Neurovirulence of yellow fever 17DD vaccine virus to rhesus monkeys

The yellow fever 17D virus is attenuated and used for human vaccination. Two of its substrains, 17D-204 and 17DD, are used for vaccine production. One of the remarkable properties of this vaccine is limited viral replication in the host but with significant dissemination of the viral mass, yielding a robust and long-lived neutralizing antibody response. The vaccine has excellent records of efficacy and safety and is cheap, used as a single dose, and there are well-established production methodology and quality control procedures which include the monkey neurovirulence test (MNTV). The present study aims at a better understanding of YF 17DD virus attenuation and immunogenicity in the MNVT with special emphasis on viremia, seroconversion, clinical and histological lesions scores, and their intrinsic variability across the tests. Several MNVTs were performed using the secondary seed lot virus 17DD 102/84 totaling 49 rhesus monkeys. Viremia was never higher than the accepted limits established in international requirements, and high levels of neutralizing antibodies were observed in all animals. None of the animals showed visceral lesions. We found that the clinical scores for the same virus varied widely across the tests. There was a direct correlation between the clinical scores in animals with clinical signs of encephalitis and a higher degree of central nervous system (CNS) histological lesions, with an increase of lesions in areas of the CNS such as the substantia nigra, nucleus caudatus, intumescentia cervicalis, and intumescentia ventralis. The histological scores were shown to be less prone to individual variations and had a more homogeneous value distribution among the tests. Since 17DD 102/84 seed virus has been used for human vaccine production and immunization for 16 years with the vaccine being safe and efficacious, it demonstrates that the observed variations across the MNVTs do not influence its effect on humans.     

Studies on dengue 2 virus infection in cyclophosphamide-treated rhesus monkeys

Dengue 2 virus (D2V) replication has been demonstrated in cultured primate mononuclear phagocytes, mitogen treated lymphocytes and lymphoblastoid cells. To determine which of these cell types might play an important role in sustaining infection in vivo, nine rhesus monkeys were immunosuppressed with cyclophosphamide and then infected with D2V. Maintenance doese which held total white blood cell counts to <3000/mm³ ablated both primary and secondary antibody responses. Six successfully immunosuppressed animals circulated virus and infected monocytes in blood for prolonged periods. Virus was recovered from lymphatic organs and visualized in tissue mononuclear leukocytes in two subjects dying during the experimental period. The results argue against the hypothesis that lymphoblasts play an important role in dengue virus infection but are consistent with the possibility that mononuclear phagocytes are the site of viral replication in vivo.This research was supported by a contract from the U.S. Army Medical Research and Development Command, DADA 17-73-C3083. We wish to thank Susan Cate, Jill Kelly and Patricia Iwamoto for excellent technical assistance     

Serotype-specific and cross-reactive neutralizing antibody responses in cynomolgus monkeys after infection with multiple dengue virus serotypes

Neutralizing antibody responses were examined in monkeys after dengue virus infections. In monkeys that had been infected once or twice with DENV-2, neutralizing antibody was cross-reactive with all four serotypes after secondary or tertiary infection with DENV-3. In monkeys that had been inoculated with DENV-1 and DENV-2 in the primary and secondary infections, neutralizing antibody titers did not increase after tertiary infection with DENV-3. These results indicate that antibody responses after secondary and tertiary infections with different serotypes are cross-reactive with all four serotypes, consistent with what has been observed in humans, and suggest that monkeys are useful for determining neutralizing antibody responses.     

Respiratory responses to Ascaris antigen in rhesus and cynomolgus monkeys

Responses to Ascaris antigen were evaluated in rhesus and cynomolgus monkeys. Of 19 cynomolgus monkeys tested, 15 were found to have cutaneous reactivity to Ascaris: 13 of these responded to Ascaris aerosols with changes in respiratory function including an increase in respiration rate, decrease in tidal volume and peak expiratory flow rate, decrease in dynamic compliance, and an increase in resistance. Four of the six rhesus monkeys studied with cutaneous reactivity to Ascaris responded to Ascaris aerosols: the changes in respiratory function observed after the Ascaris challenge in these monkeys were similar to those observed in the cynomolgus monkeys. Responses to repetitive Ascaris challenges were obtained in monkeys of both species with a recovery period of 30 to 60 min between challenges. In monkeys of both species that reacted to Ascaris aerosols, blood pressure was elevated transiently; changes in heart rate were variable. Changes in cardiovascular measurements were not observed in monkeys that did not respond to Ascaris aerosols. Thus, it appears that the cynomolgus monkey responds to an aerosol Ascaris challenge in a manner similar to the rhesus monkey and is an additional suitable model for the study of allergic respiratory responses.     

Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus

Two molecularly engineered, live-attenuated West Nile virus (WN) vaccine candidates were highly attenuated and protective in rhesus monkeys. The vaccine candidates are chimeric viruses (designated WN/DEN4) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue 4 virus (DEN4) with or without a deletion of 30 nucleotides (Delta 30) in the 3' noncoding region of DEN4. Viremia in WN/DEN4- infected monkeys was reduced 100-fold compared to that in WN- or DEN4-infected monkeys. WN/DEN4-3'Delta 30 did not cause detectable viremia, indicating that it is even more attenuated for monkeys. These findings indicate that chimerization itself and the presence of the Delta 30 mutation independently contribute to the attenuation phenotype for nonhuman primates. Despite their high level of attenuation in monkeys, the chimeras induced a moderate-to-high titer of neutralizing antibodies and prevented viremia in monkeys challenged with WN. The more attenuated vaccine candidate, WN/DEN4-3'Delta 30, will be evaluated first in our initial clinical studies.     

Neurovirulence in cynomolgus monkeys of enterovirus 71 isolated from a patient with hand,foot and mouth disease

Six cynomolgus monkeys were inoculated subcutaneously with enteroviurs 71 (E71), isolated from the stools of a patient with hand, foot and mouth disease (HFMD). Clinical symptoms were observed in three of the six monkeys. One monkey showed complete paralysis of the lower extremities and two animals showed weakness in the hind limbs 4 to 7 days after inoculation. Lesions were found in the central nervous system (CNS) of all monkeys. Mild to moderate vascular lesions, perivascular cuffings, degeneration and disappearance of the neurons and meningial lymphocytic infiltration were observed in the grey and/or white matter of the spinal cord, medulla oblongata, cerebral cortex and brain stem. No virus was recovered from the CNS or liver of any of the six monkeys. However, serum neutralizing antibody titers had risen in monkeys inoculated with E71.     

Comparative haematological and plasma chemistry values in purpose-bred squirrel, cynomolgus and rhesus monkeys

Haematological and blood chemistry data have been compared for three species of purpose-bred primates, Macaca mulatta (rhesus monkey), Macaca fascicularis (cynomolgus monkey), Saimiri sciureus (squirrel monkey). These species were housed over a 9-month period in identical conditions (diet, tap water, room temperature and relative humidity, and lighting regime) in our primate facility. Blood and urine assays were conducted using the same pre-analytical conditions (blood sampling procedure, anticoagulant and storage of sample), and analytical methods (reagent and equipment).The results indicated that squirrel, rhesus and cynomolgus monkeys have essentially biologically similar values for all of the parameters examined. However, haemoglobin level, reticulocyte, plasma total cholesterol, triglyceride, albumin and urea nitrogen values, and urinary osmolality in cynomolgus monkeys were statistically lower than those of rhesus monkeys. Erythrocyte count, plasma ALT, calcium and potassium in cynomolgus monkeys were statistically higher than those of rhesus monkeys. Erythrocyte count, haemoglobin level, haematocrit, reticulocyte, plasma total bilirubin and chloride and urinary osmolality in cynomolgus monkeys were statistically lower than those of squirrel monkeys. Leucocyte count, plasma total protein, albumin and calcium in cynomolgus monkeys were statistically higher than those of squirrel monkeys. Squirrel monkeys showed marked deviations in four assays from the other two species: ratio of lymphocytes to neutrophils, AST, ALT and urine volume.The results obtained in this study will be used as baseline data for haematology and clinical chemistry characteristics for three species of purpose-bred monkeys.     

Infection of rhesus monkeys and chimpanzees with Epstein-Barr virus

Summary Seventy-two nonhuman primates were entered into a long-term study to evaluate the pathogenicity of Epstein-Barr virus (EBV). Infectious virus was inoculated into 42 rhesus monkeys, 4 chimpanzees and 1 cynomolgus monkey. Immunostimulation or immunosuppression was attempted in 34 of these animals to enhance the oncogenic potential of the virus. Eleven inoculated animals were followed for more than 3 years and two were observed for 8 years. No tumors were observed in any of the animals; however, serological evaluation of the 47 inoculated primates and 25 matched controls indicated that at least 14 rhesus monkeys and the cynomolgus monkey were successfully infected with EBV. The potential use of rhesus monkeys as a model for EBV-induced disease in humans is discussed.With 2 FiguresSupported in part by Contracts NO 1-CP-4333 and NO 1-CP-61029 with the Carcinogenesis Extramural Program, Division of Cancer Cause and Prevention.     

Properties of a measles virus neuropathic for rhesus monkeys

Summary A highly hamster brain adapted strain of measles virus, capable of producing encephalitis in monkeys, inoculated into Vero cell cultures replicated in a suppressed growth cycle. No virus was released into the medium and the infected cells lacked specific virus determinants for hemadsorption. A complete replicative cycle was achieved after six subcultures of Vero cells and was accompanied by a partial drop in neurovirulence. However, hamster neuropathogenicity persisted throughout 26 Vero passages and was markedly higher than in non-brain passaged strains. High neurovirulence was associated with pronounced giant cell cytopathic effect and thermolability. On continuous passage these three properties dissociated at different rates indicating that they are independent properties of measles virus. The importance of their coincidence for the initiation of an encephalitic process in the brain of primates is stressed.     

Circular viral DNA detection and junction sequence analysis from PBMC of SHIV-infected cynomolgus monkeys with undetectable virus plasma RNA

Extrachromosomal forms of human immunodeficiency virus (HIV)-1 can be detected in peripheral blood mononuclear cell (PBMC) from HIV-infected patients in the absence of detectable viral replication and are thought to be a sign of active but cryptic virus replication. No information, however, are available on whether these forms are also present in animal models for acquired immunodeficiency syndrome (AIDS) and on their relation with other methods of detection of virus replication. To this aim, a polymerase chain reaction (PCR) approach was used to detect and analyze unintegrated circular 2-LTR-containing forms in PBMC of simian human immunodeficiency virus (SHIV)89.6P infected cynomolgus monkeys with RNA levels ranging between 1.8 x 10(6) and less than 50 copies/ml of plasma. 2-LTR forms were detected in 96.5% of monkeys' samples above 50 copies/ml of plasma, whereas they were present in 75.8% of monkeys' samples below 50 copies/ml of plasma. Persistence of unintegrated viral DNA in monkeys with undetectable plasma RNA could indicate either stability in non-dividing cells or ongoing low levels of viral replication in dividing cells.     

The sensitivity of rhesus and cynomolgus macaques to the human hepatitis A virus

Hepatitis A infection characterized by virus excretion in feces, synthesis of specific IgM antibody, increased activity of alanine aminotransferase in the blood serum, and a complex of morphological lesions in the liver typical of acute hepatitis was reproduced in M. fascicularis (M. f.) and Macaca rhesus (M. r.) using 2 strains of hepatitis A virus (HAV) isolated from human patients. The incubation period varying from 9 to 23 (mean 16) days in M. f. and from 12 to 35 (mean 18) days in M. r. in primary infection shortened to 1-12 (mean 10) and 3-6 (mean 5) days in the process of virus passage from monkey to monkey. The disease was observed to run both manifest forms (except jaundice) typical of human HA and an inapparent form in which the level of enzymes remained within normal limits but HAV could be detected in feces, anti-HAV-IgM in the blood serum, and morphologically acute hepatitis in the liver. Immune electron microscopy of both the initial material and in monkey feces at the levels of all three passages revealed complexes consisting of spherical viral particles 27-29 nm in size coated with antibodies. The immune complexes formed upon addition to the fecal extracts under study of IgG isolated both from human convalescent sera and from sera of experimentally infected monkeys collected in the acute stage of the illness.     

Antibody-dependent enhancement of dengue virus infection in vitro by undiluted sera from monkeys infected with heterotypic dengue virus

Two monkeys (Macaca fascicularis) each were infected with dengue virus type 1 (DENV-1) and type 2 (DENV-2). High levels of neutralizing antibody to homotypic serotype were detected from day 10 to week 58 after infection. Levels of cross-reactive neutralizing antibody to other serotypes were at lower levels or undetectable. Serum samples collected from day 10 to week 58 enhanced infection by homotypic and heterotypic serotypes of DENV when diluted, demonstrating antibody-dependent enhancement (ADE). The ADE activities to heterotypic and homotypic dengue virus infections peaked at dilutions of 1:10–1:100 and 1:100–1:1,000, respectively. Serum samples collected enhanced heterotypic dengue virus infection without any dilution. The results indicate that sera from infected monkeys have an ability to enhance heterotypic dengue virus infection in vitro without dilution, although some of these sera also possess neutralizing activity.     

Neurovirulence of influenza virus in mice I. Neurovirulence of recombinants between virulent and avirulent virus strains

The neurovirulence of influenza A/WSN (HONI) virus in mice was studied using recombinants between neurovirulent WSN and nonneurovirulent A/Hong Kong (HK, H3N2) viruses. Parental derivation of genes in recombinants were analyzed by the electrophoresis of viral RNA in urea-polyacrylamide gel. The neurovirulence was tested by intracerebral inoculation of recombinants into mice. It was found that five large genes, P₃, P₁, P₂, HA, and NP, were not essential for the virulence, because recombinants having these five genes derived from HK parent were virulent. Recombinants in which any of three remaining WSN genes, NA, M, and NS, was replaced with the one from HK parent, failed to kill mice. Therefore, these three genes were responsible for the difference of neurovirulence between the two virus strains. However, when tested in mice immunosuppressed by the administration of cyclophosphamide, recombinants containing either M or NS protein from HK parent were virulent, but viruses containing HK neuraminidase were still avirulent. Viruses containing HK neuraminidase appeared incapable of multiplying in the mouse brain, while those containing either M or NS protein derived from HK virus multiplied to a limited extent. It was suggested that WSN neuraminidase was the principal determining factor of the neurovirulence of WSN virus, without which no virus multiplication occurred, while M and probably NS proteins of WSN virus played a role of helper or accessory virulence factor(s), enabling the efficient virus replication.     

Immunogenicity of wild and attenuated varicella-zoster virus strains in rhesus monkeys

Thirty susceptible rhesus monkeys were inoculated with cell-free varicella-zoster virus strain OKA or strain KMcC. Both wild and attenuated strains were used. No clinical signs characteristic of human varicella were seen in any of the animals. Virus was not isolated from throat swabs, blood, or cerebrospinal fluid. Antibodies were measured by an enhanced plaque neutralization test. The wild and attenuated OKA strains produced comparable levels of antibodies for 3 months after inoculation. Attenuated KMcC strains produced lower titers than the wild strain. On rechallenge 3 months after primary inoculation animals boostered with the attenuated OKA strain developed significantly higher antibody titers than animals receiving the wild strain. Animals primed and challenged with the attenuated KMcC strains showed significantly lower antibody titers than animals which received the wild strain. The results indicate that the immunogenicity of attenuated OKA and KMcC strains in rhesus monkeys parallels the experience obtained with these strains in humans.     

PCR detection of dengue virus using dried whole blood spotted on filter paper

Whole blood dried onto filter paper constitutes a potentially useful material for molecular testing of viruses, including dengue. In order to assess the stability of viral RNA, we carried out dengue-RNA detection in whole blood infected with dengue virus that had been previously spotted onto filter paper. Filter papers were stored at room temperature, 4 and -70 degrees C and processed for PCR assay at intervals of 2, 4, 6 and 9 weeks. Our results demonstrated that dengue-RNA was stable in filter paper for 9 weeks at all tested temperatures. Furthermore, we evaluated these conditions using frozen sera and dried blood samples onto filter paper from 52 patients with confirmed clinical diagnosis of dengue infection. PCR results showed a 100% specificity and 93% sensitivity for dried blood samples. This storage method facilitates the transportation and analysis by nucleic acid amplification techniques even when freezing conditions are not available.     

Activation of cytomegalovirus infection in immunosuppressed cynomolgus monkeys inoculated with varicella-zoster virus

A systemic activated cytomegalovirus (CMV) infection was fortuitously detected in almost all monkeys which had been immunosuppressed with antithymocyte globulin (ATG), cyclophosphamide (CY), and cortisone acetate (CS) before and after experimental inoculation with varicella-zoster virus (VZV). They developed exudative pneumonia, and the lesions in visceral organs and tissues contained cytomegalic cells with intranuclear inclusion bodies, in which viral antigens, specific for CMV, but not inoculated VZV, were detected by immunofluorescence. Serological study of paired sera from these monkeys ascertained preexisting CMV infection. Under the present experimental conditions, this infection was highly reproducible and always occurred within three, but not two, weeks of immunosuppression in monkeys inoculated with VZV. We therefore examined the host factors involved in activation of latent CMV. The immunocompetence of the host was destroyed almost completely with treatment of ATG, CY, and CS, but not with combinations of two of these agents, revealing the systemic depletion of lymphoid cells in tissues including the thymus medulla. Although the role of VZV in the induction of CMV remains uncertain, the heterologous VZV inoculum may have produced some effects equivalent to the allogeneic reaction to release latent CMV. These monkeys may represent an animal model of "opportunistic" CMV infection in immunocompromised and/or allografted humans.