Pinhal Virus, a New Arenavirus Isolated from Calomys tener in Brazil.

Arenavirus Sabiá was originally isolated from a fatal human infection in Brazil, and after the occurrence of the second fatal human case in São Paulo state, epidemiologic and virologic studies were performed in the area where the patient lived, aiming at the identification of the Sabiá natural rodent reservoir. A broadly cross-reactive enzyme-linked immunosorbent assay (ELISA) was used to screen for antibody-positive samples. Antibodies to arenavirus were detected in two of the 55 samples of Calomys tener, and from these results, samples of rodents were analyzed by a broad RT-PCR assay. RT-PCR amplification detected arenavirus sequences in five of the 55 C. tener samples, and sequencing showed that this virus is a distinct form of Sabiá virus. Thus, we describe here the evidence for the circulation of a new arenavirus in Brazil (proposed name Pinhal virus) and its genetic characterization compared to other arenaviruses. This study also suggests C. tener as a probable rodent reservoir for this virus and associates this new virus with the lineage C of New World arenaviruses. Although we have defined some characteristics of this virus, so far, there is no evidence of its involvement in human disease.

Co-circulation of Clade C New World Arenaviruses: New geographic distribution and host species.

Clade C, of the New World Arenaviruses, is composed of only the Latino and Oliveros viruses and, besides the geographic range of their rodent reservoirs, the distribution of these viruses has been restricted to Bolivia and Argentina. In this study, the genetic detection and phylogenetic analysis of the complete S segment sequences of sympatric arenaviruses from Brazil revealed a new geographic distribution of clade C arenaviruses, as well as the association of Oliveros virus with a new rodent reservoir.

Pinhal virus, a new arenavirus isolated from calomys tener in Brazil

Arenavirus Sabiá was originally isolated from a fatal human infection in Brazil, and after the occurrence of the second fatal human case in São Paulo state, epidemiologic and virologic studies were performed in the area where the patient lived, aiming at the identification of the Sabiá natural rodent reservoir. A broadly cross-reactive enzyme-linked immunosorbent assay (ELISA) was used to screen for antibody-positive samples. Antibodies to arenavirus were detected in two of the 55 samples of Calomys tener, and from these results, samples of rodents were analyzed by a broad RT-PCR assay. RT-PCR amplification detected arenavirus sequences in five of the 55 C. tener samples, and sequencing showed that this virus is a distinct form of Sabiá virus. Thus, we describe here the evidence for the circulation of a new arenavirus in Brazil (proposed name Pinhal virus) and its genetic characterization compared to other arenaviruses. This study also suggests C. tener as a probable rodent reservoir for this virus and associates this new virus with the lineage C of New World arenaviruses. Although we have defined some characteristics of this virus, so far, there is no evidence of its involvement in human disease.

Ocozocoautla de espinosa virus and hemorrhagic fever, Mexico.

Arenavirus RNA was isolated from Mexican deer mice (Peromyscus mexicanus) captured near the site of a 1967 epidemic of hemorrhagic fever in southern Mexico. Analyses of nucleotide and amino acid sequence data indicated that the deer mice were infected with a novel Tacaribe serocomplex virus (proposed name Ocozocoautla de Espinosa virus), which is phylogenetically closely related to Tacaribe serocomplex viruses that cause hemorrhagic fever in humans in South America.

Genetic diversity among Bolivian arenaviruses.

Machupo virus and Chapare virus are members of the Tacaribe serocomplex (virus family Arenaviridae) and etiological agents of hemorrhagic fever in humans in Bolivia. The nucleotide sequences of the complete Z genes, a large fragment of the RNA-dependent RNA polymerase genes, the complete glycoprotein precursor genes, and the complete nucleocapsid protein genes of 8 strains of Machupo virus were determined to increase our knowledge of the genetic diversity among the Bolivian arenaviruses. The results of analyses of the predicted amino acid sequences of the glycoproteins of the Machupo virus strains and Chapare virus strain 200001071 indicated that immune plasma from hemorrhagic fever cases caused by Machupo virus may prove beneficial in the treatment of Bolivian hemorrhagic fever but not hemorrhagic fever caused by Chapare virus.

Genetic diversity between and within the arenavirus species indigenous to western Venezuela.

The results of analyses of Z, RNA-dependent RNA polymerase, glycoprotein precursor, and nucleocapsid protein gene sequence data suggested that Guanarito virus was the most common cause of Venezuelan hemorrhagic fever in a 7-year period in the 1990s and that the evolution of Pirital virus in association with Sigmodon alstoni (Alston's cotton rat) has occurred at a significantly higher rate than the evolution of Guanarito virus in association with Zygodontomys brevicauda (short-tailed cane mouse) on the plains of western Venezuela. The results of analyses of the primary structures of the glycoproteins of the 8 strains of Guanarito virus isolated from humans suggested that these strains would be highly cross-reactive in neutralization assays. Thus, passive antibody therapy may prove beneficial in the treatment of human disease caused by strains of Guanarito virus that are enzootic in the region in which Venezuelan hemorrhagic fever is endemic.

Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia.

A small focus of hemorrhagic fever (HF) cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá). RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.

Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the Mexican woodrat (Neotoma mexicana).

The results of analyses of glycoprotein precursor and nucleocapsid protein gene sequences indicated that an arenavirus isolated from a Mexican woodrat (Neotoma mexicana) captured in Arizona is a strain of a novel species (proposed name Skinner Tank virus) and that arenaviruses isolated from Mexican woodrats captured in Colorado, New Mexico, and Utah are strains of Whitewater Arroyo virus or species phylogenetically closely related to Whitewater Arroyo virus. Pairwise comparisons of glycoprotein precursor sequences and nucleocapsid protein sequences revealed a high level of divergence among the viruses isolated from the Mexican woodrats captured in Colorado, New Mexico, and Utah and the Whitewater Arroyo virus prototype strain AV 9310135, which originally was isolated from a white-throated woodrat (Neotoma albigula) captured in New Mexico. Conceptually, the viruses from Colorado, New Mexico, and Utah and strain AV 9310135 could be grouped together in a species complex in the family Arenaviridae, genus Arenavirus.

Phylogeny of the Venezuelan arenaviruses.

Guanarito virus (the etiologic agent of Venezuelan hemorrhagic fever, VHF) and Pirital virus coexist in the region of Venezuela in which Venezuelan hemorrhagic fever is endemic. The purpose of this study was to extend our knowledge of the evolutionary relationship between these two arenaviruses. We determined that the large genomic segments of Guanarito virus and Pirital virus are similar in size and identical in structural organization to the large genomic segments of other South American arenaviruses. For example, the Z proteins and RNA-dependent RNA polymerases of Guanarito virus and Pirital virus are encoded in nonoverlapping open reading frames of opposite polarities. Phylogenetic analyses of Z protein gene nucleotide sequences and RNA-dependent RNA polymerase gene nucleotide sequences grouped Pirital virus with Pichindé virus (a South American arenavirus which, like Pirital virus, does not appear to be pathogenic for humans) and placed the Pirital-Pichindé lineage in a sister relationship to a lineage represented by Guanarito virus and the three other arenaviruses known to cause hemorrhagic fever in South America. These results are concordant with the results of studies on the phylogeny of the arenavirus small genomic segment. Thus, the exchange of genomic elements between Guanarito virus and Pirital virus via recombination or reassortment likely did not contribute to the emergence of Venezuelan hemorrhagic fever.

Natural nidality in Bolivian hemorrhagic fever and the systematics of the reservoir species.

Zoonoses within wild reservoir host populations often occur focally obeying Pavlovskii's rules of "natural nidality". What appears to be a clear example is Bolivian hemorrhagic fever (BHF), a disease endemic to northeastern Bolivia. The etiological agent is Machupo virus (MACV, Arenaviridae). The vertebrate reservoir, identified 30 years ago, was Calomys callosus a wild rodent common to open biomes in the lowlands of southeastern South America. The lack of concordance between the occurrence of MACV and the range of its rodent host has puzzled cadres of researchers and could be used as an exemplar of natural nidality. Here, we show that the populations of rodents responsible for the maintenance and transmission of MACV are an independent monophyletic lineage, different from those in other areas of South America. Therefore a clearer understanding of the systematics of the host species explains the apparent natural nidality of BHF. Similar studies may prove to be informative in other zoonoses.

Guanarito virus (Arenaviridae) isolates from endemic and outlying localities in Venezuela: sequence comparisons among and within strains isolated from Venezuelan hemorrhagic fever patients and rodents.

Despite intensive surveillance, Venezuelan hemorrhagic fever (VHF), caused by Guanarito (GTO) virus, has been detected in only a small region of western Venezuela. To determine whether VHF is associated with a particular regional GTO virus strain(s), 29 isolates from rodents and humans throughout the surrounding regions were analyzed by partial sequencing of the nucleocapsid protein gene. Phylogenetic trees delineated nine distinct GTO genotypes that differ by 4-17% in nucleotides and up to 9% in amino acid sequences; most appeared to be restricted to discrete geographic regions, although a few genotypes were isolated in several locations. Each genotype included at least one strain recovered from a rodent, but only two genotypes were isolated from VHF cases. The presence outside of the endemic/epidemic region of two genotypes isolated also from VHF cases suggests that human pathogenic viruses occur outside of the endemic zone, but do not frequently infect people and/or cause apparent disease there. VHF does not appear to be associated with a GTO virus genotype that is restricted to a certain rodent species. When quasispecies diversity was examined, rodent isolates had higher sequence variation than human isolates. One rodent isolate included a mixture of two phylogenetically distinct genotypes, suggesting a dual infection.

Genetic characterization and phylogeny of Sabiá virus, an emergent pathogen in Brazil.

Sabiá virus, one of five arenaviruses from South America known to cause hemorrhagic fever in humans, emerged in 1990 when it was isolated from a fatal case in Sao Paulo, Brazil. Subsequently, it has caused two laboratory-acquired infections. Its natural distribution and host are still unknown. Using viral RNA and multiple polymerase chain reaction products as templates, the nucleotide sequence of the small (S) RNA segment of Sabiá virus, which codes for the nucleocapsid (N) and glycoprotein precursor, was determined. This virus shares an ambisense genome in common with other arenaviruses, although it has a unique predicted three stem--loop structure in the S RNA intergenic region. Phylogenetic analysis of a portion of the N gene sequence confirmed that Sabiá virus is distinct from all other members of the Arenaviridae and shares a progenitor with Junin, Machupo, Tacaribe, and Guanarito viruses.

Characterization of Oliveros virus, a new member of the Tacaribe complex (Arenaviridae: Arenavirus).

Oliveros virus is an agent isolated in cell culture from Bolomys obscurus (Rodentia, Muridae, Sigmodontinae) captured on the central Argentine pampa. Oliveros virus was shown to be related to members of the Tacaribe complex of the family Arenaviridae by immunofluorescent antibody (IFA) tests, electrophoretic pattern of viral proteins, and morphology as observed by electron microscopy. It was distinct from 12 other arenaviruses by a combination of plaque-reduction neutralization tests, comparison of endpoint titers among cross-IFA tests, and comparison of viral RNA sequence data. This agent is the third new arenavirus from South America described within the last three years.

New arenavirus isolated in Brazil.

A new arenavirus, called Sabiá, was isolated in Brazil from a fatal case of haemorrhagic fever initially thought to be yellow fever. Antigenic and molecular characterisation indicated that Sabiá virus is a new member of the Tacaribe complex. A laboratory technician working with the agent was also infected and developed a prolonged, non-fatal influenza-like illness. Sabiá virus is yet another arenavirus causing human disease in South America.

Experimental neuroinvasiveness of wild and laboratory Junin virus strains.

The neuroinvasiveness of Candid 1 and XJCL3 laboratory strains and CbalV4454 and CbaFHA5069 wild strains of Junin virus was studied in albino mice, guinea pigs, and a South American wild rodent, Calomys musculinus (Cm), of different ages inoculated by a non-neural route. Infectivity in brain, blood and organs, as well as lethality, were determined. The results with the 3 hosts indicate that Junin virus neuroinvasiveness is virus-strain-dependent, host species- and age-dependent, with the Candid 1 strain proving to be the least neuroinvasive of the strains studied. The lethal efficiency index (log PFU/LD50) in 2-day old albino mice and the neuroinvasiveness index (Log PFU/ND50) in 6 +/- 1 day-old Cm of the various strains using the intraperitoneal (ip) route could therefore be useful markers of Junin virus neuroinvasiveness. Moreover, different patterns of infection were established using the results of the presence of infectious virus in brain and viraemia in the 3 hosts. In nearly all cases, virus neuroinvasion was present without detectable viraemia (virus in plasma). Current evidence leads to the assumption that virus might reach the brain associated with the white cells in blood (undetectable by conventional isolation methods) or by another possible mechanism of neuroinvasion which is not haematogenous.

Molecular organization of Junin virus S RNA: complete nucleotide sequence, relationship with other members of the Arenaviridae and unusual secondary structures.

In this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5' end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3'-terminal sequences and may form a very stable panhandle structure (delta G-242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (delta G -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function.

Antigenic relationships between attenuated and pathogenic strains of Junin virus.

Antigenic relationships between attenuated and pathogenic strains of Junin virus (JV) were investigated. Five strains of either human or rodent origin were tested by cross-neutralization assay with hyperimmune antisera, raised in rabbits, against each strain. Polyclonal antisera could be used to distinguish among these JV strains, as the titer values differed significantly with ratios of homologous to heterologous titers, which ranged from 1.3 to 22.3. This demonstrates, independent of their virulence, a heterogeneity among the JV strains tested. The relatedness among JV strains was expressed quantitatively through a dendrogram based on taxonomic distance coefficients. The field strains of JV were grouped into two clusters, according to their geographic origin.

[Differentiation among strains of Junín virus by intraperitoneal infection in the rat]. / Diferenciación de cepas de virus Junín por la infección intraperitoneal en la rata.

The 2-day-old rat is known to resist intracerebral infection with the XJ prototype strain of Junin virus, but 95-100% mortality results when infected with the attenuated XJC13 strain. When this animal was inoculated by intraperitoneal route, behaviour was diametrically opposite: the XJ strain proved lethal, while de XJC13 led to low mortality. Studies on mortality, virus titer in different organs, and anti-viral humoral response in 2-day-old rats infected with Junin virus strains were carried out in order to use this system as a new attenuation marker. Mortality rates recorded for rats inoculated with either strain, were markedly different, being 84% in the XJ-infected group and barely reaching 17% in the XJC13 group. Brain viral titers were higher in the former group than the latter (10(5.26) PFU/ml vs. 10(3) PFU/ml at day 17 pi). For this reason, viral replication may be used as a virulence marker in this experimental model. Antibody levels were also higher in the XJ group most likely due to greater viral replication. The above findings support the use of the 2-day-old rat as a biologic attenuation marker since susceptibility to infection is strain-dependent.