Differential distribution of antibodies to different viruses in young animals in the free-ranging rhesus macaques of Cayo Santiago

BACKGROUND: The breeding colony of free-ranging rhesus macaques was established in 1938 in Cayo Santiago (CS) with animals collected in northern India. The seroprevalence to cercopithecine herpesvirus type 1 (B virus) and simian retroviruses has been studied previously. RESULTS: This is the first report on the seropositivity to different viruses using samples collected shortly after removing animals (n = 245) from CS. All samples were negative for measles, simian immunodeficiency virus and simian type D retroviruses. The overall prevalence of antibodies was around 50% for simian T-lymphotropic virus I (STLV-I). For B virus, the prevalence was 38%. CONCLUSIONS: Data obtained showed marked differences in the antibody distribution to B virus and STLV-I within the free-ranging colony of rhesus macaques. Implication of these data for the Specific Pathogen Free program at the Caribbean Primate Research Center are also discussed.     

Seroprevalence of specific viral infections in confiscated orangutans (Pongo pygmaeus)

A serological survey of confiscated orangutans was conducted to determine the prevalence of specific viral infections cross reacting with human viruses. Antibodies specific for human hepatitis A (HAV) and B (HBV) viruses, herpes simplex viruses (HSV), and human T-lymphotropic virus (HTLV types I and II), as well as for the simian type D retroviruses (SRV types 1 to 3) and simian immunodeficiency virus (SIV) were tested in samples from 143 orangutans. Results revealed a high prevalence of potential pathogens. The most prevalent viral infection found was HBV (59.4% prevalence) of which 89.4% of infected individuals seroconverted to the non-infectious state and 10.6% remained as chronic carriers. Antibodies to HAV, HSV, HTLV-1, and SRV were also detected but at a lower prevalence. There was no evidence of lentiviral infections in this group of animals. The results confirm the importance of quarantine and the need for diagnostic differentiation of virus infections to determine if they are of human origin or unique orangutan viruses.     

A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques

To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.     

Vaccine-induced control of viral shedding following rhesus cytomegalovirus challenge in rhesus macaques

The use of animal models of human cytomegalovirus (HCMV) infection is critical to refine HCMV vaccine candidates. Previous reports have demonstrated that immunization of rhesus monkeys against rhesus cytomegalovirus (RhCMV) can reduce both local and systemic replication of RhCMV following experimental RhCMV challenge. These studies used prime/boost combinations of DNA expression plasmids alone or DNA priming and boosting with either inactivated virion particles or modified vaccinia virus Ankara (MVA) expressing the same antigens. Viral outcomes included reduced RhCMV replication at the site of subcutaneous inoculation and RhCMV viremia following intravenous inoculation. Since shedding of cytomegalovirus from mucosal surfaces is critical for horizontal transmission of the virus, DNA priming/MVA boosting was evaluated for the ability to reduce oral shedding of RhCMV following subcutaneous challenge. Of six rhesus monkeys vaccinated exclusively against RhCMV glycoprotein B (gB), phosphoprotein 65 (pp65), and immediate-early 1 (IE1), half showed viral loads in saliva that were lower than those of control monkeys by 1 to 3 orders of magnitude. Further, there was a strong association of memory pp65 T cell responses postchallenge in animals exhibiting the greatest reduction in oral shedding. These results highlight the fact that a DNA/MVA vaccination regimen can achieve a notable reduction in a critical parameter of viral replication postchallenge. The recently completed clinical trial of a gB subunit vaccine in which the rate of HCMV infection was reduced by 50% in the individuals receiving the vaccine is consistent with the results of this study suggesting that additional immunogens are likely essential for maximum protection in an outbred human population.     

Experimental coinfection of rhesus macaques with rhesus cytomegalovirus and simian immunodeficiency virus: pathogenesis

Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.     

人工饲养中国猕猴中SRV、STLV和BV的流行病学调查

非人灵长类动物是十分重要的生物医学资源。由于与人类在生理生化、免疫、遗传等方面近似,猕猴是重要的非人灵长类实验动物之一。然而,猕猴作为自然宿主,易感染D型逆转录病毒(simian type D retrovirus,SRV)和T淋巴细胞白血病病毒(simian T lymphotropic virus,STLV)这两种逆转录病毒,并可能会影响AIDS猕猴动物模型等的研究结果。猴B病毒(ceropithecine herpesvirus1,BV)对猕猴及动物从业人员均有危害。云南省拥有较大规模的中国猕猴繁殖种群。基于以上原因,建立SPF级别的中国猕猴种群十分必要。该文应用PCR技术筛查了人工饲养种群中411只中国猕猴的SRV、STLV和BV感染流行情况。结果表明:SRV、STLV和BV的阳性感染率分别为19.71%(81/411)、13.38%(55/411)和23.11%(95/411)。同时比较分析了不同性别及年龄组中国猕猴的病毒感染情况。该研究将有助于建立SPF级别的中国猕猴繁殖种群。     

Declining prevalence of hepatitis A virus antibodies among children from low socioeconomic groups reinforces the need for the implementation of hepatitis A vaccination in Brazil

Age-related seroprevalence studies that have been conducted in Brazil have indicated a transition from a high to a medium endemicity of hepatitis A virus (HAV) infection in the population. However, most of these studies have focused on urban populations that experience lower incidence rates of HAV infection. In the current study, the prevalence of anti-HAV antibodies was investigated in children with a low socioeconomic status (SES) that live on the periphery of three capital cities in Brazil. A total of 1,162 dried blood spot samples were collected from individuals whose ages ranged from one-18 years and tested for anti-HAV antibodies. A large number of children under five years old (74.1-90%) were identified to be susceptible to HAV infection. The anti-HAV antibody prevalence reached ≥ 50% among those that were 10-14 years of age or older. The anti-HAV prevalence rates observed were characteristics of regions with intermediate level of hepatitis A endemicity. These data indicated that a large proportion of children with a low SES that live at the periphery of urban cities might be at risk of contracting an HAV infection. The hepatitis A vaccine that is currently offered in Brazil is only available for high-risk groups or at private clinics and is unaffordable for individuals with a lower SES. The results from this study suggest that the hepatitis A vaccine should be included in the Brazilian National Program for Immunisation.     

Prevalence of hepatitis A virus infection in Afro-Brazilian isolated communities in Central Brazil

To investigate hepatitis A virus (HAV) infection rates among isolated African-descendant communities in Central Brazil, 947 subjects were interviewed about demographic characteristics in all 12 isolated Afro-descendant communities existing in the state of Mato Grosso do Sul, Central Brazil, between March 2002 and November 2003. Blood samples were collected and sera were tested for HAV antibodies (total and IgM anti-HAV) by enzyme-linked immunosorbent assay. The overall prevalence of HAV infection was 75.6% (95% CI: 72.7-78.3), ranging from 55.4 to 97.3%, depending on the communities studied. The prevalence of anti-HAV increased significantly with age, from 13.8% in the age 0-5 age group to 96.6% in those older than 40 years. The findings point out an intermediate endemicity of HAV infection in some Afro-Brazilian isolated communities in Central Brazil. In addition, the high proportion of susceptible young subjects could be target of future HAV vaccination programs.     

Use of specific-pathogen-free (SPF) rhesus macaques to better model oral pediatric cytomegalovirus infection

Background Congenital human cytomegalovirus (HCMV) infection can result in lifelong neurological deficits. Seronegative pregnant woman often acquire primary HCMV from clinically asymptomatic, but HCMV‐shedding children. Methods Potential age‐related differences in viral and immune parameters of primary RhCMV infection were examined in an oral rhesus CMV infection model in specific pathogen free macaques. RhCMV shedding was measured by real time PCR in plasma, saliva and urine. Immune parameters, including neutralizing and binding antibodies and RhCMV‐specific T cell responses, were assessed in longitudinally collected blood samples. Results The oral RhCMV infection model in infant SPF rhesus macaques demonstrated that (i) the susceptibility to oral RhCMV infection declines with age, and (ii) infant macaques shed RhCMV more persistently and at higher titers compared to adult macaques. (iii) Conclusions The oral infant RhCMV infection model appears to reflect viral pathogenesis in human HCMV‐infected children. Larger studies are needed to define immune parameters associated with better control of RhCMV in adult compared to young animals.     

Hepatitis a outbreak in a public school in Rio de Janeiro,Brazil

From June 1 to July 1 1999, an outbreak involving 25 cases of hepatitis A occurred in a public school in Rio de Janeiro, Brazil. Since these cases were notified to the State Health Department, the National Reference Center for Hepatitis Viruses (CNRHV) was required to investigate the extent of hepatitis A virus (HAV) dissemination. Blood samples from all students were tested for IgM and total anti-HAV antibodies using a commercial enzyme-linked immunoassay (ELISA). At the same time, a questionnaire was completed in order to identify possible risk factors for HAV infection. The environmental investigation showed that there was no fecal contamination of the water supply. The epidemiological investigation demonstrated that almost 50% of this population was susceptible to HAV infection and probably person-to-person transmission was the principal mode of virus dissemination. In this situation, a massive vaccination campaign could control the HAV infection.     

Pathogenesis of Experimental Rhesus Cytomegalovirus Infection

Human cytomegalovirus (HCMV) establishes and maintains a lifelong persistence following infection in an immunocompetent host. The determinants of a stable virus-host relationship are poorly defined. A nonhuman primate model for HCMV was used to investigate virological and host parameters of infection in a healthy host. Juvenile rhesus macaques (Macaca mulatta) were inoculated with rhesus cytomegalovirus (RhCMV), either orally or intravenously (i.v. ), and longitudinally necropsied. None of the animals displayed clinical signs of disease, although hematologic abnormalities were observed intermittently in i.v. inoculated animals. RhCMV DNA was detected transiently in the plasma of all animals at 1 to 2 weeks postinfection (wpi) and in multiple tissues beginning at 2 to 4 wpi. Splenic tissue was the only organ positive for RhCMV DNA in all animals. The location of splenic cells expressing RhCMV immediate-early protein 1 (IE1) in i.v. inoculated animals changed following inoculation. At 4 to 5 wpi, most IE1-positive cells were perifollicular, and at 25 wpi, the majority were located within the red pulp. All animals developed anti-RhCMV immunoglobulin M (IgM) antibodies within 1 to 2 wpi and IgG antibodies within 2 to 4 wpi against a limited number of viral proteins. Host reactivity to RhCMV proteins increased in titer (total and neutralizing) and avidity with time. These results demonstrate that while antiviral immune responses were able to protect from disease, they were insufficient to eliminate reservoirs of persistent viral gene expression.     

Morphologic and immunophenotypic characteristics of malignant lymphomas in SIV-infected rhesus macaques

Eight cases of extranodal non-Hodgkin's lymphoma in simian immunodeficiency virus (SIV)-infected rhesus macaques, aged 4-9 years, were phenotypically and immunologically characterized, using the updated Kiel classification, in order to determine both the differences and the similarities between these types of lymphoma in immunodeficient rhesus macaques (Macaca mulatta) and man. The high-grade malignant tumors were of B-cell origin, with a predilection for extranodal growth in viscera and periorbital tissues. Immunophenotypical characterization showed that the monkey lymphomas were similar in many aspects to human immunodeficiency virus-associated lymphomas. The number of Ki67 positive cells varied from case to case and ranged from 50 to 90%. A serological screening for the simian equivalent of the Epstein-Barr virus (sEBV) by immunofluorescence assay revealed a prevalence of 92% of the sEBV antibodies in our cohort. The presence of Ebstein-Barr virus nuclear antigen (EBNA-2) could be demonstrated by immunohistochemistry in four out of eight cases. In situ hybridization revealed the presence of small EBV-encoded RNAs (EBER-1, EBER-2) in six of the eight cases. Further studies should define the precise role of herpesvirus infection for lymphomagenesis in SIV-induced immunodeficiency.     

Incidence and molecular characterization of hepatitis A viruses in Korean surface water between 2007 and 2010

Several bodies of surface water in Korea were surveyed for the presence of hepatitis A virus (HAV) between 2007 and 2010. Of 265 surface water samples, 9 (3.4%) were HAV‐positive. HAVs were mainly detected in the summer (3/62, 4.8%) and spring (4/96, 4.2%) seasons. Comparing different water sources, the highest prevalence (6.6%) of positive samples was seen in lake water, four HAV‐positive samples being from lakes. Comparing prevalence rates across the four representative Korean basin systems, no HAVs were found in the Han or Nakdong river basins. The highest HAV prevalence was found in samples from the Yeongsan river and other basins (6.3%); the Geum/Seom river was also found to have a high HAV prevalence (5.7%). HAVs from the nine positive samples were then sequenced and analyzed phylogenetically. Two of the HAVs belong to genotype IA and fall within the same cluster as HAVs 6‐3(ASAN4) (EU049548), KANSAN‐PS1 (EU049554), and ASAN‐KM (EU049563), which were collected from the stools of patients with gastroenteritis in Korea. The seven other HAV nucleotide sequences belong to the genotype IB cluster. This is the first nationwide surveillance of HAV in major Korean water sources.     

Development of Breeding Populations of Rhesus Macaques (Macaca mulatta) That Are Specific Pathogen-free for Rhesus Cytomegalovirus

Development of breeding colonies of rhesus macaques (Macaca mulatta) that are specific pathogen-free (SPF) for rhesus cytomegalovirus (RhCMV) is relatively straightforward and requires few modifications from current SPF programs. Infants separated from the dam at or within a few days of birth and cohoused with similarly treated animals remain RhCMV seronegative indefinitely, provided they are never directly or indirectly exposed to a RhCMV-infected monkey. By systematically cohousing seronegative animals into larger social cohorts, breeding populations of animals SPF for RhCMV can be established. The additional costs involved in expanding the current definition of SPF status to include RhCMV are incremental compared with the money already being spent on existing SPF efforts. Moreover, the large increase in research opportunities available for RhCMV-free animals arguably would far exceed the development costs. Potential new areas of research and further expansion of existing research efforts involving these newly defined SPF animals would have direct implications for improvements in human health.Abbreviations: HCMV, human cytomegalovirus; NHP, nonhuman primate; RhCMV, rhesus cytomegalovirus; SPF, specific pathogen-freeThe impetus for expanding the current SPF definition to include RhCMV is 2-fold. The first is the increasing number of studies involving infection of rhesus macaques with RhCMV as a nonhuman primate (NHP) model of human infection with human cytomegalovirus (HCMV). The second is the recognition that the current SPF protocols result in animals that are also uninfected with RhCMV, such that a relatively minor change in derivation can generate monkeys that meet the current SPF definition and that are uninfected with other endemic viruses, including RhCMV and simian foamy virus.     

猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。     

Seroepidemiological markers of enterically transmitted viral hepatitis A and E in individuals living in a community located in the North Area of Rio de Janeiro,RJ, Brazil

We investigated the seroprevalence of hepatitis A virus (HAV) and hepatitis E virus (HEV) infection in subjects living in the community of Manguinhos, Rio de Janeiro, Brazil, and assisted at the Health Unit of Escola Nacional de Saúde Pública, Funda??o Oswaldo Cruz. After formal consent, individuals were submitted to an interview using a standardized questionnaire. Anti-HAV and anti-HEV antibodies were detected by ELISA. Statistical analysis was carried out using the Epi-Info 6.04b software, to investigate possible associations between serological markers and risk factors. Results were regarded as significant when p value < 0.05. Although a high prevalence of anti-HAV was observed (87%), almost 50% of subjects under the age of 10 were susceptible to HAV infection, an unexpected rate in endemic areas. This fact could be attributed to improvements in environmental sanitation, occurring in this area in the last years. The increasing proportion of susceptible people may result in outbreaks of HAV infection, since the virus still circulates in this area, as verified by the detection of anti-HAV IgM in some individuals. No statistical association was met between HAV infection and the risk factors here assessed. The anti-HEV IgG prevalence found in this population was 2.4%, consistent with the one found in non-endemic areas.     

Optimization of a Novel Non-invasive Oral Sampling Technique for Zoonotic Pathogen Surveillance in Nonhuman Primates

Free-ranging nonhuman primates are frequent sources of zoonotic pathogens due to their physiologic similarity and in many tropical regions, close contact with humans. Many high-risk disease transmission interfaces have not been monitored for zoonotic pathogens due to difficulties inherent to invasive sampling of free-ranging wildlife. Non-invasive surveillance of nonhuman primates for pathogens with high potential for spillover into humans is therefore critical for understanding disease ecology of existing zoonotic pathogen burdens and identifying communities where zoonotic diseases are likely to emerge in the future. We developed a non-invasive oral sampling technique using ropes distributed to nonhuman primates to target viruses shed in the oral cavity, which through bite wounds and discarded food, could be transmitted to people. Optimization was performed by testing paired rope and oral swabs from laboratory colony rhesus macaques for rhesus cytomegalovirus (RhCMV) and simian foamy virus (SFV) and implementing the technique with free-ranging terrestrial and arboreal nonhuman primate species in Uganda and Nepal. Both ubiquitous DNA and RNA viruses, RhCMV and SFV, were detected in oral samples collected from ropes distributed to laboratory colony macaques and SFV was detected in free-ranging macaques and olive baboons. Our study describes a technique that can be used for disease surveillance in free-ranging nonhuman primates and, potentially, other wildlife species when invasive sampling techniques may not be feasible.     

The chronic course of spontaneous and experimental hepatitis A in rhesus monkeys with viral persistence]

The prolonged (up to 2 years) complex observation of 11 rhesus macaques (Macaca mulatta) with spontaneous hepatitis A and 14 rhesus macaques with experimental hepatitis A developing after their intravenous and/or oral infection with human hepatitis A virus (HAV). Both natural and experimental infection took a chronic course (15-18 months). In 13 monkeys showing morphological changes in the liver during the whole period of the disease elevated enzyme levels in the blood and virus shedding in feces were periodically observed. Only one monkey had acute hepatitis A which lasted 1.5 months. In 11 monkeys the disease took an undulating course with 1-2 relapses when virological, biochemical and morphological signs of the disease could be detected. Seroconversion was observed in all monkeys. Anti-HAV IgM antibodies were retained for not more than 6-7 months and total anti-HAV antibodies, during the whole period of observation. Relapses were found to induce no antibody formation. Evidence on the prolonged (up to 12-16 months) persistence of HAV in primates was obtained for the first time.     

Serological markers of hepatitis B virus and cytomegalovirus infections in Norwegians with coagulation factor defects

The prevalence of serological markers for present and past hepatitis B virus (HBV) infection and antibodies against cytomegalovirus (CMV) among Norwegians with coagulation factor defects was examined in serum samples collected before virus-inactivated coagulation concentrates came into use. Sera collected in 1985/86 from 324 of 377 (86%) registered persons with such defects were available. Three persons were chronic carriers of HBsAg. The prevalence of HBV antibodies was 28% compared with about 5% in the general population. The highest prevalence rate was found among patients with severe haemophilia A (44%) and in patients with haemophilia B (39%). The prevalence of anti-CMV antibodies was 75% which is similar to that found in the general Norwegian population.