Evidence of Hantavirus Infection in Microtus Ochrogaster in St. Louis County, Missouri.

Abstract: Between 31 October and 4 December 1994, 252 rodents were collected from The Washington University Tyson Biological Research Center (TBRC), St. Louis County, Missouri. Blood samples from 101 rodents were tested by enzyme-linked immunosorbent assay for antibodies reactive with Sin Nombre and other related hantaviruses. Antibody was detected in two of 58 tested prairie voles, Microtus ochrogaster. This is the second record of prairie voles being positive for hantavirus in Missouri. Given the relative specificity of hantavirus-host relationships, and the absence of infection of any other species in the study area, we suspect that the virus we have detected is most likely to be Bloodland Lake virus, which as been described as being associated with prairie voles. Our data, when combined with that of other studies showing hantavirus infection in prairie voles, suggest that the species is infected throughout a wide portion of its range.

Key Words: Microtus ochrogaster; Hantavirus; Bloodland Lake virus; Missouri

Introduction

The hantaviruses constitute a genus in the family Bunyaviridae. Unlike other genera in the family, an arthropod vector does not transmit hantaviruses. The virus is shed in the saliva, urine and feces of infected rodents. Humans may be infected by inhalation of infectious aerosols, or direct contact of infectious materials with mucous membranes or broken skin (Tsai, 1987). Hantaviruses are circulated in nature among small mammals and have a worldwide distribution. With a single exception, all known hantaviruses are associated with three subfamilies of rodent hosts of the family Muridae. Several viruses associated with the old-world subfamily Murinae cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe. Viruses associated with the new-world subfamily Sigmodontinae (including Sin Nombre virus, associated with Peromyscus maniculatus) cause hantavirus with pulmonary syndrome (HPS) in the Americas. A third group of hantaviruses is associated with voles (subfamily Arvicolinae) throughout the northe rn hemisphere. Puumala virus, associated with Bank voles, Cleithrionomys glareolus, causes a mild form of HFRS in Europe and Asia; however, none of the North American arvicoline viruses are known to cause any form of human disease (Schmaljohn and Hjelle, 1997).

Three hantaviruses have been described among arvicoline species in North America. These include Prospect Hill virus associated with the meadow vole, Microtus pennsylvanicus (Lee et al, 1982), Isla Vista virus, associated with the California vole, M. californicus (Song et al., 1995), and Bloodland Lake virus, associated with the prairie vole, M. ochrogaster (Hjelle et al, 1995). In addition a possibly distinct hantavirus has been described from montane vole M. montanus in Nevada (Rowe et al., 1995)

Materials and methods

Between 31 October and 4 December 1994, 252 rodents were collected from the Washington University Tyson Biological Research Center (TBRC) as part of a mammal survey of the center. TBRC is a 2000 acre (809 ha) area devoted to the study of environmental issues located in western St. Louis County, Missouri (38 31' N; 90 33' W) in the lower valley of the Meramec River. The entire facility is part of the Ozark Border Natural Division, which consists of the area to the north and east of the Ozark Plateau (Nigh et al., 1992). The vegetation is predominanfly oak-hickory forest. Only 15 percent of TBRC is open grassland or work areas, the remaining 85 percent is forested (Zimmerman and Wagner, 1979).

Sherman live traps (H. B. Sherman Traps, Tallahassee, Florida) baited with mixed birdseed, were used to collect rodents. Trap sites were selected on the basis of preferred rodent habitat and visible evidence of rodent activity. Blood was collected from the retrorbital capillary plexus of each animal, using a capillary tube. Sera were separated by centrifugation and frozen at --70C until shipped on dry ice to the Centers for Disease Control and Prevention, Atlanta, Ga., for testing.

Rodent blood samples were tested by enzyme-linked immunosorbent assay for antibodies reactive with hantavirus according to a standard protocol (Feldmann et al, 1993). This assay will detect but not distinguish among antibodies of a wide variety of hantaviruses associated with sigmodontine and arvicoline rodents (Mills et al., 1997).

Results

A total of 252 rodents were trapped during 1350 trap nights for an overall trap success of 18.59%. Of this 101 alive and adult specimens were tested for hantavirus (Table 1). The other 151 specimens were not tested because they had died in the traps or were subadults. Two of the 101 specimens had antibody reaction with Sin Nombre virus (SNV) and related hantaviruses. Both were adult male prairie voles, Microtus Ochrogaster, with scrotal testes. A total of 3.4% of prairie voles and 8% of male specimens had antibody (Table 1). Seropositive voles were found at only one of 5 trap sites where the species was captured. This represents an 8.6% antibody prevalence for the site and a 20% prevalence for males from this site. Our data are consistent with other studies which have shown that male rodents tend to have a higher infection rate for hantavirus (Mills et al., 1997).

This is the second documented occurrence of hantavirus infection in prairie voles from Missouri. The first instance was at Fort Leonard Wood, Pulaski County, Missouri where the virus was sequenced and later named Bloodland Lake virus (GENBANK Accession number U19303). Given the relative specificity of hantavirus-host relationships, and the absence of infection of any other species in the study area, we suspect that the virus we have detected is mostly to be Bloodland Lake virus. This virus has been described as being associated with the prairie vole (Hjelle et al., 1995). Amplification of the viral genome (by polymerase chain reaction) and sequencing would be necessary to confirm the specific identification of the virus. Antibodies to hantavirus in prairie voles have been described from western Nebraska (Mills et al., 1998). Assuming that all of this infection represents Bloodland Lake virus, these data, combined with our demonstration of antibodies in prairie voles from eastern Missouri, indicate the specie s is infected throughout a wide portion of its range.

Discussion

Although hantaviruses from North American arvicoline rodents have never been associated with human disease, antibodies reactive with Prospect Hill virus have been documented in American mammalogists, who have had extensive exposure to meadow voles (Tsal et al., 1985). These results, however, must be reassessed in the light of recent discoveries of numerous hantaviruses in North America and the low specificity of serological assays. Mild proteinuria without fever or hemorrhage occurs in cynomolgus monkeys, Macaca fascicularis, inoculated with Prospect Hill virus (Amyx et al., 1984). The extent to which infection with arvicoline hantaviruses in humans resembles experimental infection in monkeys, or even infection with a unnamed hemorrhagic virus causing nephropathia epidemica, remains to be determined (Lee et al., 1982).

The prairie vole is mostly endemic to the great central grasslands of North America, from the central Prairie Provinces of Canada, south to Oklahoma and from the Rocky Mountains east to the edge of the eastern deciduous forest and beyond (Jones et al., 1983).

Acknowledgments

Dr. Jerry Choate, Fort Hays State University, confirmed identification of specimens. Specimens collected are housed at the Tyson Research Area Mammal Collection, Washington University, St. Louis, Missouri. Hantavirus serology was performed in the laboratory of Dr. Tom Ksiazek, Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, Ga. LTC Cecil Chambliss, Gene Cogorno, Gerald Ellis, and LTC Randy Osowski assisted in collecting specimens. Missouri Department of Conservation provided the collecting permits.

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SPECIES                    # COLLECTED  # TESTED  # POSITIVE
Microtus ochrogaster          138          58         2
Microtus pinetorum              8           6         0
Peromyscus maniculatus         39          15         0
Peromyscus leucopus            39          14         0
Reithrodontomys megalotis      20           8         0
Mus musculus                    1           0         0
Blarina hylophaga               7           0         0
TOTALS                        252         101         2
SPECIES                    % POSITIVE
Microtus ochrogaster          3.4
Microtus pinetorum             0
Peromyscus maniculatus         0
Peromyscus leucopus            0
Reithrodontomys megalotis      0
Mus musculus                   0
Blarina hylophaga              0
TOTALS                        0.2