Specific Detection of Two Divergent Simian Arteriviruses Using RNAscope In Situ Hybridization

Simian hemorrhagic fever (SHF) is an often lethal disease of Asian macaques. Simian hemorrhagic fever virus (SHFV) is one of at least three distinct simian arteriviruses that can cause SHF, but pathogenesis studies using modern methods have been scarce. Even seemingly straightforward studies, such as examining viral tissue and cell tropism in vivo, have been difficult to conduct due to the absence of standardized SHFV-specific reagents. Here we report the establishment of an in situ hybridization assay for the detection of SHFV and distantly related Kibale red colobus virus 1 (KRCV-1) RNA in cell culture. In addition, we detected SHFV RNA in formalin-fixed, paraffin-embedded tissues from an infected rhesus monkey (Macaca mulatta). The assay is easily performed and can clearly distinguish between SHFV and KRCV-1. Thus, if further developed, this assay may be useful during future studies evaluating the mechanisms by which a simian arterivirus with a restricted cell tropism can cause a lethal nonhuman primate disease similar in clinical presentation to human viral hemorrhagic fevers.     

Exceptional Simian Hemorrhagic Fever Virus Diversity in a Wild African Primate Community

Simian hemorrhagic fever virus (SHFV) is an arterivirus that causes severe disease in captive macaques. We describe two new SHFV variants subclinically infecting wild African red-tailed guenons (Cercopithecus ascanius). Both variants are highly divergent from the prototype virus and variants infecting sympatric red colobus (Procolobus rufomitratus). All known SHFV variants are monophyletic and share three open reading frames not present in other arteriviruses. Our data suggest a need to modify the current arterivirus classification.     

Novel, divergent simian hemorrhagic fever viruses in a wild Ugandan red colobus monkey discovered using direct pyrosequencing

Background

Simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but its distribution in wild primates has remained obscure. Here, we describe the discovery and genetic characterization by direct pyrosequencing of two novel, divergent SHFV variants co-infecting a single male red colobus monkey from Kibale National Park, Uganda.

Methodology/Principal Findings

The viruses were detected directly from blood plasma using pyrosequencing, without prior virus isolation and with minimal PCR amplification. The two new SHFV variants, SHFV-krc1 and SHFV-krc2 are highly divergent from each other (51.9% nucleotide sequence identity) and from the SHFV type strain LVR 42-0/M6941 (52.0% and 51.8% nucleotide sequence identity, respectively) and demonstrate greater phylogenetic diversity within SHFV than has been documented within any other arterivirus. Both new variants nevertheless have the same 3′ genomic architecture as the type strain, containing three open reading frames not present in the other arteriviruses.

Conclusions/Significance

These results represent the first documentation of SHFV in a wild primate and confirm the unusual 3′ genetic architecture of SHFV relative to the other arteriviruses. They also demonstrate a degree of evolutionary divergence within SHFV that is roughly equivalent to the degree of divergence between other arterivirus species. The presence of two such highly divergent SHFV variants co-infecting a single individual represents a degree of within-host viral diversity that exceeds what has previously been reported for any arterivirus. These results expand our knowledge of the natural history and diversity of the arteriviruses and underscore the importance of wild primates as reservoirs for novel pathogens.     

Zoonotic Potential of Simian Arteriviruses

Wild nonhuman primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae. Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild nonhuman primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be “preemergent” zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur.     

Enzyme-linked immunosorbent assay for detection of antibodies against simian hemorrhagic fever virus

Better assays are needed for the detection of simian hemorrhagic fever virus (SHFV), which induces persistent infection without overt signs of disease in most old world monkeys, but causes a fatal hemorrhagic fever in macaques. An enzyme-linked immunosorbent assay (ELISA) is described here that is useful in identifying primates previously exposed to SHFV. This assay involves testing serum samples against SHFV and cell antigens to obtain an ODvirus-to-ODcell ratio that eliminates potential high background values associated with primate serum. High correlation was found using this assay, compared with that found with the current "gold standard" indirect immunofluorescence assay (IFA). However, this ELISA is less time consuming, less subjective, and not as prone to human error than the SHFV-IFA.     

Elimination of persistent simian hemorrhagic fever (SHF) virus infection in patas monkeys

Devastating epizootics of simian hemorrhagic fever (SHF) have been iatrogenically initiated in captive colonies of macaque monkeys by strains of SHF virus emanating from asymptomatic persistently infected patas monkeys. We have found that persistently infected patas monkeys can be cleared of their infection by superinfection with strains of SHF virus which cause acute infections in this species. All 20 persistently infected animals subjected to this procedure have been cleared of their infection within 3 months. These animals were shown to be virus free by the most sensitive in vitro and in vivo tests currently available and periodic tests of serum from these animals over several years have shown them to remain virus free. Superinfection has in some cases caused some adverse clinical symptoms (anorexia, lethargy, facial edema, dehydration, and mild subcutaneous hemorrhages), but with supportive care, no fatal infections have occurred. Thus, superinfection with acute strains of SHF virus is a highly effective method of eliminating persistent infection in patas monkeys.     

Molecular characterization of the 3' terminus of the simian hemorrhagic fever virus genome.

The 3' end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and sequenced. Adjacent to the 3' poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two overlapping reading frames (ORFs). The ultimate 3' ORF of the viral genome encodes the capsid protein, and the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26 nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3' coterminal. In its virion morphology and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydrogenase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.     

Monkey viral pathology in the Sukhum colony and modeling human viral infections

The data characterizing spontaneous infections of Old World monkeys: measles, poliomyelitis, hepatitis A (HPA), encephalomyocarditis, coronavirus infection, simian hemorrhagic fever (SHF), are presented. The experimental infections were reproduced with the isolated pathogens. On these models, pathogenesis and epidemiology of these diseases were studied. The efficiency of poliomyelitis, measles and HPA vaccines is shown. The priority of data on the discovery of earlier unknown disease—SHF and “Sukhumi” virus—are emphasized. Several important pathogenic mechanisms common for various hemorrhagic fevers were studied on experimental SHF of macaques. This model is uniquely safe and adequate for the assessment of therapy of hemorrhagic fevers dangerous for humans.     

Autoantibodies against golgi apparatus induced by arteriviruses.

Members of the genus Arterivirus within the monogeneric family Arteriviridae are lactate dehydrogenase-elvating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), equine arteritis virus (EAV) and simian hemorrhagic fever virus. In LDV-infected mice the appearance of autoantibodies against Golgi-antigen dominated the early immune response. Shared antigenicity between LDV and Golgi-antigen of normal cells could not be demonstrated. Monoclonal antibodies (MAbs) reacted either with LDV or with Golgi-antigen but not with both. Immunization of mice with the porcine arterivirus PRRSV, however, led to the establishment of MAbs that recognized the structural glycoprotein GP3 as well as Golgi-antigen of normal porcine cells indicating molecular mimicry of viral and cellular antigen. In addition to cross-reactive antibodies MAbs solely reactive with Golgi-antigen were observed. After immunization of mice with EAV, the equine arterivirus, clones were isolated producing Golgi-antigen recognizing autoantibodies. Morphogenesis of arteriviruses occurs in the Golgi region. The autoimmune responses following immunization with arteriviruses may offer an approach for determining the mechanism by which such responses develop and become of biologic importance.     

Differential Responses of Disease-Resistant and Disease-Susceptible Primate Macrophages and Myeloid Dendritic Cells to Simian Hemorrhagic Fever Virus Infection

Simian hemorrhagic fever virus (SHFV) causes a fatal hemorrhagic fever in macaques but an asymptomatic, persistent infection in baboons. To investigate factors contributing to this differential infection outcome, the targets of SHFV infection, macrophages (MΦs) and myeloid dendritic cells (mDCs), were differentiated from macaque and baboon peripheral blood monocytes and used to compare viral replication and cell responses. SHFV replicated in >90% of macaque MΦs but in only ∼10% of baboon MΦs. Although SHFV infected ∼50% of macaque and baboon mDCs, virus replication was efficient in macaque but not in baboon mDCs. Both types of macaque cultures produced higher virus yields than baboon cultures. A more efficient type I interferon response and the production of proinflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-12/23(p40), tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 1α (MIP-1α), in response to SHFV infection were observed in macaque but not baboon cultures, suggesting less efficient counteraction of these responses by viral proteins in macaque cells. Baboon cultures produced higher levels of IL-10 than macaque cultures both prior to and after SHFV infection. In baboon but not macaque cell cultures, SHFV infection upregulated IL-10R1, a subunit of the IL-10 receptor (IL-10R), and also SOCS3, a negative regulator of proinflammatory cytokine production. Incubation of macaque cultures with human IL-10 before and/or after SHFV infection decreased production of IL-6, IL-1β, and MIP-1α but not TNF-α, suggesting a role for IL-10 in suppressing SHFV-induced proinflammatory cytokine production in macaques.     

A 68-Nucleotide Sequence within the 3′ Noncoding Region of Simian Hemorrhagic Fever Virus Negative-Strand RNA Binds to Four MA104 Cell Proteins

The 3′ noncoding region (NCR) of the negative-strand RNA [3′(−)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3′ region, designated 3′(−)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5′ leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3′(−)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3′ end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3′(−)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3′(−)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3′(−)209 RNA also bind to the LDV-C 3′(−)NCR RNA and equine arteritis virus 3′(−)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3′(−)NCR and SHFV 3′(−)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.     

Method to detect asymptomatic carriers of simian hemorrhagic fever virus

Evidence was obtained that mononuclear phagocytic cells are the target cells for simian hemorrhagic fever virus replication. Using peritoneal macrophages from rhesus monkeys in an in vitro, 18 of 20 asymptomatic chronically infected patas monkeys were detected from coded samples. The two chronically infected patas monkeys not detected by the test, nevertheless, contained virus. This was determined by inoculating macrophage cultures with plasma from macaques dying as a result of inoculation with plasma from these chronically infected animals. in addition to virus found in chronically infected animals, all isolates of simian hemorrhagic fever virus tested previously described epizootics lytically infected rhesus monkey macrophages. These data suggested that the highly fatal nature of simian hemorrhagic fever in macaques was related to the extreme sensitivity of their mononuclear phagocytic cells to infection and lysis.     

Successful topical respiratory tract immunization of primates against Ebola virus

Ebola virus causes outbreaks of severe viral hemorrhagic fever with high mortality in humans. The virus is highly contagious and can be transmitted by contact and by the aerosol route. These features make Ebola virus a potential weapon for bioterrorism and biological warfare. Therefore, a vaccine that induces both systemic and local immune responses in the respiratory tract would be highly beneficial. We evaluated a common pediatric respiratory pathogen, human parainfluenza virus type 3 (HPIV3), as a vaccine vector against Ebola virus. HPIV3 recombinants expressing the Ebola virus (Zaire species) surface glycoprotein (GP) alone or in combination with the nucleocapsid protein NP or with the cytokine adjuvant granulocyte-macrophage colony-stimulating factor were administered by the respiratory route to rhesus monkeys--in which HPIV3 infection is mild and asymptomatic--and were evaluated for immunogenicity and protective efficacy against a highly lethal intraperitoneal challenge with Ebola virus. A single immunization with any construct expressing GP was moderately immunogenic against Ebola virus and protected 88% of the animals against severe hemorrhagic fever and death caused by Ebola virus. Two doses were highly immunogenic, and all of the animals survived challenge and were free of signs of disease and of detectable Ebola virus challenge virus. These data illustrate the feasibility of immunization via the respiratory tract against the hemorrhagic fever caused by Ebola virus. To our knowledge, this is the first study in which topical immunization through respiratory tract achieved prevention of a viral hemorrhagic fever infection in a primate model.     

Aggressive organ penetration and high vector transmissibility of epidemic dengue virus-2 Cosmopolitan genotype in a transmission mouse model

Dengue virus (DENV) causes dengue fever and severe hemorrhagic fever in humans and is primarily transmitted by Aedes aegypti and A. albopictus mosquitoes. The incidence of DENV infection has been gradually increasing in recent years due to global urbanization and international travel. Understanding the virulence determinants in host and vector transmissibility of emerging epidemic DENV will be critical to combat potential outbreaks. The DENV serotype 2 (DENV-2), which caused a widespread outbreak in Taiwan in 2015 (TW2015), is of the Cosmopolitan genotype and is phylogenetically related to the virus strain linked to another large outbreak in Indonesia in 2015. We found that the TW2015 virus was highly virulent in type I and type II interferon-deficient mice, with robust replication in spleen, lung, and intestine. The TW2015 virus also had high transmissibility to Aedes mosquitoes and could be effectively spread in a continuous mosquitoes-mouse-mosquitoes-mouse transmission cycle. By making 16681-based mutants carrying different segments of the TW2015 virus, we identified the structural pre-membrane (prM) and envelope (E) genes as key virulence determinants in the host, with involvement in the high transmissibility of the TW2015 virus in mosquitoes. The transmission mouse model will make a useful platform for evaluation of DENV with high epidemic potential and development of new strategies against dengue outbreaks.     

Rift Valley fever virus

Rift Valley fever is considered to be one of the most important viral zoonoses in Africa. In 2000, the Rift valley fever virus spread to the Arabian Peninsula and caused two simultaneous outbreaks in Yemen and Saudi Arabia. It is transmitted to ruminants and to humans by mosquitoes. The viral agent is an arbovirus, which belongs to the Phlebovirus genus in the Bunyaviridae family. This family of viruses comprises more than 300 members grouped into five genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tospovirus. Several members of the Bunyaviridae family are responsible for fatal hemorrhagic fevers: Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), Hantaan, Sin Nombre and related viruses (Hantavirus), and recently Garissa, now identified as Ngari virus (Orthobunyavirus). Here are reviewed recent advances in Rift Valley fever virus, its epidemiology, molecular biology and focus on recent data on the interactions between viral and cellular proteins, which help to understand the molecular mechanisms utilized by the virus to circumvent the host cellular response.     

Changes in Rodent Abundance and Weather Conditions Potentially Drive Hemorrhagic Fever with Renal Syndrome Outbreaks in Xi’an,China, 2005–2012

BackgroundIncreased risks for hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus have been observed since 2005, in Xi’an, China. Despite increased vigilance and preparedness, HFRS outbreaks in 2010, 2011, and 2012 were larger than ever, with a total of 3,938 confirmed HFRS cases and 88 deaths in 2010 and 2011.ConclusionsIn addition to a strong seasonal pattern, HFRS incidence was correlated with rodent density and rainfall, indicating that they potentially drive the HFRS outbreaks. Future work should aim to determine the mechanism underlying the seasonal pattern and autocorrelation. However, this model can be useful in risk management to provide early warning of potential outbreaks of this disease.     

Problems in the Laboratory Isolation of Simian Hemorrhagic Fever Viruses and Isolation of the Agent Responsible for the Sussex/69 Epizootic

At least six epizootics of simian hemorrhagic fever have occurred at four different primate centers. Although these diseases could easily be transmitted to other monkeys of the Macaca species, difficulty has been encountered in isolating the causative virus in cell culture. The results of this study have shown that the isolation of simian hemorrhagic fever virus strains in cell culture is dependent upon the use of a susceptible MA-104 cell strain and that the ability of such strains to support the replication of these viral agents may vary. By using this information we have been able to isolate a viral agent in cell culture from materials derived from the Sussex/69 epizootic.     

Human Alkhumra hemorrhagic Fever: Emergence,history and epidemiological and clinical profiles

Alkhumra hemorrhagic fever (AHF) is a severe, often fatal hemorrhagic disease in humans. It is caused by Alkhumra hemorrhagic fever virus (AHFV), a newly described flavivirus first isolated in 1995 in Alkhumra district, south of Jeddah City, Saudi Arabia. It is transmitted from infected livestock animals to humans by direct contact with infected animals or by tick bites. In the recent past, the incidence of AHF has increased, with a total of 604 confirmed cases have been reported in Saudi Arabia between 1995 and 2020. Yet, no specific treatment or control strategies have been developed and implemented against this infection. Hence, the likelihood of increased prevalence or the occurrence of outbreaks is high, particularly in the absence of appropriate prevention and control strategies. This narrative review presents an overview of the current knowledge and future concerns about AHF globally.     

Simian Varicella in Old World Monkeys

Simian varicella virus (SVV) causes a natural erythematous disease in Old World monkeys and is responsible for simian varicella epizootics that occur sporadically in facilities housing nonhuman primates. This review summarizes the biology of SVV and simian varicella as a veterinary disease of nonhuman primates. SVV is closely related to varicella–zoster virus, the causative agent of human varicella and herpes zoster. Clinical signs of simian varicella include fever, vesicular skin rash, and hepatitis. Simian varicella may range from a mild infection to a severe and life-threatening disease, and epizootics may have high morbidity and mortality rates. SVV establishes a lifelong latent infection in neural ganglia of animals in which the primary disease resolves, and the virus may reactivate later in life to cause a secondary disease corresponding to herpes zoster. Prompt diagnosis is important for control and prevention of epizootics. Antiviral treatment for simian varicella may be effective if administered early in the course of infection.Abbreviations: FEAU, 1-(2′-deoxy-2′-flouro-β-D-arabinofuranosyl)-5-iodouracil, IE, immediate early, ORF, open reading frame, PBL, peripheral blood lymphocyte, SVV, simian varicella virus, VZV, varicella–zoster virusSimian varicella is a natural erythematous disease of Old World primates (Superfamily Cercopithecoidea, Subfamily Cercopithecinae), involving particularly patas (Erythrocebus patas), African green or vervet (Chlorocebus aethiops), and various species of macaque (Macaca spp.) monkeys. Epizootics of simian varicella occur sporadically in facilities housing nonhuman primates. These outbreaks are sometimes associated with high morbidity and mortality and the loss of valuable research animals. Simian varicella virus (SVV; Cercopithecine herpesvirus 9), a primate herpesvirus, is the etiologic agent of the disease. SVV is antigenically and genetically related to varicella–zoster virus (VZV; Human herpesvirus 3), the cause of human varicella (chickenpox) and herpes zoster (shingles). The clinical similarities between simian and human varicella and the relatedness of SVV and VZV, indicate that SVV infection of nonhuman primates is a useful model for study of varicella pathogenesis and development of antiviral therapies. A previous comprehensive review emphasized simian varicella as an experimental model for VZV infections.²² This review focuses on simian varicella as a veterinary disease of nonhuman primates. Simian varicella outbreaks and their epidemiology are considered, and the etiologic agent, clinical manifestations, pathogenesis, diagnosis, treatment, and control of the disease are discussed.     

Ngari virus is a Bunyamwera virus reassortant that can be associated with large outbreaks of hemorrhagic fever in Africa

Two isolates of a virus of the genus Orthobunyavirus (family Bunyaviridae) were obtained from hemorrhagic fever cases during a large disease outbreak in East Africa in 1997 and 1998. Sequence analysis of regions of the three genomic RNA segments of the virus (provisionally referred to as Garissa virus) suggested that it was a genetic reassortant virus with S and L segments derived from Bunyamwera virus but an M segment from an unidentified virus of the genus Orthobunyavirus. While high genetic diversity (52%) was revealed by analysis of virus M segment nucleotide sequences obtained from 21 members of the genus Orthobunyavirus, the Garissa and Ngari virus M segments were almost identical. Surprisingly, the Ngari virus L and S segments showed high sequence identity with those of Bunyamwera virus, showing that Garissa virus is an isolate of Ngari virus, which in turn is a Bunyamwera virus reassortant. Ngari virus should be considered when investigating hemorrhagic fever outbreaks throughout sub-Saharan Africa.