgus macaques and 4 of 4 pig-tailed macaques receiving the same inoculum died acutely within 9 days. Clinical disease in macaques that died was characterized by diffuse lymphadenopathy within 5 days of inoculation and severe diarrhea beginning 1 to 3 days before death. Anorexia, lymphopenia (1000 cells/mm3), and mild hypoalbuminemia preceded onset of diarrhea by 24 h. Viral p27 was detected in circulation by day 6 postinfection, with all animals dying acutely having detectable serum p27 and no detectable humoral response. Acute lethality was attributed to severe metabolic acidosis (pH7.20) which was observed 24-48 h prior to death in the pig-tailed and cynomolgus macaques. Immunohistochemistry revealed numerous SIV antigen-positive lymphocytes and macrophages in the lymph nodes, spleen, gut-associated lymphoid tissues and gastrointestinal lamina propria. Histopathologic lesions included marked to severe hyperplasia of the T-cell-dependent areas in lymphoid tissues and diffuse nonulcerative lymphohistiocytic gastroenteritis. Surviving rhesus developed strong humoral immune responses to the major SIV proteins.

In animals, multisystemic eosinophilic disease is a rare condition characterized by eosinophilic and lymphoplasmacytic infiltrates in various organs. This disorder resembles the human disease known as hypereosinophilic syndrome, a condition defined by prolonged peripheral eosinophilia in the absence of recognizable etiology and associated with end-organ damage. In this report we describe a research-naïve, colony-born, juvenile female owl monkey (Aotus vociferans) who presented clinically with severe respiratory distress and histologically with multiple end-organ infiltration with phenotypically mature eosinophils, plasma cells, and lymphocytes. No tumors or infectious agents were noted either macroscopically or microscopically. Cultures from lung samples revealed no bacteria or fungi. Histologic examination of lung, heart, thymus, liver, spleen, kidney, adrenal, pancreas, stomach, small intestine, and colon revealed no migrating nematode larvae, other parasites, or foreign material that might trigger eosinophilia, nor was there any evidence of or history consistent with an allergic etiology. Given that we ruled out most exogenous and endogenous triggers of eosinophilia, the signs, symptoms, and pathologic findings support the diagnosis of multisystemic eosinophilic disease. To our knowledge, this report is the first description of presumptive hypereosinophilic syndrome in a nonhuman primate.

Spironucleus muris is an unacceptable infectious agent for most rodent colonies. Exposure of sentinel mice to dirty bedding and examination of sentinel intestinal smears was not sufficient for identification of the extent of spironucleosis within 1 mouse room. Clinical abnormalities were not reported in the animals housed in the room despite extensive breeding and a preponderance of mice genetically engineered to have nonfunctional T and B cells. In addition, researchers reported that the infection had not altered their research data. During investigation of the outbreak, direct intestinal smears performed on related animals (conspecifics, offspring, or siblings) revealed that immunodeficient mice often tested negative whereas the immunocompetent cohort tested positive. In this study, we used culled colony animals and compared direct intestinal exam test results with histologic results. The comparison showed the extent of false negatives that may occur when direct intestinal exam alone is used to detect this protozoon. Sensitivity of the direct intestinal exam for detection of S. muris was calculated to be 71%, while histology sensitivity was 91%. In light of the study results and an extensive literature review, we revised our health surveillance plan so that the age and duration of exposure to dirty bedding among sentinel mice is varied at the time of testing.

The purpose of this study was to develop a rapid DNA isolation method and a sensitive and specific PCR assay for detecting Spironucleus muris in mouse tissue and fecal samples. A PCR assay based on the carboxy terminus of the elongation factor 1α gene was developed; the PCR product was confirmed by nucleic acid sequencing and nested PCR. The new PCR assay then was used to test feces from animals that had been screened for S. muris by using direct intestinal examination and histology. The PCR assay was determined to be a more sensitive test than either direct intestinal examination or intestinal histology. To our knowledge, this assay represents the first use of a PCR-based diagnostic screening method to confirm the presence of S. muris in murine tissue and fecal samples.

The pathology of 33 moustached tamarins (Saguinus mystax) previously used in hepatitis A and GB virus studies is reported. Chronic lesions in colon, heart, and kidney were common in the monkeys and appeared not to be due to the experimental exposures. Colitis cystica profunda (CCP), a disease that affects humans and is characterized by the presence of mucin-filled epithelial downgrowths and cysts in the colonic submucosa, was found in 24 of the 33 (72.7%) tamarins. Interstitial myocardial fibrosis was present in 22 (66.6%) animals, and various degrees of membranoproliferative glomerulonephritis occurred in 28 (84.8%) monkeys. In addition, 28 (84.8%) tamarins demonstrated diffuse hepatocellular vacuolation with mild lymphoplasmacytic infiltrates, possibly as a result of the experimental infections, and peliosis hepatis occurred in 7 (21.2%) animals. The etiology of CCP is unknown, and no reliable animal models are available because most cases in animals are reported only sporadically. Myocardial fibrosis in tamarins has not been reported previously, and all current animal models require experimental manipulation of the animal to mimic the human disease. The results from this study suggest that captive S. mystax has high incidence of spontaneous CCP, myocardial fibrosis, and membranoproliferative glomerulonephritis. This species may be a spontaneous animal model for pathogenesis and experimental therapy studies of the analogous human diseases.

A rhesus macaque (Macaca mulatta) infected with simian-human immunodeficiency virus (SHIV) while undergoing AIDS research, required a comprehensive physical examination when it presented with slight peripheral edema, hypoalbuminemia, and proteinuria. Many of the clinical findings were consistent with nephrotic syndrome, which is an indication of glomerular disease, but the possibility of concurrent disease needed to be considered because lentiviral induced immune deficiency disease manifests multiple clinical syndromes. The animal was euthanized when its condition deteriorated despite supportive care that included colloidal fluid therapy. Histopathology confirmed membranoproliferative glomerulonephritis, the result of immune complex deposition most likely due to chronic SHIV infection. Clinical symptoms associated with this histopathology in SHIV-infected macaques have not previously been described. Here we offer suggestions for the medical management of this condition, which entails inhibition of the renin–angiotensin–aldosterone system and diet modifications.

Murine norovirus (MNV) is a common viral infection of mice in many research facilities. MNV infects hematopoietic cells and alters their cellular morphology. Because of MNV's probable effects on the systemic immune response of infected mice the decision was made to eradicate the virus from 2 rooms containing infected animals in our vivarium. Two different eradication methods were selected. One room, in which most of the indirectly exposed sentinels had antibodies to MNV, was depopulated and thoroughly cleaned prior to repopulation. In the other room, in which only 13% of the sentinels had positive MNV titers, selective testing was used, and MNV-positive animals were removed. Data from surveillance of the sentinel mice exposed to dirty bedding indicate that the test-and-removal method was ineffective in eliminating MNV from the room, whereas sentinel mice in the room that underwent depopulation and cleaning prior to repopulation have not shown any evidence of MNV since December 2006.

Murine norovirus (MNV) is a recently discovered infectious agent in mice and may be the most common naturally occurring infection of laboratory mice in North America. In 2005, we surveyed the Swiss Webster female sentinel mice in our institute's research facilities. Of the 4 facilities surveyed, 3 had sentinel mice that were positive for MNV antibodies, whereas our largest facility (which only receives mice directly from select vendors or by embryo rederivation directly into the facility) was apparently MNV-free. However, testing of sentinel mice in this large facility 1 y later found that 2% of the animals had developed MNV-specific antibodies. In a recently opened fifth facility, a serologic survey in 2006 identified MNV-antibody-positive Tac:SW sentinel mice that had received bedding from experimental mice on the same rack quadrant. Reverse transcription- polymerase chain reaction analysis of feces from the cages of these mice showed evidence for shedding of MNV. These sentinel mice were used to study the fecal excretion, antibody development, gross lesions upon necropsy, histopathology, and immunohistochemistry of the viral infection. None of the MNV-antibody-positive sentinel mice exhibited clinical signs or gross lesions, but these mice excreted virus in feces and developed antibodies to MNV. Histopathologic lesions consisted only of a few hepatic inflammatory foci in each liver section, some of which were immunoreactive with antibodies to MNV. MNV viral antigens also were present in the mesenteric lymph nodes.

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis -reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.

The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (≥6.0 log 10 ) in the upper respiratory tract of hamsters and to moderate titers (≥3.6 log 10 ) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPV-infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro.

A recombinant live-attenuated chimeric human parainfluenza virus type 1 (HPIV1) candidate vaccine was previously generated by replacing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein open reading frames (ORFs) of the HPIV3 candidate vaccine, rHPIV3 cp 45, with those of wild-type HPIV1. Previously, this recombinant chimeric virus, designated rHPIV3–1 cp 45, exhibited a greater level of the temperature sensitivity of replication in vitro and a greater level of attenuation of replication in the respiratory tract of immunized hamsters when compared to its HPIV3 cp 45 parent virus. In the present study, rHPIV3–1 cp 45 was evaluated for its level of attenuation and efficacy in African green monkeys ( Cercopithecus aethiops ), a primate in which both HPIV1 and HPIV3 wild-type viruses replicate efficiently. The rHPIV3–1 cp 45 candidate vaccine was as restricted in replication in the upper and lower respiratory tract as its thoroughly characterized rHPIV3 cp 45 parent indicating that the attenuating mutations present in the rHPIV3 cp 45 backbone specified an appropriate level of attenuation of rHPIV3–1 cp 45 for primates. The level to which rHPIV3–1 cp 45 replicated in the respiratory tract of African green monkeys was also sufficient to induce a strong immune response to HPIV1 and provided protection against challenge with wild-type HPIV1. These results provide a basis for further evaluation of this HPIV1 candidate vaccine in humans.

The recombinant dengue virus type-4 vaccine candidate 2AA30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3'-untranslated region of the viral genome. A clinical trial to evaluate the safety and immunogenicity of a single dose of 2Adelta30 was conducted with 20 adult human volunteers. The vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. Viremia was detectable in 14 volunteers at a mean level of 1.6 log10 plaque-forming units/ml of serum, although all 20 volunteers seroconverted with a seven-fold or greater increase in serum neutralizing antibody titer on day 28 post-vaccination (mean titer = 1:580). A mild, asymptomatic, macular rash developed in 10 volunteers, and a transient elevation in the serum level of alanine aminotransferase was noted in five volunteers. The low level of reactogenicity and high degree of immunogenicity of this vaccine candidate warrant its further evaluation and its use to create chimeric vaccine viruses expressing the structural genes of dengue virus types 1, 2, and 3.

The distribution and levels of simian immunodeficiency virus (SIV) in tissues and plasma were assessed in naturally infected African green monkeys (AGM) of the vervet subspecies ( Chlorocebus pygerythrus ) by limiting-dilution coculture, quantitative PCR for viral DNA and RNA, and in situ hybridization for SIV expression in tissues. A wide range of SIV RNA levels in plasma was observed among these animals (

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F H HN H ) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F H HN H replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F H HN H conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F H HN H as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.

Human parainfluenza virus (HPIV) types-1, -2, and -3 are significant causes of both upper and lower respiratory tract disease in infants and children. Although there are two live attenuated vaccines for the prevention of HPIV-3 disease in phase 1 clinical trials, vaccines are not currently available for prevention of HPIV-1 or -2 disease. Our laboratory is developing candidate vaccines for the prevention of HPIV-1, -2, and -3 disease, and a suitable nonhuman primate model is needed for evaluation of these vaccine candidates prior to administration to humans. We evaluated the replication of HPIV-1 and -2 in six different species of nonhuman primates and found both viruses to replicate most efficiently in African green monkeys and chimpanzees. We then compared the replication of HPIV-3 in African green monkeys to that in rhesus macaques, which we have used previously, and found that HPIV-3 replicated to higher titer in African green monkeys. In summary, African green monkeys provide a very useful nonhuman primate for the evaluation of HPIV-1, -2, and -3 vaccine candidates, especially for the evaluation of various combinations of these PIV vaccines and for vaccine strategies that employ sequential immunization.

Measles remains a major cause of childhood mortality, with questions about virus virulence and pathogenesis still requiring answers. Rhesus macaques were infected with 5 different culture-adapted strains of measles virus, including 2 from patients with progressive vaccine-induced disease, and a sixth nonculture-adapted strain, Bilthoven. All caused infection detectable by reverse transcriptase-polymerase chain reaction and induction of antibody. Chicago-1 and Bilthoven induced viremias detectable by leukocyte cocultivation. Bilthoven induced Koplik's spots, conjunctivitis, and rash. Lymphopenia and depressed interleukin (IL)-2 production were followed by monocytosis and eosinophilia. All monkeys, including 41 involved in a primate facility outbreak, showed suppressed responses to phytohemagglutinin. As the rash resolved production of IL-2, IL-1beta, tumor necrosis factor-alpha, IL-6, and IL-5 mRNA increased. Monkeys are useful for studies of measles immunopathogenesis, but virus strains must be carefully chosen. Increased virulence of vaccine strains isolated from immunocompromised infants with fatal infections was not evident.

Two parainfluenza virus type 3 (PIV3) vaccine candidates-cp45, a live attenuated derivative of the JS wild type (wt), and a replication-defective vaccinia virus recombinant expressing the hemagglutinin-neuraminidase or fusion glycoprotein of human PIV3 (modified vaccinia virus Ankara [MVA]/PIV3 recombinants)-were evaluated in rhesus monkeys to determine whether successful immunization could be achieved in the presence of passively transferred PIV3 antibodies. The cp45 virus, administered intranasally (in) and intratracheally (it) in the presence of high levels of PIV3 antibodies, replicated efficiently in the nasopharynx and protected against challenge with wt human PIV3. The MVA recombinants, administered in, it, and intramuscularly in the absence of passive antibody, conferred protection against replication of PIV3 wt challenge virus, but this was largely abrogated when immunization occurred in the presence of passive antibodies. Because immunization for the prevention of HPIV3 disease must occur in early infancy when maternal antibodies are present, the live attenuated cp45 virus continues to be a promising vaccine candidate for this age group.

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466–473, 1998; A. Bukreyev et al., J. Virol. 71:8973–8982, 1997; and this study). The resulting viruses, designated rA2ΔNS2 and rA2ΔSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2ΔNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2ΔSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The ΔSH mutation was incorporated into a recombinant form of the cpts 248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.

Respiratory syncytial virus (RSV) cpts 530/1030 is an attenuated, temperature-sensitive subgroup A vaccine candidate derived previously from cold-passaged RSV ( cp RSV) by two sequential rounds of chemical mutagenesis and biological selection. Here, cpts 530/1030 was shown to be highly attenuated in the upper and lower respiratory tracts of seronegative chimpanzees. However, evaluation in seropositive children showed that it retains sufficient replicative capacity and virulence to preclude its direct use as a live attenuated vaccine. Nucleotide sequence analysis of the genome of cpts 530/1030 showed that it had acquired two nucleotide substitutions (compared to its cpts 530 parent), both of which were in the L gene: a silent mutation at nucleotide position 8821 (amino acid 108) and a missense mutation at nucleotide position 12458 resulting in a tyrosine-to-asparagine change at amino acid 1321, herein referred to as the 1030 mutation. It also contained the previously identified 530 missense mutation at nucleotide 10060 in the L gene. The genetic basis of attenuation of cpts 530/1030 was defined by the introduction of the 530 and 1030 mutations into a cDNA clone of cp RSV, from which recombinant RSV was derived and analyzed to determine the contribution of each mutation to the temperature sensitivity ( ts ) and attenuation ( att ) phenotypes of cpts 530/1030. The 530 mutation, derived from cpts 530, was previously shown to be responsible for the ts and att phenotypes of that virus. In the present study, the 1030 mutation was shown to be responsible for the increased temperature sensitivity of cpts 530/1030. In addition, the 1030 mutation was shown to be responsible for the increased level of attenuation of cpts 530/1030 in the upper and lower respiratory tracts of mice. The 530 and 1030 mutations were additive in their effects on the ts and att phenotypes. It was possible to introduce the 1030 mutation, but not the 530 mutation, into an attenuated vaccine candidate with residual reactogenicity in very young infants, namely, cpts 248/404, by use of reverse genetics. The inability to introduce the 530 mutation into the cpts 248/404 virus was shown to be due to its incompatibility with the 248 missense mutation at the level of L protein function. The resulting rA2cp248/404/1030 mutant virus was more temperature sensitive and more attenuated than the cpts 248/404 parent virus, making it a promising new RSV vaccine candidate created by use of reverse genetics to improve upon an existing vaccine virus.

The viral accessory protein Vpx is required for productive in vitro infection of macrophages by simian immunodeficiency virus from sooty mangabey monkeys (SIV(SM)). To evaluate the roles of Vpx and macrophage infection in vivo, we inoculated pigtailed macaques intravenously or intrarectally with the molecularly cloned, macrophage tropic, acutely pathogenic virus SIV(SM) PBj 6.6, or accessory gene deletion mutants (deltaVpr or deltaVpx) of this virus. Both wild-type and SIV(SM) PBj deltaVpx viruses were readily transmitted across the rectal mucosa. A subsequent 'stepwise' process of local amplification of infection and dissemination was observed for wild-type virus, but not for SIV(SM) PBj deltaVpx, which also showed considerable impairment of the overall kinetics and extent of its replication. In animals co-inoculated with equivalent amounts of wild-type and SIV(SM) Pbj deltaVpx intravenously or intrarectally, the deltaVpx mutant was at a strong competitive disadvantage. Vpx-dependent viral amplification at local sites of initial infection, perhaps through a macrophage-dependent mechanism, may be a prerequisite for efficient dissemination of infection and pathogenic consequences after exposure through either mucosal or intravenous routes.

Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1.

An infectious molecular clone of simian immunodeficiency virus SIVsm was derived from a biological isolate obtained late in disease from an immunodeficient rhesus macaque (E543) with SIV-induced encephalitis. The molecularly cloned virus, SIVsmE543-3, replicated well in macaque peripheral blood mononuclear cells and monocyte-derived macrophages and resisted neutralization by heterologous sera which broadly neutralized genetically diverse SIV variants in vitro. SIVsmE543-3 was infectious and induced AIDS when inoculated intravenously into pig-tailed macaques (Macaca nemestrina). Two of four infected macaques developed no measurable SIV-specific antibody and succumbed to a wasting syndrome and SIV-induced meningoencephalitis by 14 and 33 weeks postinfection. The other two macaques developed antibodies reactive in Western blot and virus neutralization assays. One macaque was sacrificed at 1 year postinoculation, and the survivor has evidence of immunodeficiency, characterized by persistently low CD4 lymphocyte subsets in the peripheral blood. Plasma samples from these latter animals neutralized SIVsmE543-3 but with much lower efficiency than neutralization of other related SIV strains, confirming the difficulty by which this molecularly cloned virus is neutralized in vitro. SIVsmE543-3 will provide a valuable reagent for studying SIV-induced encephalitis, mapping determinants of neutralization, and determining the in vivo significance of resistance to neutralization in vitro.

Like the other hepatitis viruses, hepatitis E virus (HEV) has been difficult to study because of limitations in cell culture systems and small animal models. Much of what we know has come from epidemiological studies in developing countries and, more recently, in industrialized countries. However, the epidemiology is very different in these two settings: hepatitis E in developing countries is epidemic as well as sporadic, principally water-borne, most likely to cause disease in older children and young adults and relatively severe, especially in pregnant women; in industrialized countries the disease is sporadic, principally food-borne, most common in the elderly and probably associated with mostly inapparent infections. These differences are believed to be genotypically determined. To examine the biological parameters of hepatitis E, we have studied HEV infections in nonhuman primates, which are surrogates of man. Infections with HEV genotypes 1–3 were compared in rhesus and cynomolgus macaques and chimpanzees. In general, the biological characteristics of the different HEV genotypes mirrored their epidemiological characteristics.

The dynamics of plasma viremia were explored in a group of 12 simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) that had received prior immunization with either nonrecombinant or trivalent (gag-pol, env) SIV-recombinant vaccinia viruses. Three distinct patterns of viral replication observed during and following primary viremia accounted for significant differences in survival times. High-level primary plasma viremia with subsequently increasing viremia was associated with rapid progression to AIDS (n = 2). A high-level primary plasma virus load with a transient decline and subsequent progressive increase in viremia in the post-acute phase of infection was associated with progression to AIDS within a year (n = 6). Low levels of primary plasma viremia followed by sustained restriction of virus replication were associated with maintenance of normal lymphocyte subsets and intact lymphoid architecture (n = 4), reminiscent of the profile observed in human immunodeficiency virus type 1-infected long-term nonprogressors. Three of four macaques that showed this pattern had been immunized with an SIV recombinant derived from the attenuated vaccinia virus, modified vaccinia virus Ankara. These data link the dynamics and extent of virus replication to disease course and suggest that sustained suppression of virus promotes long-term, asymptomatic survival of SIV-infected macaques. These findings also suggest that vaccine modulation of host immunity may have profound beneficial effects on the subsequent disease course, even if sterilizing immunity is not achieved.

The use of a recombinant virus containing a heterologous gene of another microorganism as a live vaccine was suggested more than 10 years ago (Mackett et al, 1982; Panicali and Paoletti, 1982). Vaccinia virus was considered for such a purpose because of its success as a smallpox vaccine and ease and economy of production, distribution and administration (Fermer et al., 1988). The extensive experimental use of recombinant vaccinia viruses was facilitated by the construction of plasmid transfer vectors containing a vaccinia virus promoter, one or more convenient restriction endonuclease sites for inserting a foreign gene, flanking DNA sequences for homologous recombination into a non-essential site of the vaccinia virus genome and for selection and/or screening of recombinant viruses (Chakrabarti et al., 1985; Mackett et al., 1984). Humoral and cell mediated immune responses to an expressed foreign protein and protection of experimental animals against challenge with the corresponding pathogen were demonstated in a variety of animal model systems (Cox et al., 1992; Moss, 1991).

Previous studies suggested that simian immunodeficiency viruses isolated from African green monkeys (SIVagm) are relatively nonpathogenic. The report describes the isolation and biologic and molecular characterization of a pathogenic SIVagm strain derived from a naturally infected African green monkey. This virus induced an AIDS-like syndrome characterized by early viremia, frequent thrombocytopenia, severe lymphoid depletion, opportunistic infections, meningoencephalitis, and death of five of eight macaques within 1 year after infection. An infectious clone derived from this isolate reproduced the immunodeficiency disease in pig-tailed (PT) macaques, providing definitive proof of the etiology of this syndrome. Although the virus was highly pathogenic in PT macaques, no disease was observed in experimentally infected rhesus macaques and African green monkeys despite reproducible infection of the last two species. Whereas infection of PT macaques was associated with a high viral load in plasma, peripheral blood mononuclear cells, and tissues, low-level viremia and infrequent expression in lymph nodes of rhesus macaques and African green monkeys suggest that differences in pathogenicity are associated with the extent of in vivo replication. The availability of a pathogenic molecular clone will provide a useful model for the study of viral and host factors that influence pathogenicity.