Characterization of Haartman Institute snake virus-1 (HISV-1) and HISV-like viruses-The representatives of genus Hartmanivirus, family Arenaviridae.

The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular inclusion bodies typical for the reptarenavirus-induced boid inclusion body disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed.

When Viruses Don't Go Viral: The Importance of Host Phylogeographic Structure in the Spatial Spread of Arenaviruses.

Many emerging infections are RNA virus spillovers from animal reservoirs. Reservoir identification is necessary for predicting the geographic extent of infection risk, but rarely are taxonomic levels below the animal species considered as reservoir, and only key circumstances in nature and methodology allow intrinsic virus-host associations to be distinguished from simple geographic (co-)isolation. We sampled and genetically characterized in detail a contact zone of two subtaxa of the rodent Mastomys natalensis in Tanzania. We find two distinct arenaviruses, Gairo and Morogoro virus, each spatially confined to a single M. natalensis subtaxon, only co-occurring at the contact zone's centre. Inter-subtaxon hybridization at this centre and a continuum of quality habitat for M. natalensis show that both viruses have the ecological opportunity to spread into the other substaxon's range, but do not, strongly suggesting host-intrinsic barriers. Such barriers could explain why human cases of another M. natalensis-borne arenavirus, Lassa virus, are limited to West Africa.

[Arenavirus research and antiviral candidate].

Arenavirus is a genetic term for viruses belonging to the family Arenaviridae and is presented from lymphocytic choriomeningitis virus (LCMV), which shows almost no pathogenicity to humans, to Lassa virus, Junin virus, Machupo virus, Chapare virus, Lujo virus, Sabia virus, and Guanarito virus, which shows high pathogenicity to humans. These viruses except for LCMV are risk group 4 pathogens specified by World Health Organization. Based on this designation, it is designated as Class I pathogens in Japan. Although there have been no reports excluding one imported case of the Lassa fever patient, it is not surprising whenever imported cases occur in our country. Considering the disease severity and mortality rate, it is an urgent matter to develop vaccines and therapeutic drugs in endemic areas, and maintenances of these are also important in countries other than endemic areas. However, basic research on highly pathogenic arenavirus infections and development of therapeutic drugs are not easily progressed, because handling in highly safe research facilities is indispensable. In this article, we will outline the current knowledge from the recent basic research on arenavirus to the development situation of antivirals against arenaviruses.

Arenavirus Quasispecies and Their Biological Implications.

The family Arenaviridae currently comprises over 20 viral species, each of them associated with a main rodent species as the natural reservoir and in one case possibly phyllostomid bats. Moreover, recent findings have documented a divergent group of arenaviruses in captive alethinophidian snakes. Human infections occur through mucosal exposure to aerosols or by direct contact of abraded skin with infectious materials. Arenaviruses merit interest both as highly tractable experimental model systems to study acute and persistent infections and as clinically important human pathogens including Lassa (LASV) and Junin (JUNV) viruses, the causative agents of Lassa and Argentine hemorrhagic fevers (AHFs), respectively, for which there are no FDA-licensed vaccines, and current therapy is limited to an off-label use of ribavirin (Rib) that has significant limitations. Arenaviruses are enveloped viruses with a bi-segmented negative strand (NS) RNA genome. Each genome segment, L (ca 7.3 kb) and S (ca 3.5 kb), uses an ambisense coding strategy to direct the synthesis of two polypeptides in opposite orientation, separated by a noncoding intergenic region (IGR). The S genomic RNA encodes the virus nucleoprotein (NP) and the precursor (GPC) of the virus surface glycoprotein that mediates virus receptor recognition and cell entry via endocytosis. The L genome RNA encodes the viral RNA-dependent RNA polymerase (RdRp, or L polymerase) and the small (ca 11 kDa) RING finger protein Z that has functions of a bona fide matrix protein including directing virus budding. Arenaviruses were thought to be relatively stable genetically with intra- and interspecies amino acid sequence identities of 90-95 % and 44-63 %, respectively. However, recent evidence has documented extensive arenavirus genetic variability in the field. Moreover, dramatic phenotypic differences have been documented among closely related LCMV isolates. These data provide strong evidence of viral quasispecies involvement in arenavirus adaptability and pathogenesis. Here, we will review several aspects of the molecular biology of arenaviruses, phylogeny and evolution, and quasispecies dynamics of arenavirus populations for a better understanding of arenavirus pathogenesis, as well as for the development of novel antiviral strategies to combat arenavirus infections.

Vaccination strategies against highly pathogenic arenaviruses: the next steps toward clinical trials.

Vaccination is one of the most valuable weapons against infectious diseases and has led to a significant reduction in mortality and morbidity. However, for most viral hemorrhagic fevers caused by arenaviruses, no prophylactic vaccine is available. This is particularly problematic as these diseases are notoriously difficult to diagnose and treat. Lassa fever is globally the most important of the fevers caused by arenaviruses, potentially affecting millions of people living in endemic areas, particularly in Nigeria. Annually, an estimated 300,000 humans are infected and several thousands succumb to the disease. The successful development of the vaccine "Candid#1" against Junin virus, the causative agent of Argentine hemorrhagic fever, proved that an effective arenavirus vaccine can be developed. Although several promising studies toward the development of a Lassa fever vaccine have been published, no vaccine candidate has been tested in human volunteers or patients. This review summarizes the immunology and other aspects of existing experimental arenavirus vaccine studies, discusses the reasons for the lack of a vaccine, and proposes a plan for overcoming the final hurdles toward clinical trials.

Past, present, and future of arenavirus taxonomy.

Until recently, members of the monogeneric family Arenaviridae (arenaviruses) have been known to infect only muroid rodents and, in one case, possibly phyllostomid bats. The paradigm of arenaviruses exclusively infecting small mammals shifted dramatically when several groups independently published the detection and isolation of a divergent group of arenaviruses in captive alethinophidian snakes. Preliminary phylogenetic analyses suggest that these reptilian arenaviruses constitute a sister clade to mammalian arenaviruses. Here, the members of the International Committee on Taxonomy of Viruses (ICTV) Arenaviridae Study Group, together with other experts, outline the taxonomic reorganization of the family Arenaviridae to accommodate reptilian arenaviruses and other recently discovered mammalian arenaviruses and to improve compliance with the Rules of the International Code of Virus Classification and Nomenclature (ICVCN). PAirwise Sequence Comparison (PASC) of arenavirus genomes and NP amino acid pairwise distances support the modification of the present classification. As a result, the current genus Arenavirus is replaced by two genera, Mammarenavirus and Reptarenavirus, which are established to accommodate mammalian and reptilian arenaviruses, respectively, in the same family. The current species landscape among mammalian arenaviruses is upheld, with two new species added for Lunk and Merino Walk viruses and minor corrections to the spelling of some names. The published snake arenaviruses are distributed among three new separate reptarenavirus species. Finally, a non-Latinized binomial species name scheme is adopted for all arenavirus species. In addition, the current virus abbreviations have been evaluated, and some changes are introduced to unequivocally identify each virus in electronic databases, manuscripts, and oral proceedings.

Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease.

Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae.

Two novel arenaviruses detected in pygmy mice, Ghana.

Two arenaviruses were detected in pygmy mice (Mus spp.) by screening 764 small mammals in Ghana. The Natal multimammate mouse (Mastomys natalensis), the known Lassa virus reservoir, was the dominant indoor rodent species in 4 of 10 sites, and accounted for 27% of all captured rodents. No rodent captured indoors tested positive for an arenavirus.

Detection of novel divergent arenaviruses in boid snakes with inclusion body disease in The Netherlands.

Arenaviruses are bi-segmented negative-stranded RNA viruses, which were until recently only detected in rodents and humans. Now highly divergent arenaviruses have been identified in boid snakes with inclusion body disease (IBD). Here, we describe the identification of a new species and variants of the highly divergent arenaviruses, which were detected in tissues of captive boid snakes with IBD in The Netherlands by next-generation sequencing. Phylogenetic analysis of the complete sequence of the open reading frames of the four predicted proteins of one of the detected viruses revealed that this virus was most closely related to the recently identified Golden Gate virus, while considerable sequence differences were observed between the highly divergent arenaviruses detected in this study. These findings add to the recent identification of the highly divergent arenaviruses in boid snakes with IBD in the United States and indicate that these viruses also circulate among boid snakes in Europe.

Novel arenavirus, Zambia.

To investigate arenavirus in Zambia, we characterized virus from the kidneys of 5 arenavirus RNA-positive rodents (Mastomys natalensis) among 263 captured. Full-genome sequences of the viruses suggested that they were new strains similar to Lassa virus-related arenaviruses. Analyzing samples from additional rodents and other species can elucidate epizootiologic aspects of arenaviruses.

Transmission of Guanarito and Pirital viruses among wild rodents, Venezuela.

Samples from rodents captured on a farm in Venezuela in February 1997 were tested for arenavirus, antibody against Guanarito virus (GTOV), and antibody against Pirital virus (PIRV). Thirty-one (48.4%) of 64 short-tailed cane mice (Zygodontomys brevicauda) were infected with GTOV, 1 Alston's cotton rat (Sigmodon alstoni) was infected with GTOV, and 36 (64.3%) of 56 other Alston's cotton rats were infected with PIRV. The results of analyses of field and laboratory data suggested that horizontal transmission is the dominant mode of GTOV transmission in Z. brevicauda mice and that vertical transmission is an important mode of PIRV transmission in S. alstoni rats. The results also suggested that bodily secretions and excretions from most GTOV-infected short-tailed cane mice and most PIRV-infected Alston's cotton rats may transmit the viruses to humans.

Alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses.

We screened ∼2,200 compounds known to be safe in people for the ability to reduce the amount of virion-associated hepatitis B virus (HBV) DNA in the culture medium of producer cells. These efforts led to the discovery of an alkylated porphyrin, chlorophyllide, as the compound that achieved the greatest reduction in signal. Here we report that chlorophyllide directly and quantitatively disrupted HBV virions at micromolar concentrations, resulting in the loss of all detectable virion DNA, without detectably affecting cell viability or intracellular viral gene products. Chemophores of chlorophyllide were also tested. Chlorin e6, a metal-free chlorophyllide-like molecule, showed the strongest antiviral activity against HBV as well as profound antiviral effects on other enveloped viruses, such as hepatitis C virus (HCV), human immunodeficiency virus (HIV), dengue virus (DENV), Marburg virus (MARV), Tacaribe virus (TCRV), and Junin viruses (JUNV). Remarkably, chlorin e6 inactivated DENV at subnanomolar-level concentrations. However, the compound had no antiviral effect against encephalomyocarditis virus and adenovirus, suggesting that chlorin e6 may be less active or inactive against nonenveloped viruses. Although other porphyrin derivatives have been previously reported to possess antiviral activity, this is the first analysis of the biochemical impact of chlorophyllide and chlorin e6 against HBV and of the dramatic anti-infectivity impact upon DENV. The possible application of this family of compounds as antiviral agents, as microbicides and systemic virus neutralizing agents, is discussed.

A new arenavirus in a cluster of fatal transplant-associated diseases.

BACKGROUND: Three patients who received visceral-organ transplants from a single donor on the same day died of a febrile illness 4 to 6 weeks after transplantation. Culture, polymerase-chain-reaction (PCR) and serologic assays, and oligonucleotide microarray analysis for a wide range of infectious agents were not informative. METHODS: We evaluated RNA obtained from the liver and kidney transplant recipients. Unbiased high-throughput sequencing was used to identify microbial sequences not found by means of other methods. The specificity of sequences for a new candidate pathogen was confirmed by means of culture and by means of PCR, immunohistochemical, and serologic analyses. RESULTS: High-throughput sequencing yielded 103,632 sequences, of which 14 represented an Old World arenavirus. Additional sequence analysis showed that this new arenavirus was related to lymphocytic choriomeningitis viruses. Specific PCR assays based on a unique sequence confirmed the presence of the virus in the kidneys, liver, blood, and cerebrospinal fluid of the recipients. Immunohistochemical analysis revealed arenavirus antigen in the liver and kidney transplants in the recipients. IgM and IgG antiviral antibodies were detected in the serum of the donor. Seroconversion was evident in serum specimens obtained from one recipient at two time points. CONCLUSIONS: Unbiased high-throughput sequencing is a powerful tool for the discovery of pathogens. The use of this method during an outbreak of disease facilitated the identification of a new arenavirus transmitted through solid-organ transplantation.

Experimental Morogoro Virus Infection in Its Natural Host, Mastomys natalensis.

Natural hosts of most arenaviruses are rodents. The human-pathogenic Lassa virus and several non-pathogenic arenaviruses such as Morogoro virus (MORV) share the same host species, namely Mastomys natalensis (M. natalensis). In this study, we investigated the history of infection and virus transmission within the natural host population. To this end, we infected M. natalensis at different ages with MORV and measured the health status of the animals, virus load in blood and organs, the development of virus-specific antibodies, and the ability of the infected individuals to transmit the virus. To explore the impact of the lack of evolutionary virus-host adaptation, experiments were also conducted with Mobala virus (MOBV), which does not share M. natalensis as a natural host. Animals infected with MORV up to two weeks after birth developed persistent infection, seroconverted and were able to transmit the virus horizontally. Animals older than two weeks at the time of infection rapidly cleared the virus. In contrast, MOBV-infected neonates neither developed persistent infection nor were able to transmit the virus. In conclusion, we demonstrate that MORV is able to develop persistent infection in its natural host, but only after inoculation shortly after birth. A related arenavirus that is not evolutionarily adapted to M. natalensis is not able to establish persistent infection. Persistently infected animals appear to be important to maintain virus transmission within the host population.

Special Issue "Arenaviruses 2020".

Rodent-borne arenaviruses have been traditionally predominantly associated with certain muroid species from Mastomys/Praomys genera (African arenaviruses) or with species that belong to murid subfamily Cricetidae (New World arenaviruses) [...].

Nosocomial outbreak of novel arenavirus infection, southern Africa.

A nosocomial outbreak of disease involving 5 patients, 4 of whom died, occurred in South Africa during September-October 2008. The first patient had been transferred from Zambia to South Africa for medical management. Three cases involved secondary spread of infection from the first patient, and 1 was a tertiary infection. A novel arenavirus was identified. The source of the first patient's infection remains undetermined.

Endangered wild salmon infected by newly discovered viruses.

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.