Pathology of Whitewater Arroyo viral infection in the white-throated woodrat (Neotoma albigula).

The white-throated woodrat is a principal host of Whitewater Arroyo (WWA) virus, an arenavirus, in the western United States. The purpose of the present study was to investigate the pathology of WWA infection in this species. Twenty-one animals (eight newborn, seven juvenile, and six adult) were inoculated with WWA virus and killed at varying intervals after inoculation. The most striking histological findings were lymphocytic meningitis and perivascular lymphocytic cuffing in the brains of the animals killed on day 85, 113 or 121. Arenaviral antigen was detected immunohistochemically in the brain of each affected animal, suggesting that the inflammatory lesions in the brain were caused by WWA virus. Comparisons of the results of tests for infectious virus and antigen in brain and other solid tissues indicated that immunohistochemistry may be a useful method for detection of WWA viral antigen in post-mortem specimens.

Experimental infection of Neotoma albigula (Muridae) with Whitewater Arroyo virus (Arenaviridae).

The Whitewater Arroyo virus (WWA) is a newly described North American arenavirus. The purpose of this study was to elucidate the biology of this virus in its natural rodent host, Neotoma albigula (white-throated woodrat). Thirteen adult, 7 juvenile, and 8 newborn woodrats each were inoculated subcutaneously with 1,000 cell culture infectious dose50 of the WWA virus prototype strain AV 9310135. All 28 animals became infected (as measured by the recovery of infectious virus and/or seroconversion) and no overt illness was associated with infection. Infection and virus shedding in the adult animals were transient (less than 59 days) whereas virus shedding in animals inoculated at birth persisted through 164 days of age. These results indicate that the duration of WWA virus infection in N. albigula is dependent upon the animal's age at the onset of infection and that neonatal infection can result in chronic (perhaps lifelong) virus shedding.

Geographical distribution of rodent-associated hantaviruses in Texas.

The purpose of this study was to increase our knowledge of the geographic distribution and natural host range of hantaviruses in Texas, southeastern New Mexico, and Mexico. Blood samples from 3,225 wild rodents, representing 34 species, were tested for hantavirus antibody (IgG), using an enzyme-linked immunosorbent assay. Hantavirus antibody was found in one or more rodents from each of 13 counties in Texas, Otero County in southeastern New Mexico, and Mexico State (central Mexico). The 133 antibody-positive rodents included seven Peromyscus species (P. attwateri, P. boylii, P. hylocetes, P. leucopus, P. maniculatis, P. melanotis, and P. pectoralis), Sigmodon hispidus, Oryzomys palustris, two Reithrodontomys species (R. fulvescens and R. megalotis), Neotoma albigula, and Perognathus merriami. This study provides further evidence that rodent-associated hantaviruses are geographically widely distributed in Texas. The discovery of antibody in P. hylocetes and P. melanotis is evidence that peromyscine rodents in Mexico are naturally associated with viruses belonging to the genus Hantavirus.

Transmission of an arenavirus in white-throated woodrats (Neotoma albigula), southeastern Colorado, 1995-1999.

From 1995 to 1999, we conducted longitudinal studies of white- throated woodrats (Neotoma albigula) in southeastern Colorado. Forty-five (42.9%) of 105 female and 15 (26.8%) of 56 male N. albigula had antibodies against Whitewater Arroyo virus (WWAV). Sixteen female and three male N. albigula seroconverted during the study period, most of them during July-November, when population densities are highest. Analyses of longevity data, minimum numbers alive and infected, movements, and weight data suggest that the dominant mode of WWAV transmission among white-throated woodrats in Colorado is direct contact. WWAV was recently reported to cause fatal infection in humans. Our findings will lead to better assessment of the public health threat posed by infected woodrats and may be useful in predicting periods of increased risk for human infection.

Geographic distribution and genetic diversity of Whitewater Arroyo virus in the southwestern United States.

The purpose of this study was to extend our knowledge of the geographic distribution and genetic diversity of the arenavirus(es) associated with Neotoma species (woodrats) in the southwestern United States. Infectious arenavirus was recovered from 14 (3.3%) of 425 woodrats. The virus-positive species included N. albigula in New Mexico and Oklahoma, N. cinerea in Utah, N. mexicana in New Mexico and Utah, and N. micropus in Texas. Analyses of viral nucleocapsid protein gene sequence data indicated that all the isolates were strains of the Whitewater Arroyo virus, an arenavirus previously known only from northwestern New Mexico. Analyses of the sequence data also indicated that there can be substantial genetic diversity among strains of Whitewater Arroyo virus from conspecific woodrats collected from different localities and substantial genetic diversity among strains from different woodrat species collected from the same locality.

Extreme genetic diversity among Pirital virus (Arenaviridae) isolates from western Venezuela.

Pirital-like virus isolates from rodents collected in a variety of habitats within a six-state area of central Venezuela were analyzed genetically by amplifying a portion of the nucleocapsid protein gene using RT-PCR. Comparisons of the sequences from 30 selected Pirital-like virus isolates demonstrated up to 26% divergence in nucleotide sequences and up to 16% divergence in deduced amino acid sequences. Within the Pirital monophyletic group, 14 distinct lineages or genotypes, differing by at least 6% in nucleotide sequences, were identified. Although sample sizes were small for some lineages, many of the different genotypes were sampled in only one region or locality, suggesting allopatric divergence. Complement fixation tests with representatives of the most divergent Pirital virus lineages failed to delineate multiple species or subtypes within the Pirital clade. These results indicate that the previously proposed 12% nucleocapsid protein amino acid sequence divergence cutoff value for delineating arenavirus species is not appropriate for the entire family. When individual clones were examined from PCR amplicons, a mean of 0.17% sequence diversity vs the consensus sequences was detected, suggesting diverse quasispecies populations within infected rodent hosts. Possible explanations for the extreme genetic diversity within and among Pirital virus populations in infected rodents are discussed.

The Whitewater Arroyo virus: natural evidence for genetic recombination among Tacaribe serocomplex viruses (family Arenaviridae).

The Tacaribe serocomplex (family Arenaviridae) comprises three phylogenetic lineages, designated A, B, and C. The sequence of a 3278-nt fragment of the small genomic segment of the Whitewater Arroyo (WWA) virus was determined to extend our knowledge on the phylogenetic relationship of this newly discovered North American Tacaribe complex virus to other arenaviruses. Independent analyses of full-length nucleoprotein (N) and glycoprotein precursor (GPC) amino acid sequences indicated that the WWA virus N and GPC genes are descended from a lineage A virus and lineage B virus, respectively. The different phylogenetic histories of the N and GPC genes indicate that the WWA virus genome is a product of recombination between two Tacaribe complex viruses.

Nucleotide sequence of the pirital virus (family Arenaviridae) small genomic segment.

Pirital virus is a newly discovered South American member of the family Arenaviridae. We determined that the complete nucleotide sequence of the small genomic segment of Pirital virus is 3393 nt long, and encodes the viral nucleoprotein (N) and glycoprotein precursor (GPC) (561 aa and 509 aa, respectively) in nonoverlapping open reading frames of opposite polarities. The N and GPC genes are separated by an intergenic region that is 80 nt long; the predicted secondary structure of this region includes a single hairpin stabilized by 11 G-C and 8 A-U base pairs. Independent analyses of N and GPC amino acid sequence data confirmed that Pirital virus is related to Pichindé virus and belongs to the lineage A of the New World (Tacaribe complex) arenaviruses. The analysis of genetic distances between Pirital virus and other arenaviruses confirmed that Pirital virus is a distinct species within the family Arenaviridae.

Arenavirus antibody in rodents indigenous to coastal southern California.

The purpose of this study was to extend our knowledge on the geographic and natural rodent host ranges of New World arenaviruses in California. Sera from 1,094 sigmodontine and 112 murine rodents were tested for antibody against Whitewater Arroyo and Amapari viruses. Antibody was found in 55 (4.6%) of the 1,206 rodents: 4 from northwestern San Diego County, 3 from Los Angeles County, and 48 from Orange County. The antibody-positive rodents included 8 (7.8%) of 103 Neotoma fuscipes, 1 (0.6%) of 180 Neotoma lepida, 1 (3.1%) of 32 Peromyscus boylii, 8 (11.0%) of 73 Peromyscus californicus, 1 (1.2%) of 85 Peromyscus eremicus, 30 (8.5%) of 353 Peromyscus maniculatus, and 6 (2.2%) of 268 Reithrodontomys megalotis. This study provides the first evidence that New World arenaviruses occur in Los Angeles and Orange counties and northwestern San Diego County, and the first evidence that Peromyscus and Reithrodontomys species are naturally infected with New World arenaviruses.

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.

Experimental infection of the cane mouse Zygodontomys brevicauda (family Muridae) with guanarito virus (Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever.

Chronic infections in specific rodents appear to be crucial to the long-term persistence of arenaviruses in nature. The cane mouse, Zygodontomys brevicauda, is a natural host of Guanarito virus (family Arenaviridae), the etiologic agent of Venezuelan hemorrhagic fever. The purpose of this study was to elucidate the natural history of Guanarito virus infection in Z. brevicauda. Thirty-nine laboratory-reared cane mice each were inoculated subcutaneously with 3.0 log10 plaque-forming units of the Guanarito virus prototype strain INH-95551. No lethality was associated with infection in any animal, regardless of age at inoculation. The 13 newborn, 14 weanling, and 8 of the 12 adult animals developed chronic viremic infections characterized by persistent shedding of infectious virus in oropharyngeal secretions and urine. These findings indicate that Guanarito virus infection in Z. brevicauda can be chronic and thus support the concept that this rodent species is the natural reservoir of Guanarito virus.

Natural rodent host associations of Guanarito and pirital viruses (Family Arenaviridae) in central Venezuela.

The objective of this study was to elucidate the natural rodent host relationships of Guanarito and Pirital viruses (family Arenaviridae) in the plains of central Venezuela. Ninety-two arenavirus isolates from 607 animals, representing 10 different rodent species, were characterized to the level of serotype. The 92 isolates comprised 19 Guanarito virus strains and 73 Pirital virus strains. The 19 Guanarito virus isolates were from Zygodontomys brevicauda; 72 (98.6%) of the 73 Pirital virus isolates were from Sigmodon alstoni. These results indicate that the natural rodent associations of these 2 sympatric arenaviruses are highly specific and that Z brevicauda and S. alstoni are the principal rodent hosts of Guanarito and Pirital viruses, respectively.

Isolation and genetic characterization of a hantavirus (Bunyaviridae: Hantavirus) from a rodent, Oligoryzomys microtis (Muridae), collected in northeastern Peru.

This paper describes the isolation and partial genetic characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected within the urban area of Iquitos, Loreto Department, Peru. The virus, designated HTN-007, exhibited the highest degree of genetic similarity to Rio Mamore virus, which was originally described from the same rodent species in eastern Bolivia. Comparison of small and medium segment nucleotide sequence data from HTN-007 and Rio Mamore virus revealed 87% and 85% sequence identity, respectively. Based on these analyses, HTN-007 appears to be a variant of Rio Mamore virus. As such, it represents the first successful isolation of Rio Mamore virus and the first evidence for the existence of a hantavirus in Peru. Serologic studies done by immunofluorescence on blood samples of 56 O. microtis trapped at the collection site indicated that 21.4% had antibodies to hantavirus. In view of the proximity of this rodent species to humans and the close phylogenetic relationship of Rio Mamore virus to hantaviruses that have been associated with human disease, Rio Mamore virus may be a hantavirus of some public health importance in tropical South America.

Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996.

Ebola (subtype Reston [EBO-R]) virus infection was detected in macaques imported into the United States from the Philippines in March 1996. Studies were initiated in the Philippines to identify the source of the virus among monkey-breeding and export facilities, to establish surveillance and testing, and to assess the risk and significance of EBO-R infections in humans who work in these facilities. Over a 5-month period, acutely infected animals were found at only one facility, as determined using Ebola antigen detection. Three of 1732 monkeys and 1 of 246 animal handlers tested had detectable antibodies; all were from the same facility, which was the source of infected monkeys imported to the United States. Virus transmission, which was facilitated by poor infection-control practices, continued for several months in one facility and was stopped only when the facility was depopulated. None of the 246 employees of the facilities or 4 contacts of previously antibody-positive individuals reported an Ebola-like illness. This investigation suggests that human EBO-R infection is rare.

Venezuelan hemorrhagic fever: clinical and epidemiological studies of 165 cases.

Epidemiological and clinical data are presented on 165 cases of Venezuelan hemorrhagic fever (VHF), a newly emerging viral zoonosis caused by Guanarito virus (of the family Arenaviridae). The disease is endemic in a relatively circumscribed area of central Venezuela. Since its first recognition in 1989, the incidence of VHF has peaked each year between November and January, during the period of major agricultural activity in the region of endemicity. The majority of cases have involved male agricultural workers. Principal symptoms among the patients with VHF included fever, malaise, headache, arthralgia, sore throat, vomiting, abdominal pain, diarrhea, convulsions, and a variety of hemorrhagic manifestations. The majority of patients also had leukopenia and thrombocytopenia. The overall fatality rate among the 165 cases was 33.3%, despite hospitalization and vigorous supportive care.

Isolation, characterization and geographic distribution of Caño Delgadito virus, a newly discovered South American hantavirus (family Bunyaviridae).

Rodents collected from the Venezuelan llanos (plains) during field studies of viral hemorrhagic fever were tested for evidence of hantavirus infection. Hantavirus antibody was found in one (7.7%) of 13 Oryzomys bicolor, one (3.4%) of 29 Rattus rattus, 10 (6.0%) of 166 Sigmodon alstoni and one (2.2%) of 45 Zygodontomys brevicauda. Hantavirus-specific RNA was detected in lung tissues from four antibody-positive rodents: two S. alstoni from Portuguesa State and one S. alstoni each from Cojedes and Barinas States. A hantavirus isolate (herein identified as VHV-574) was recovered from lung tissue from a hantavirus RNA-positive S. alstoni collected from Portuguesa State. The results of serological tests and analyses of small and medium RNA segment nucleotide sequence data indicated that VHV-574 represents a novel hantavirus (proposed name 'Caño Delgadito') that is distinct from all previously characterized hantaviruses. The results of analyses of nucleotide sequence data from the four hantavirus RNA-positive S. alstoni suggested that Caño Delgadito virus is widely distributed in the Venezuelan llanos.

Isolation and characterization of Whitewater Arroyo virus, a novel North American arenavirus.

Rodents are principal hosts for each of the well-characterized arenaviruses. Prior to the present study, Tamiami (TAM) virus was the sole arenavirus known to be indigenous to North America; it has been isolated only from southern Florida where its primary host is the cotton rat Sigmodon hispidus. Recently, arenavirus antibody was found in Neotoma albigula woodrats collected from the southwestern United States. The purpose of the present study was to isolate and characterize the arenavirus associated with N. albigula. Three isolates of a novel arenavirus (proposed name "Whitewater Arroyo," WWA) were recovered from two arenavirus antibody-positive N. albigula collected from Whitewater Arroyo in McKinley County, New Mexico. Two-way serologic tests indicated that WWA virus is antigenically distinct from other arenaviruses but most closely related to TAM virus. Phylogenetic analysis of nucleocapsid protein gene sequence data showed that WWA virus is a novel arenavirus that is genetically most closely related to TAM virus. The recovery of WWA virus from antibody-positive N. albigula suggests that WWA virus infection in this species can be chronic and thus that N. albigula is a reservoir host of the virus.

Ecology of Jamestown Canyon virus (Bunyaviridae: California serogroup) in coastal California.

This paper reports the first isolation of Jamestown Canyon (JC) virus from coastal California and the results of tests for antibody to JC virus in mammals living in coastal California. The virus isolation was made from a pool of 50 Aedes dorsalis females collected as adults from Morro Bay, San Luis Obispo County, California. The virus isolate was identified by two-way plaque reduction-serum dilution neutralization tests done in Vero cell cultures. Sera from the mammals were tested for antibody to JC virus by a plaque-reduction serum dilution neutralization method. A high prevalence of JC virus-specific antibody was found in horses and cattle sampled from Morro Bay. This finding is additional evidence for the presence of a virus antigenically identical or closely related to JC virus in Morro Bay and indicates that the vectors of the virus in Morro Bay feed on large mammals. A high prevalence of virus-specific antibody was also found in horses sampled from Marin and San Diego counties. This finding suggests that viruses antigenically identical or closely related to JC virus are geographically widespread in coastal California.

Geographic distribution and serologic and genomic characterization of Morro Bay virus, a newly recognized bunyavirus.

More than 75,000 immature mosquitoes in three genera were collected from coastal California, reared to the adult stage, and tested for virus by plaque assay in Vero cell cultures. Twenty-six strains of Morro Bay (MB) virus, a newly recognized member of the California (CAL) serogroup, were isolated from Aedes squamiger, a pestiferous salt marsh mosquito species restricted to intertidal salt marshes in coastal California and Baja California. The geographic distribution of the isolates was 10 from San Luis Obispo County, one each from Santa Barbara and Orange Counties, and 14 from San Diego County. No virus isolations were made from 23,157 Ae. squamiger collected north of San Luis Obispo County (midpoint in the geographic range of this species in California). Thus, MB virus infection in Ae. squamiger appears to be restricted to the southern range of this species in California. Serum dilution neutralization tests indicated that MB virus represents a novel subtype of the California encephalitis (CE) serotype within the CAL serogroup. Comparative analyses of genomic sequence data from four geographically distinct MB virus isolates indicated that the isolates are genetically similar to each other and distinct from other CE serotype bunyaviruses. Phylogenetic analysis of nucleocapsid protein gene sequence data indicated that MB virus represents a distinct lineage within the CE serotype and thus supports the serologic classification of MB virus as a distinct CAL serogroup virus.

Prevalence of antibodies to arenaviruses in rodents from the southern and western United States: evidence for an arenavirus associated with the genus Neotoma.

The objectives of this study were to extend our knowledge of the geographic distribution and rodent host range of arenaviruses in North America. Sera from wild rodents collected from the southern and western United States were tested for antibody against Tamiami, Pichinde, Junin, and lymphocytic choriomeningitis viruses, using an indirect fluorescent antibody test. Antibody to at least one arenavirus was found in 220 (3.1%) of 7,106 rodents tested. The antibody-positive animals included Mus musculus from Florida and Texas; Neotoma albigula from Arizona, Colorado, and New Mexico; N. fuscipes and N. lepida from California: N. mexicana from Arizona, New Mexico, and Utah; N. stephensi from Arizona and New Mexico; and Oryzomys palustris and Sigmodon hispidus from Florida. Sigmodon hispidus seropositive for Tamiami virus were found only in Florida (156 [27.0%] of 578 tested), although 463 hispid cotton rats from outside that state were examined. High-titered antibodies to Tamiami virus were present in sera from S. hispidus, (geometric mean antibody titer [GMAT] of 1:792), whereas sera from Neotoma spp. reacted at high titer to both Tamiami (GMAT = 1:905) and Pichinde (GMAT = 1:433) viruses. The results suggest that arenaviruses are widely distributed in the southern United States and that one or more indigenous arenaviruses are associated with Neotoma spp. in North America.