Experimental infection of Japanese macaques with simian retrovirus 5.

Recently, a large number of Japanese macaques (Macaca fuscata) died of an unknown hemorrhagic syndrome at Kyoto University Primate Research Institute (KUPRI) and an external breeding facility for National Institute for Physiological Sciences (NIPS). We previously reported that the hemorrhagic syndrome of Japanese macaques at KUPRI was caused by infection with simian retrovirus 4 (SRV-4); however, the cause of similar diseases that occurred at the external breeding facility for NIPS was still unknown. In this study, we isolated SRV-5 from Japanese macaques exhibiting thrombocytopenia and then constructed an infectious molecular clone of the SRV-5 isolate. When the SRV-5 isolate was inoculated into two Japanese macaques, severe thrombocytopenia was induced in one of two macaques within 22 days after inoculation. Similarly, the clone-derived virus was inoculated into the other two Japanese macaques, and one of two macaques developed severe thrombocytopenia within 22 days. On the other hand, the remaining two of four macaques survived as asymptomatic carriers even after administering an immunosuppressive agent, dexamethasone. As determined by real-time PCR, SRV-5 infected a variety of tissues in Japanese macaques, especially in digestive and lymph organs. We also identified the SRV-5 receptor as ASCT2, a neutral amino acid transporter in Japanese macaques. Taken together, we conclude that the causative agent of hemorrhagic syndrome occurred at the external breeding facility for NIPS was SRV-5.

Identification of novel simian endogenous retroviruses that are indistinguishable from simian retrovirus (SRV) on current SRV diagnostic assays.

Simian betaretroviruses include the well-known exogenous simian retroviruses (SRV-1 through SRV-8), and some closely related simian endogenous retroviruses (SERV). Here, we characterized two new viral genomes, which appear to represent novel SERVs but have characteristics of both SRV and SERV highlighting the need to develop new assays providing molecular and serologic differentiation of SERV and SRV to avoid false positives.

Evidence of simian retrovirus type D by polymerase chain reaction.

BACKGROUND: Over the past few years, there have been reports of finding Simian retrovirus type D (SRV) in macaque colonies where some animals were characterized as antibody positive but virus negative raising questions about how SRV was transmitted or whether there is a variant strain detected by antibody but not polymerase chain reaction (PCR) in current use. METHODS: We developed a three-round nested PCR assay using degenerate primers targeting the pol gene to detect for SRV serotypes 1-5 and applied this newly validated PCR assay to test macaque DNA samples collected in China from 2010 to 2015. RESULTS: Using the nested PCR assay validated in this study, we found 0.15% of the samples archived on FTA® cards were positive. CONCLUSIONS: The source of SRV infection identified within domestic colonies might have originated from imported macaques. The multiplex nested PCR assay developed here may supplement the current assays for SRV.

High Prevalences and a Wide Genetic Diversity of Simian Retroviruses in Non-human Primate Bushmeat in Rural Areas of the Democratic Republic of Congo.

Like the majority of emerging infectious diseases, HIV and HTLV are of zoonotic origin. Here we assess the risk of cross-species transmissions of their simian counterparts, SIV and STLV, from non-human primates (NHP) to humans in the Democratic Republic of Congo (DRC). A total of 331 samples, derived from NHP bushmeat, were collected as dried blood spots (DBS, n = 283) or as tissue samples (n = 36) at remote forest sites mainly in northern and eastern DRC. SIV antibody prevalences in DBS were estimated with a novel high throughput immunoassay with antigens representing the actual known diversity of HIV/SIV lineages. Antibody-positive samples were confirmed by PCR and sequence analysis. Screening for STLV infection was done with universal primers in tax, and new strains were further characterized in LTR. SIV and STLV infection in tissue samples was done by PCR only. Overall, 5 and 15.4% of NHP bushmeat was infected with SIV and STLV, respectively. A new SIV lineage was identified in Allen's swamp monkeys (Allenopithecus nigroviridis). Three new STLV-1 subtypes were identified in Allen's swamp monkeys (Allenopithecus nigroviridis), blue monkeys (Cercopithecus mitis), red-tailed guenons (Cercopithecus ascanius schmidti) and agile mangabeys (Cercocebus agilis). SIV and STLV prevalences varied according to species and geographic region. Our study illustrates clearly, even on a small sample size from a limited number of geographic areas, that our knowledge on the genetic diversity and geographic distribution of simian retroviruses is still limited and that humans continue to be exposed to relative high proportions on infected NHP bushmeat.

A novel simian retrovirus subtype discovered in cynomolgus monkeys (Macaca fascicularis).

A new simian retrovirus (SRV) subtype was discovered in China and the USA from Cambodian-origin cynomolgus monkeys. Histopathological examination from necropsied animals showed multifocal lymphoplasmacystic and histocytic inflammation. The complete genome sequences demonstrated that the US virus isolates were nearly identical (99.91-99.93 %) and differed only slightly (99.13-99.16 % identical) from the China isolate. Phylogenetic analysis showed that the new virus isolates formed a distinct branch of SRV-1 through -7, and therefore were named this subtype, SRV-8. This SRV-8 variant was also phylogenetically and serologically more closely related to SRV-4 than any other SRV subtype.

Evaluation of Vero-cell-derived simian endogenous retrovirus infection in humans by detection of viral genome in clinicopathological samples and commercialized vaccines and by serology of Japanese general population.

BACKGROUND: Vero cells are used in laboratories for the isolation of viruses and the production of vaccines. Recently, the sequence of simian endogenous retrovirus (SERV) was identified in Vero cells (SERVagm-Vero), with homology to exogenously transmitted, pathogenic simian retroviruses (SRVs). Although SERVagm-Vero was shown to be noninfectious to human cells in vitro, SERV infection in humans is controversial. In this study, we evaluated the status of SERV infection in humans by detecting the viral genome in clinicopathological samples and commercialized vaccines, and with a serological survey of the Japanese general population. METHODS: Real-time polymerase chain reaction (PCR) and reverse transcription-PCR were used to detect the SERVagm-Vero genome. We also examined the seroprevalence of SERV in 1000 individuals in the Japanese population with an enzyme-linked immunoabsorbent assay (ELISA) using mixed SERVagm-Vero gag, pol, and env proteins as antigens. RESULTS: Real-time PCR failed to detect SERVagm-Vero genomic fragments in 783 human clinicopathological samples, and all 13 human cell lines tested were negative for the SERVagm-Vero genome. Thirteen commercialized vaccines, including five Vero-based vaccines, were also negative for the SERVagm-Vero genome on real-time PCR and reverse transcription-PCR. Eight (0.8%) were seropositive on ELISA, and western blotting showed that all eight sera contained anti-pol antibodies. All SERV-seropositive individuals were born before 1965, suggesting that SERV infection in Japan might not be associated with vaccine, because more than 90% of Japanese children born from 1964 to 2012 have received live poliovirus vaccines containing virus produced in Vero cells since the 1980s. CONCLUSION: We have confirmed that the vaccines we use today are free of SERVagm-Vero. Moreover, SERV infection in humans is very rare and unlikely to be associated with vaccines in the Japanese general population.

Experimental evaluation of the zoonotic infection potency of simian retrovirus type 4 using humanized mouse model.

During 2001-2002 and 2008-2011, two epidemic outbreaks of infectious hemorrhagic disease have been found in Japanese macaques (Macaca fuscata) in Kyoto University Primate Research Institute, Japan. Following investigations revealed that the causative agent was simian retrovirus type 4 (SRV-4). SRV-4 was isolated by using human cell lines, which indicates that human cells are potently susceptible to SRV-4 infection. These raise a possibility of zoonotic infection of pathogenic SRV-4 from Japanese macaques into humans. To explore the possibility of zoonotic infection of SRV-4 to humans, here we use a human hematopoietic stem cell-transplanted humanized mouse model. Eight out of the twelve SRV-4-inoculated humanized mice were infected with SRV-4. Importantly, 3 out of the 8 infected mice exhibited anemia and hemophagocytosis, and an infected mouse died. To address the possibility that SRV-4 adapts humanized mouse and acquires higher pathogenicity, the virus was isolated from an infected mice exhibited severe anemia was further inoculated into another 6 humanized mice. However, no infected mice exhibited any illness. Taken together, our findings demonstrate that the zoonotic SRV-4 infection from Japanese macaques to humans is technically possible under experimental condition. However, such zoonotic infection may not occur in the real society.

Emergence of infectious malignant thrombocytopenia in Japanese macaques (Macaca fuscata) by SRV-4 after transmission to a novel host.

We discovered a lethal hemorrhagic syndrome arising from severe thrombocytopenia in Japanese macaques kept at the Primate Research Institute, Kyoto University. Extensive investigation identified that simian retrovirus type 4 (SRV-4) was the causative agent of the disease. SRV-4 had previously been isolated only from cynomolgus macaques in which it is usually asymptomatic. We consider that the SRV-4 crossed the so-called species barrier between cynomolgus and Japanese macaques, leading to extremely severe acute symptoms in the latter. Infectious agents that cross the species barrier occasionally amplify in virulence, which is not observed in the original hosts. In such cases, the new hosts are usually distantly related to the original hosts. However, Japanese macaques are closely related to cynomolgus macaques, and can even hybridize when given the opportunity. This lethal outbreak of a novel pathogen in Japanese macaques highlights the need to modify our expectations about virulence with regards crossing species barriers.

Nonhuman primate retroviruses from Cambodia: high simian foamy virus prevalence, identification of divergent STLV-1 strains and no evidence of SIV infection.

Nonhuman primates (NHPs) carry retroviruses such as simian immunodeficiency viruses (SIV), simian T-cell lymphotropic viruses (STLV) and simian foamy viruses (SFV). Here, we revisited NHPs from Cambodia to assess the prevalence and diversity of these retroviruses using updated viral detection tools. We screened blood from 118 NHPs consisting of six species (Macaca fascicularis (n=91), Macaca leonine (n=8), Presbytis cristata (n=3), Nycticebus coucang (n=1), Hylobates pileatus (n=14), and Pongo pygmaeus) (n=1) by using a Luminex-based multiplex serology assay that allows the detection of all known SIV/HIV and SFV lineages. We also used highly sensitive PCR assays to detect each simian retrovirus group. Positive PCR products were sequenced and phylogenetically analyzed to infer evolutionary histories. Fifty-three of 118 (44.9%) NHPs tested positive for SFV by serology and 8/52 (15.4%), all from M. fascicularis, were PCR-confirmed. The 8 novel SFV sequences formed a highly supported distinct lineage within a clade composed of other macaque SFV. We observed no serological or molecular evidence of SIV infection among the 118 NHP samples tested. Four of 118 (3.3%) NHPs were PCR-positive for STLV, including one M. fascicularis, one P. cristata, and two H. pileatus. Phylogenetic analyses revealed that the four novel STLV belonged to the PTLV-1 lineage, outside the African radiation of PTLV-1, like all Asian PTLV identified so far. Sequence analysis of the whole STLV-1 genome from a H. pileatus (C578_Hp) revealed a genetic structure characteristic of PTLV. Similarity analysis comparing the STLV-1 (C578_Hp) sequence with prototype PTLVs showed that C578_Hp is closer to PTLV-1s than to all other types across the entire genome. In conclusion, we showed a high frequency of SFV infection but found no evidence of SIV infection in NHPs from Cambodia. We identified for the first time STLV-1 in a P. cristata and in two H. pileatus.

Isolation and DNA characterization of a simian retrovirus 5 from a Japanese monkey (Macaca fuscata).

An SRV-like virus was isolated from a colony-born Japanese monkey. To identify this SRV-like virus, we designed universal primers at regions that were conserved among the reported SRV sequences in the 5'-LTR and the short ORF and we obtained plasmid clones containing the complete gag, prt, pol and env genes. The full-length sequences of the isolate were determined from the plasmids and by direct sequencing. Sequence comparisons and phylogenetic analyses indicated that this SRV-like virus had a sequence identical to the reported 626 bp of SRV-5. In this study, we isolated SRV5/JPN/2005/V1 from a Japanese monkey and characterized the full-length SRV-5 sequence.

Simian retroviruses in African apes.

It is now well established that simian immunodeficiency viruses (SIVs) from chimpanzees (SIVcpz) and gorillas (SIVgor) from west Central Africa are at the origin of HIV-1/AIDS. Apes are also infected with other retroviruses, notably simian T-cell lymphotropic viruses (STLVs) and simian foamy viruses (SFVs), that can be transmitted to humans. We discuss the actual knowledge on SIV, STLV and SFV infections in chimpanzees, gorillas, and bonobos. We especially elaborate on how the recent development of non-invasive methods has allowed us to identify the reservoirs of the HIV-1 ancestors in chimpanzees and gorillas, and increased our knowledge of the natural history of SIV infections in chimpanzees. Multiple cross-species events with retroviruses from apes to humans have occurred, but only one transmission of SIVcpz from chimpanzees in south-eastern Cameroon spread worldwide, and is responsible for the actual HIV pandemic. Frequent SFV transmissions have been recently reported, but no human-to-human transmission has been documented yet. Because humans are still in contact with apes, identification of pathogens in wild ape populations can signal which pathogens may be cause risk for humans, and allow the development of serological and molecular assays with which to detect transmissions to humans. Finally, non-invasive sampling also allows the study of the impact of retroviruses and other pathogens on the health and survival of endangered species such as chimpanzees, gorillas, and bonobos.

[Epidemiological survey of a captive Chinese rhesus macaque breeding colony in Yunnan for SRV, STLV and BV].

Nonhuman primates are critical resources for biomedical research. Rhesus macaque is a popularly used laboratory nonhuman primate that share many characteristics with humans. However, rhesus macaques are the natural host of two exogenous retroviruses, SRV (simian type D retrovirus) and STLV (simian T lymphotropic virus). SRV and STLV may introduce potentially significant confounding factors into the study of AIDS model. Moreover, B virus (ceropithecine herpesvirus 1) is likely to harm not only rhesus macaque but also humans in experiments involving rhesus macaque. Yunnan province has large-scale breeding colonies of Chinese rhesus macaque. Therefore there is an urgent need for SPF Chinese rhesus macaque colonies. Here we investigated SRV, STLV and BV infections in 411 Chinese rhesus macaque by PCR technique. The results showed that the prevalence of SRV, STLV and BV among Chinese rhesus macaque breeding colony was 19.71% (81/411), 13.38% (55/411) and 23.11% (95/411), respectively. Comparison of viruses infection in different age-groups and male/female of Chinese rhesus macaque was also analyzed. This study will contribute to establishment of SPF Chinese rhesus macaque breeding colony.

Virological and serological characterization of SRV-4 infection in cynomolgus macaques.

The nature of SRV-4 infection in cynomolgus macaques remains unclear to date. Here, we report the monitoring of 24 cynomolgus monkeys that were naturally infected with SRV-4 for virus isolation, proviral load and antibody. The results indicated that the SRV-4 antibody status was statistically correlated to environmental temperature.

Hemophagocytic syndrome in a pancytopenic simian retrovirus-infected male rhesus macaque (Macaca mulatta).

Hemophagocytic syndrome (HPS) is a macrophage hyperactivation disorder triggered by disrupted T-cell macrophage cytokine interaction. HPS has been reported in humans, dogs, cats, and cattle, and it is infrequent and poorly characterized in animals. A 16-year-old male rhesus macaque was euthanized because of severe pancytopenia, including nonregenerative anemia (hematocrit = 5.5%), neutropenia (0.29 K/µl), and thrombocytopenia (21 K/µl). Bone marrow was hypocellular with normal maturation, myeloid hypoplasia, and few megakaryocytes. There were numerous morphologically normal macrophages (12% of nucleated cells), with 6% of nucleated cells being hemophagocytic macrophages in the bone marrow. Serology was negative, but polymerase chain reaction and immunohistochemistry were positive for simian retrovirus type 2. Blood and bone marrow findings were consistent with HPS. Cytopenias are common in simian retrovirus-infected macaques, but HPS has not been reported. An association between simian retrovirus infection and HPS is undetermined, but retrovirus-associated HPS has been observed in humans.

The complete genome and genetic characteristics of SRV-4 isolated from cynomolgus monkeys (Macaca fascicularis).

At least 5 serotypes of exogenous simian retrovirus type D (SRV/D) have been found in nonhuman primates, but only SRV-1, 2 and 3 have been completely sequenced. SRV-4 was recovered once from cynomolgus macaques in California in 1984, but its genome sequences are unknown. Here we report the second identification of SRV-4 and its complete genome from infected cynomolgus macaques with Indochinese and Indonesian/Indochinese mixed ancestry. Phylogenetic analysis demonstrated that SRV-4 was distantly related to SRV-1, 2, 3, 5, 6 and 7. SRV/D-T, a new SRV/D recovered in 2005 from cynomolgus monkeys at Tsukuba Primate Center in Japan, clustered with the SRV-4 isolates from California and Texas and was shown to be another occurrence of SRV-4 infection. The repeated occurrence of SRV-4 in cynomolgus monkeys in different areas of the world and across 25years suggests that this species is the natural host of SRV-4.

Simian retroviruses: infection and disease--implications for immunotoxicology research in primates.

Non-human primates have assumed an important role in preclinical safety assessment studies, particularly in the evaluation of biopharmaceutical and immunomodulatory therapies. Naturally occurring simian retrovirus infections may adversely affect the suitability of primates for use in such studies. Various species of non-human primates are the natural hosts for six exogenous retroviruses, representing five genera within the family Retroviridae. Retroviruses establish persistent infections with a broad spectrum of pathogenic potential, ranging from nonpathogenic to highly pathogenic, depending on the variety of the host, virus, and environmental factors. In the context of immunotoxicology, in which the research objective is to specifically evaluate the effect of drugs or biologics on the immune system, the immune modulatory effects of simian retroviruses, which may be subtle or profound, may introduce significant confounding into the studies of immunotoxic effects utilizing non-human primates. Latent or subclinical retrovirus infections are common and research-related procedures may lead to virus reactivation or overt disease. Adverse effects of undetected retrovirus infections on preclinical research include the loss of experimental subjects (and potentially of statistical power) due to increased morbidity and mortality, virus-induced clinical abnormalities, histologic lesions, alteration of physiologic parameters and biologic responses, and interference with in vitro assays and/or cytolytic destruction of primary cell cultures. The aim of this review is to provide an overview of the key biological, clinical, and pathological features of several important simian retroviruses, with emphasis on viruses infecting macaques and other primate species commonly used in preclinical research, and a discussion of the implications of these infections for immunotoxicology and other preclinical research in primates. Adequate pre-study retrovirus screening is essential to exclude retrovirus-infected primates from research protocols.

Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in human and other primate cell lines.

The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections.

Detection of SRV/D shedding in body fluids of cynomolgus macaques and comparison of partial gp70 sequences in SRV/D-T isolates.

We previously reported the isolation of a novel subtype of SRV/D-Tsukuba (SRV/D-T) from two cynomolgus monkeys (Macaca facicularis) in the breeding colony of Tsukuba Primate Research Center (TPRC). We surveyed for SRV/D infection in the TPRC cynomolgus colony using SRV/D-specific PCR primer sets designed based on the entire gag region sequence. The only SRV/D subtype detected in the colony was SRV/D-T with a positive infection rate of 22.4% (n = 49). It has been reported that the mode of transmission of SRV/D is via contact with virus shed in the body fluids. In this report, to investigate the infection route of SRV/D-T in monkeys at TPRC, we performed virus isolation and PCR for detection of the SRV/D genome from peripheral blood mononuclear cells (PBMCs), plasma, saliva, urine, and feces. Virus isolation and PCR detection were positive in plasma, saliva, urine, and fecal samples from all monkeys on which virus was isolated from PBMCs. This suggests that the spread of SRV/D-T infection in TPRC is via contact with virus shed in saliva, urine, and/or feces. Also, comparison of sequences of gp70 on multiple SRV/D-T isolates revealed that there was little intra- and inter-monkey variation, suggesting that SRV/D-T is fairly stable.

Survey of captive cynomolgus macaque colonies for SRV/D infection using polymerase chain reaction assays.

The exogenous simian type D retroviruses (SRV/Ds) are prevalent in macaque monkeys and sometimes cause immunodeficiency with anemia, weight loss, and persistent unresponsive diarrhea. SRV/D isolates are classified as subtypes 1 to 6, and the entire sequences of the gag region of SRV/D-1, -2, and -3 and SRV/D-Tsukuba (SRV/D-T) have been determined. We designed specific primers in the gag region of SRV/D-T that enabled us to directly detect by polymerase chain reaction (PCR) SRV/D-T proviral DNA sequences in DNA extracted from whole blood. Using this assay and another PCR assay that detects multiple SRV/D subtypes, we performed a survey for SRV/D infection in our specific pathogen-free (SPF) and conventional colonies at Tsukuba Primate Center (TPC). In the SPF colony, no SRV/D signal was detected in any animal. On the other hand, SRV/D-T was detected in 11 of 49 animals (22.5%) in the conventional colony. SRV/D-T was the only SRV/D subtype detected. Consequently, SRV/D-T is the major SRV/D subtype present in cynomolgus monkeys at TPC.

Isolation and characterization of a new simian retrovirus type D subtype from monkeys at the Tsukuba Primate Center, Japan.

Exogenous type D simian retroviruses (SRV/D) are prevalent in captive and feral populations of various macaque monkeys. Thus far, five subtypes of SRV/Ds have been reported, three of which (SRV-1, -2 and -3) have been molecularly characterized. Two SRV/D strains (N27 and T150) were isolated from seropositive cynomolgus macaques at the Tsukuba Primate Center (TPC) in Japan, showing clinical signs of SRV/D infection, including anemia and persistent unresponsive diarrhea. Electron microscopy demonstrated that both SRV/D isolates have a virion morphology typical of type D retrovirus. The SRV/D N27 and T150 isolates were essentially the same based on sequence analysis. From homology analysis of the entire gag sequence, the N27 isolate is closely related to the other known SRV/Ds but is distinct from the three molecularly characterized SRV/Ds. Thus, we have tentatively designated the N27 and T150 viruses isolated from TPC cynomolgus macaques as SRV/D-Tsukuba (SRV/D-T).