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Logo of vbzMary Ann Liebert, Inc.Mary Ann Liebert, Inc.JournalsSearchAlerts
Vector Borne and Zoonotic Diseases
 
Vector Borne Zoonotic Dis. 2008 August; 8(4): 523–540.
PMCID: PMC2714187
NIHMSID: NIHMS115284

Diversity Among Tacaribe Serocomplex Viruses (Family Arenaviridae) Naturally Associated with the White-Throated Woodrat (Neotoma albigula) in the Southwestern United States

Abstract

Bayesian analyses of glycoprotein precursor and nucleocapsid protein gene sequences indicated that arenaviruses naturally associated with white-throated woodrats in central Arizona are phylogenetically closely related to the Whitewater Arroyo virus prototype strain AV 9310135, which originally was isolated from a white-throated woodrat captured in northwestern New Mexico. Pairwise comparisons of glycoprotein precursor and nucleocapsid protein amino acid sequences revealed extensive diversity among arenaviruses isolated from white-throated woodrats captured in different counties in central Arizona and extensive diversity between these viruses and Whitewater Arroyo virus strain AV 9310135. It was concluded that the viruses isolated from the white-throated woodrats captured in Arizona represent 2 novel species (Big Brushy Tank virus and Tonto Creek virus) and that these species should be included with Whitewater Arroyo virus in a species complex within the Tacaribe serocomplex (family Arenaviridae, genus Arenavirus).

Key Words: Arenaviridae, Arenavirus, Tacaribe serocomplex, Big Brushy Tank virus, Tonto Creek virus, Whitewater Arroyo virus

Introduction

The virus family Arenaviridae, genus Arenavirus, comprises 2 serocomplexes and 22 species (Salvato et al. 2005). The lymphocytic choriomeningitis-Lassa (Old World) serocomplex includes Lassa virus (LASV), lymphocytic choriomeningitis virus (LCMV), Ippy virus (IPPV), Mobala virus (MOBV), and Mopeia virus (MOPV). The Tacaribe (New World) serocomplex includes Bear Canyon virus (BCNV), Tami-ami virus (TAMV), and Whitewater Arroyo virus (WWAV) in North America, Tacaribe virus (TCRV) on Trinidad in the Caribbean Sea, and Allpahuayo virus (ALLV), Amapari virus (AMAV), Cupixi virus (CPXV), Flexal virus (FLEV), Guanarito virus (GTOV), Junín virus (JUNV), Latino virus (LATV), Machupo virus (MACV), Oliveros virus (OLVV), Parana virus (PARV), Pichinde virus (PICv), Pirital virus (PIRV), and Sabia virus (SABV) in South America. Catarina virus (CTNV) is a provisional member of the Tacaribe serocomplex and native to North America (Cajimat et al. 2007a).

The genomes of arenaviruses comprise 2 single-stranded RNA segments, designated large (L) and small (S) (Salvato et al. 2005). The L segment (~7.5 kb) consists of a 5 noncoding region (NCR), the Z gene, an intergenic region that separates the Z gene from the RNA-de-pendent RNA polymerase (RdRp) gene, the RdRp gene, and a 3 NCR. Similarly, the S segment (~3.5 kb) consists of a 5 NCR, the glycoprotein precursor (GP-C) gene, an intergenic region that separates the GP-C gene from the nucleocapsid (N) protein gene, the N protein gene, and a 3 NCR. Our most comprehensive knowledge of the phylogenetic history of the Tacaribe serocomplex viruses is based on the results of analyses of full-length GP-C amino acid sequences and full-length N protein amino acid sequences (Archer and Rico-Hesse 2002, Charrel et al. 2002).

Specific members of the rodent family Cricetidae (Musser and Carleton 2005) are the principal hosts of the Tacaribe serocomplex viruses for which natural host relationships have been well characterized (Childs and Peters 1993). For example, the southern plains woodrat (Neotoma micropus) in southern Texas is the principal host of CTNV (Cajimat et al. 2007a, Fulhorst et al. 2002), the hispid cotton rat (Sigmodon hispidus) in southern Florida is the principal host of TAMV (Calisher et al. 1970, Jennings et al. 1970), the drylands vesper mouse (Calomys musculinus) in central Argentina is the principal host of JUNV (Mills et al. 1992), and a vesper mouse (Calomys species) in northeastern Bolivia is the principal host of MACV (Johnson et al. 1966, Salazar-Bravo et al. 2002).

The WWAV prototype strain AV 9310135 originally was isolated from a white-throated woodrat (Neotoma albigula) captured in 1993 in McKinley County in northwestern New Mexico (Fulhorst et al. 1996). Subsequently, anti-body to strain AV 9310135 was found in 112 (26.7%) of 420 white-throated woodrats captured in 5 counties in Arizona (Abbott et al. 2004). The objectives of this study were to determine whether the arenaviruses associated with the white-throated woodrat (N. albigula) in Arizona are strains of WWAV and to further our knowledge of the biology of arenavirus infections in white-throated woodrats in nature.

Materials and Methods

Safety

All laboratory work with potentially infectious rodent tissues and all work with infectious arenavirus were performed inside a biosafety level 3 (BSL-3) laboratory located on the campus of the University of Texas Medical Branch, Galveston.

Rodents

The 420 white-throated woodrats in this study were from a previously published study on the epizootiology of arenavirus infections in woodrats in Arizona (Abbott et al. 2004). Rodents captured alongside the white-throated woodrats included 3 brush mice (Peromyscus boylii), 81 cactus mice (P. eremicus), 15 deer mice (P. maniculatus), 25 pinyon mice (P. truei), and 9 northern grasshopper mice (Onychomys leuco-gaster). The woodrats and the 133 other rodents were captured at 11 localities in 5 counties in Arizona (Table 1, Fig. 1). The rodents were euthanized in the field by exposure to a lethal dose of vaporized isoflurane. The carcasses of all the rodents were shipped on dry ice from the field to the Department of Biological Sciences, Texas Tech University. Subsequently, (1) each carcass was assigned a unique museum identification (TK) number; (2) samples of cardiac blood for antibody assay were collected from all carcasses; (3) samples of brain, kidney, and spleen for virus assay were collected from the carcasses of the antibody-positive animals; (4) samples of brain, kidney, and spleen for virus assay were collected from the carcasses of 44 of the 308 antibody-negative white-throated woodrats; (5) samples of urine for virus assay were collected from the urinary bladders of 37 of the antibody-positive woodrats; and (6) samples of heart, lung, liver, kidney, spleen, and skeletal muscle, and the skins and skeletons of all the rodents were deposited into the Natural Science Research Laboratory, Museum of Texas Tech University.

FIG. 1.
Map of Arizona showing the 11 localities at which the 420 white-throated woodrats (Neotoma albigula) were captured. At least 1 white-throated woodrat at each locality was antibody-positive to WWAV strain AV 9310135. The filled circles indicate the localities ...
Table 1.
Prevalence of Antibody-Positive White-Throated Woodrats and Virus-Positive White-Throated Woodrats Captured at 11 Localities in Central and Northeastern Arizona

Antibody assay

The blood samples from the carcasses of the rodents were tested for IgG to WWAV strain AV 9310135, using an enzyme-linked immunosorbent assay (ELISA) (Bennett et al. 2000). The test antigen was a lysate of Vero E6 cells infected with WWAV strain AV 9310135, the control (comparison) antigen was a lysate of uninfected Vero E6 cells, and serial 4-fold dilutions (from 1:80 through 1:5120) of each blood sample were tested against both antigens. Antibody (IgG) bound to antigen was detected by using a mixture of a goat anti-rat IgG peroxidase conjugate and a goat anti-P. leucopus IgG peroxidase conjugate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) in conjunction with the ABTS Microwell Peroxidase Substrate System (Kirkegaard and Perry Laboratories). Optical densities (OD) at 405 nm (reference = 490 nm) were measured with a Dynatech MR 5000 microplate reader (Dynatech Industries, Inc., McLean, VA). The adjusted OD (AOD) of a blood-antigen reaction was the optical density of the well coated with the test antigen less the OD of the well coated with the control antigen. A sample was considered positive if the AOD at 1:80 was ≥0.200, the AOD at 1:320 was ≥0.200, and the sum of the AOD for the series of 4-fold dilutions (from 1:80 through 1:5120) was ≥0.750. The criteria for positivity were based on the results of a laboratory study on the pathogenesis of WWAV strain AV 9310135 infections in experimentally infected white-throated woodrats (Fulhorst et al. 2001a). Serial 4-fold dilutions (from 1:80 through 1:1,310,720) of each antibody-positive sample were tested against the test antigen and the control antigen. The endpoint titer in a positive sample was the highest dilution for which the AOD was ≥0.200.

Virus assay

Samples of brain, kidney, and spleen from the 112 antibody-positive woodrats, samples of urine from 37 of the antibody-positive woodrats, and samples of brain, kidney, and spleen from 26 (38.8%) of the 67 antibody-negative woodrats captured at locality 4 and 18 (23.7%) of the 76 antibody-negative woodrats captured at locality 7 were tested for arenavirus (Table 1). Each of the 13 antibody-positive woodrats captured at locality 4 and each of the 9 antibody-positive woodrats captured at locality 7 were matched to 2 antibody-negative white-throated woodrats on locality, gender, and size (nose-to-rump length) to assess the prevalence of virus-positive woodrats in the group of 308 antibody-negative woodrats in this study.

The samples of brain, kidney, spleen, and urine were tested for arenavirus as described previously (Fulhorst et al. 2001a). Briefly, 10% w/v crude suspensions of the tissue samples and 10% v/v suspensions of the urine samples were inoculated onto monolayers of Vero E6 cells grown in 12.5-cm2 plastic culture flasks, the inoculated cell monolayers were maintained under a fluid overlay at 37°C for 13 or 14 days, and then arenavirus antigen in infected Vero E6 cells was revealed by using an indirect fluorescent antibody test. The primary and secondary antibodies in the indirect fluorescent antibody test were a hyperimmune mouse ascitic fluid raised against WWAV strain AV 9310135 and a goat anti-mouse IgG fluorescein conjugate (Kirkegaard and Perry Laboratories), respectively.

Genetic characterization of viruses

The nucleotide sequences of a 3318- to 3390-nt fragment of the S genomic segments of arenavirus strains AV D0150144, AV D0390060, AV D0390174, and AV D0390324 were determined. These viruses were isolated from white-throated woodrats captured at localities 8 (Gila County), 9 (Gila County), 11 (Graham County), and 10 (Graham County), respectively (Table 2). Each sequence included a 73- to 137-nt fragment of the 5 NCR, the complete GP-C gene, intergenic region, and N protein gene, and a 29- to 30-nt fragment of the 3 NCR. Total RNA was isolated from monolayers of arenavirus-in-fected Vero E6 cells on the seventh, ninth, or eleventh day after inoculation, using TRIzol® Reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA). First-strand cDNA was synthesized using Superscript II RNase H Reverse Transcriptase (Invitrogen Life Technologies, Inc.) in conjunction with oligonucleotide 19C-cons (5 -CGCACMGWGGATCCTAGGC-3) (Cajimat et al. 2007b). Amplicons were generated from 3 overlapping fragments of the arenavirus-specific first-strand cDNA using the Master Taq Kit (Eppendorf North America, Inc., Westbury, NY) in conjunction with 19C-cons and AVGPC14 (5-GGACAGCCYTCRCCRA-TKATGTGTCTGTG-3), either AVGPC45 (5-GAGTAARGARTATGAAGAGAGGC-3) and AVNP72 (5-GTTGATGTGAAGCTAAGTGC-3) or AVGPC71 (5 -AGTARAGAATATGARGAAAGACA-3) and AVNP97 (5-GATATG-CATGGWAGACARGATTT-3) or AVGPC74 (5-TGTTGGCTTGTGAGAAATGGCAG-3) and AVNP97, and then AVNP13 (5-GTTGTKTCWGGYTCYCTGAA-3) and 19C-cons. Oligonucleotides 19C-cons and AVGPC14 flanked a 1408- to 1475-nt fragment of the S segment that extended from within the 5 NCR into the GP-C gene. Oligonucleotides AVGPC45 and AVNP72 flanked a 537-nt fragment of the S segment of AV D0150144 that extended from within the GP-C gene through the stop codon of the N protein gene, AVGPC71 and AVNP97 flanked a 568-nt fragment of the S segment of AV D0390174 and a 567-nt fragment of the S segment of AV D0390324 that extended from within the GP-C gene through the stop codon of the N protein gene, and AVGPC74 and AVNP97 flanked a 656-nt fragment of the S segment of AV D0390060 that extended from within the GP-C gene through the stop codon of the N protein gene. Oligonucleotides AVNP13 and 19C-cons flanked a 1685-nt fragment that extended from within the N protein gene, through the start codon of the N protein gene, and into the 3 NCR. Both strands of each amplicon were sequenced directly, using the dye termination cycle sequencing technique (Applied Biosystems, Inc., Foster City, CA). Sequence Rx Enhancer Solution A (Invitrogen Life Technologies, Inc.) or DMSO (10% v/v) was included in some of the sequencing reactions to improve the quality of the intergenic region sequence data. The nucleotide sequences of the S segments of strains AV D0150144, AV D0390060, AV D0390174, and AV D0390324 were deposited into the GenBank nucleotide sequence database under Accession Nos. EF619033, EF619034, EF619035, and EF619036, respectively.

Table 2.
Arenaviruses Isolated from White-Throated Woodrats and Selected for Genetic Characterization

Genetic characterization of rodents

Arenavirus strains AV D0150144, AV D0390060, AV D0390174, and AV D0390324 were isolated from white-throated woodrats TK93637, TK113981, TK114533, and TK114581, respectively (Table 2). White-throated woodrat NK50020 was captured in April 1993 at a site less than 15.0 km west of the town of Gallup in McKinley County, New Mexico. The nucleotide sequences of the cytochrome b genes of TK93637, TK113981, TK114533, TK114581, and NK50020 were determined to confirm the taxonomical relationships among the white-throated woodrat in Gila County, the white-throated woodrat in Graham County, and the white-throated woodrat that inhabits the region in New Mexico in which WWAV is enzootic.

Mitochondrial DNA was isolated from samples of liver using the DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). The complete cytochrome b gene (1143 bp) of each rodent was amplified using TaqGold DNA Polymerase (Applied Biosystems, Inc.) in conjunction with oligonucleotides H15915 (Irwin et al. 1991) and MVZ05 (Smith and Patton 1993) or oligonucleotides H15149 (Irwin et al. 1991) and L14724 (Irwin et al. 1991). Both strands of each amplicon were sequenced directly, using the dye termination cycle sequencing technique (Applied Biosystems, Inc.) in conjunction with oligonucleotides MVZ05, 400R (Tiemann-Boege et al. 2000), 700L (Tiemann-Boege et al. 2000), 400F (Tiemann-Boege et al. 2000), H15149, or L14724. The nucleotide sequences of the cytochrome b genes of TK93637, TK113981, TK114533, TK114581, and NK50020 were deposited into the GenBank nucleotide sequence database under Accession Nos. EU141961, EU141963, EU141960, EU141964, and EU141962, respectively.

Data analysis

The analyses of the nucleotide sequences and amino acid sequences of the viruses included BCNV strain AV A0060209 (GenBank Accession No. AF512833), CTNV strains AV A0400135 and AV A0400212 (DQ865244 and DQ865245, respectively), TAMV strain W 10777 (AF512828), WWAV strain AV 9310135 (AF228063), ALLV strain CLHP-2472 (AY012687), AMAV strain BeAn 70563 (AF512834), CPXV strain BeAn 119303 (AF512832), FLEV strain BeAn 293022 (AF512831), GTOV strain INH-95551 (NC_005077), JUNV strains XJ13, MC2, and Romero (NC_005081, D10072, and AY619641, respectively), LATV strain MARU 10924 (AF485259), MACV strains Carvallo, Chicava, Mallele, and 9530537 (NC_005078, AY624355, AY619645, and AY571959, respectively), OLVV strain 3229-1 (U34248), PARV strain 12056 (AF485261), PICV strain An 3739 (NC_006447), PIRV strain VAV-488 (NC_005894), SABV strain SPH 114202 (NC_006317), TCRV strain TRVL 11573 (NC_004293), and LCMV strain WE (M22138). Multiple strains of CTNV, JUNV, and MACV were included in the analyses to provide an assessment of the magnitude of diversity in the GP-C and in the N protein within different New World arenavirus species. The alignments of the GP-C and N protein amino acid sequences were constructed using the computer program CLUSTAL W1.7 (Thompson et al. 1994). The alignments of the GP-C and N protein gene sequences were constructed manually based on the computer-generated alignments of the GP-C and N protein amino acid sequences, respectively. The analyses of the nucleotide sequence alignments were done using MRBAYES 3.1.2 (Huelsenbeck and Ronquist 2001) and programs in the computer software package PAUP*, version 4.0b10 (Swofford 2002). Sequence nonidentities were equivalent to uncorrected distances. The Bayesian analyses used a GTR+I+G model with a site-specific gamma distribution and the following options in MRBAYES 3.1.2: 2 simultaneous runs of 4 Markov chains, 1,000,000 generations, and sample frequency = every 1000th generation. The LCMV strain WE was the designated outgroup in the Bayesian analyses. The first 100 trees were discarded after review of the likelihood scores, convergence statistics, and potential scale reduction factors. A consensus tree (50% majority rule) was constructed from the remaining trees and clade probability values were generated to assess support for the nodes within the consensus tree.

The nucleotide sequences of the cytochrome b genes of white-throated woodrats TK93637, TK113981, TK114581, TK114533, and NK50020 were compared with the nucleotide sequences of the cytochrome b genes of 15 other woodrats captured in the United States and 5 woodrats captured in northern Mexico: NK54407 and TK54559 (N. albigula, northern Arizona, Gen-Bank Accession Nos. AF186811 and AF186807, respectively), TK50148 (N. albigula, northeastern Arizona, AF186808), NK54403 (N. albigula, south-central Arizona, AF186810), TK77854 and TK74856 (N. albigula, southwestern Arizona, AF186816 and AF376472, respectively), NK17583 (N. albigula, central Chihuahua, AF186804), NK1330 (N. albigula, northwestern Sonora, AF186814), NK56291 (N. cinerea, northwestern Colorado, AF186800), NK1335 (N. de-via, northwestern Sonora, AF307830), TK52109 (N. floridana, northeastern Texas, AF186819), TK77287 (N. fuscipes, southern California, AF376475), NK77284 (N. lepida, southern California, AF307835), TK28742 (N. leucodon, western Oklahoma, AF186815), NK64158 (N. magister, Virginia, AF294336), TK51346 (N. mexicana, southeastern Colorado, AF186821), TK31643 (N. micropus, southeastern New Mexico, AF186822), TK93390 (N. picta, southwest Guerrero, AF305569), TK77928 (N. stephensi, northeast Arizona, AF308867), and TK45042 (Hodomys alleni, western Michoacan, AF186801). The eastern woodrat (N. floridana), Allegheny woodrat (N. magister), and 9 of the other woodrats were included in the analyses to provide a metric for interpretation of the genetic distances between the white-throated woodrats captured at localities 8, 9, 10, and 11 in Arizona and white-throated woodrat NK50020. The results of phylogenetic analyses of cytochrome b gene sequences in previous studies (Edwards and Bradley 2002, Longhofer and Bradley 2006) indicated that N. albigula is phylogenetically closely related to N. floridana and N. magister. The analyses of the alignment of cytochrome b gene sequences were done using MRBAYES 3.1.2 (Huelsenbeck and Ronquist 2001) and programs in the computer software package PAUP*, version 4.0b10 (Swofford 2002). Sequence nonidentities were equivalent to un-corrected distances. The Bayesian analyses used a GTR+I+G model with a site-specific gamma distribution and the following options in MRBAYES 3.1.2: 2 simultaneous runs of 4 Markov chains, 1,000,000 generations, and sample frequency = every 1000th generation. The Allen's woodrat (H. alleni) was the designated outgroup taxon in the Bayesian analyses. The first 100 trees were discarded after review of the likelihood scores, a consensus tree (50% majority rule) was constructed from the remaining trees, and clade probability values were generated to assess support for the nodes within the consensus tree.

The statistical tests of the associations between antibody status, antibody titer, success of virus isolation, and gender and size of woodrat were restricted to the 8 localities at which 7 or more woodrats were antibody-positive: localities 3 through 5 and 7 through 11 (Table 1). The male woodrats captured at these 8 localities were assigned to 4 size classes based on their nose-to-rump lengths (measured in mm): I, 36–147 (n = 15); II, 150–174 (n = 58); III, 175–199 (n = 106); IV, 200–227 (n = 15). Likewise, the female woodrats captured at these 8 localities were assigned to 4 size classes based on their nose-to-rump lengths (measured in mm): I, 67–145 (n = 21); II, 149–166 (n = 59); III, 167-186 (n = 104); IV, 187-200 (n = 15). The class boundaries were established based on the mean and standard deviation (SD) of the noseto-rump lengths of the 194 male woodrats (174.7 and 25.1 mm, respectively) and the mean and SD of the nose-to-rump lengths of the 199 female woodrats (166.4 and 20.3 mm, respectively) captured at localities 3 through 5 and 7 through 11. The upper boundary of class I was the mean length less 1 SD, the upper boundary of class II was the mean length, the upper boundary of class III was the mean length plus 1 SD; and the upper boundary of class IV was the longest nose-to-rump length. The acceptable type I error in all statistical tests was alpha = 0.05.

Results

Antibody assay

Antibody (IgG) to WWAV strain AV 9310135 was found in blood samples from 112 (26.7%) of the 420 white-throated woodrats and none of the 133 other rodents. The antibody titers in the antibody-positive samples ranged from 320 to greater than or equal to 1,310,720.

Virus assay

Arenavirus was isolated from 17 (15.2%) of the 112 antibody-positive woodrats. The virus-positive animals included 1 captured at locality 8, 1 captured at locality 9, 14 captured at locality 10, and 1 captured at locality 11 (Table 1). The virus-positive specimens included the samples of spleen from 9 animals, brain from 12 animals, kidney from 16 animals, and urine from 4 animals (Table 3). The virus isolation attempts on the samples of spleen, brain, and kidney from the 26 antibody-negative woodrats captured at locality 4 and the virus isolation attempts on the samples of spleen, brain, and kidney from the 18 antibody-negative woodrats captured at locality 7 were negative (Table 1).

Table 3.
Genders, Size Classes, and Antibody Titers of 17 Virus-Positive White-Throated Woodrats Captured at 4 Localities in Central Arizona

Characterization of viruses

The lengths of the GP-C genes and intergenic regions of AV D0150144, AV D0390060, AV D0390174, and AV D0390324 ranged from 1455 to 1458 nt and from 72 to 77 nt, respectively, and the lengths of the N protein genes of these 4 viruses were identical, i.e., 1689 nt. Nonidentities (uncorrected distances) between the nucleotide sequences of the GP-C genes of AV D0150144, AV D0390060, AV D0390174, and AV D0390324 and the nucleotide sequences of the GP-C genes of BCNV strain A0060209, CTNV strains AV A0400135 and AV A0400212, TAMV strain W 10777, and WWAV strain AV 9310135 ranged from 28.4% to 36.3% (Table 4). Similarly, nonidentities between the nucleotide sequences of the N protein genes of AV D0150144, AV D0390060, AV D0390174, and AV D0390324 and the nucleotide sequences of the N protein genes of the 5 other North American viruses ranged from 22.2% to 27.9% (Table 4).

Table 4.
Nonidentities between the Nucleotide Sequences of the Glycoprotein Precursor Genes and between the Nucleotide Sequences of the Nucleocapsid Protein Genes of 9 North American Arenaviruses

The 2 trees generated by simultaneous Bayesian analyses of the GP-C gene sequences were identical. Likewise, the 2 trees generated by simultaneous Bayesian analyses of the N protein gene sequences were identical. The results of the analyses of the GP-C and N protein gene sequences (Fig. 2A and Fig. 2B, respectively) indicated that the 7 viruses isolated from woodrats (i.e., AV D0150144, AV D0390060, AVD0390174, AV D0390324, CTNV strains AV A0400135 and AV A0400212, and WWAV strain AV 9310135) are monophyletic and phylogenetically distinct from BCNV strain AV A0060209 and TAMV strain W 10777. The analyses of the GP-C gene sequences placed AV D0150144 in a close sister relationship to AV D0390060, AV D0390174 in a close sister relationship to AV D0390324, and WWAV strain AV 9310135 in a sister relationship to the AV D0150144–AV D0390060 lineage. However, the clade probability values for monophyly of AV D0150144, AV D0390060, and WWAV strain AV 9310135 in both analyses of the GP-C gene sequences were less than 0.95. The analyses of the N protein gene sequences placed AV D0150144 in a close sister relationship to AV D0390060, AV D0390174 in a close sister relationship to AV D0390324, and WWAV strain AV 9310135 in a sister relationship to the CTNV lineage. However, the clade probability value for monophyly of WWAV and CTNV was only 0.94 in 1 of the analyses of the N protein gene sequences.

FIG. 2.
Phylogenetic relationships among 28 New World arenaviruses based on Bayesian analyses of (A) glycoprotein precursor gene sequences and (B) nucleocapsid protein gene sequences. The lengths of the scale bars are equivalent to 100 changes. The number(s) ...

Nonidentities between the amino acid sequences of the GP-C and between the amino acid sequences of the N proteins of strains of a single species ranged from 0.8% (JUNV strains XJ13 and Romero) to 5.6% (MACV strains Chicava and 9530537) and from 0.9% (MACV strains Carvallo and Chicava) to 3.9% (JUNV strains XJ13 and MC2), respectively. Nonidentities between the sequences of the GP-C and between the sequences of the N proteins of AV D0150144 and AV D0390060 were 11.2% and 5.0%, respectively (Table 5). Similarly, non-identities between the sequences of the GP-C and between the sequences of the N proteins of AV D0390174 and AV D0390324 were 6.8% and 0.5%, respectively (Table 5). Thus, AV D0150144 and AV D0390060 could be conspecific and AV D0390174 and AV D0390324 are conspecific.

Table 5.
Nonidentities between the Predicted Amino Acid Sequences of the Glycoprotein Precursors and between the Predicted Amino Acid Sequences of the Nucleocapsid Proteins of 9 North American Arenaviruses

Nonidentities between the sequences of the GP-C and between the sequences of the N proteins of strains of different South American arenavirus species ranged from 15.8% (ALLV strain CLHP-2472 and FLEV strain BeAn 293022) to 60.3% (AMAV strain BeAn 70563 and PIRV strain VAV-488) and from 11.9% (JUNV strain XJ13 and MACV strain 9530537) to 44.9% (PICV strain An 3739 and CPXV strain BeAn 119303), respectively. Similarly, non-identities between the sequences of the GP-C and between the sequences of the N proteins of BCNV strain AV A0060209, CTNV strain AV A0400135 or AV A0400212, TAMV strain W 10777, and WWAV strain AV 9310135 ranged from 33.4% to 40.3% and from 13.3% to 21.7%, respectively (Table 5).

Nonidentities between the sequences of the GP-C of AV D0150144 and AV D0390060 and the sequences of the GP-C of BCNV strain AV A0060209, CTNV strains AV A0400135 and AV A0400212, TAMV strain W 10777, and WWAV strain AV 9310135 ranged from 28.3% to 35.7% (Table 5). Nonidentities between the sequences of the N proteins of AV D0150144 and AV D0390060 and the sequences of the N proteins of BCNV strain AV A0060209, CTNV strains AV A0400135 and AV A0400212, TAMV strain W 10777, and WWAV strain AV 9310135 ranged from 9.8% to 19.0% (Table 5). Accordingly, AV D0150144 and AV D0390060 are strains of a novel species. The name Tonto Creek virus (TTCV) is proposed to distinguish this species from WWAV and all other arenavirus species.

Nonidentities between the sequences of the GP-C of AV D0390174 and AV D0390324 and the sequences of the GP-C of strains AV D0150144 and AV D0390060, BCNV strain AV A0060209, CTNV strains AV A0400135 and AV A0400212, TAMV strain W 10777, and WWAV strain AV 9310135 ranged from 25.6% to 36.0% (Table 5). Nonidentities between the sequences of the N proteins of AV D0390174 and AV D0390324 and the sequences of the N proteins of TTCV strains AV D0150144 and AV D0390069, BCNV strain AV A0060209, CTNV strains AV A0400135 and AV A0400212, TAMV strain W 10777, and WWAV strain AV 9310135 ranged from 10.1% to 19.6% (Table 5). Thus, AV D0390174 and AV D0390324 are strains of a novel species. The name Big Brushy Tank virus (BBTV) is proposed to distinguish this species from TTCV, WWAV, and all other arenavirus species.

Characterization of rodents

The 2 trees generated by simultaneous Bayesian analyses of the cytochrome b gene sequences were identical and indicated that TK93637, TK113981, TK114533, TK114581, 7 other white-throated woodrats captured in Arizona, NK50020, and 2 white-throated woodrats captured in Mexico are monophyletic (results not shown). Further, the Bayesian analyses placed N. albigula in a sister relationship to a clade that included N. floridana and N. magister. The clade probability values for monophyly of the 14 white-throated woodrats were 1.00, the clade probability values for monophyly of N. albigula, N. floridana, and N. magister were 1.00, and the relationships among the N. albigula–N. floridana–N. magister clade and the 10 other Neotoma species included in the Bayesian analyses were similar to the results of studies published previously (Edwards and Bradley 2002, Longhofer and Bradley 2006).

In pairwise comparisons of cytochrome b gene sequences, nonidentities (uncorrected genetic distances) among the white-throated woodrats captured in Gila County, the white-throated woodrats captured in Graham County, and NK50020 ranged from 0.88% to 6.56%, and nonidentities between individuals of different woodrat species ranged from 7.65% (N. devia and N. lepida) to 16.9% (N. cinerea and N. magister). These intra- and interspecific genetic distances are similar in magnitude to the genetic distances within and between 13 woodrat species published previously (Edwards et al. 2001, Edwards and Bradley 2001, 2002).

Associations between antibody status, antibody titer, success of virus isolation, and zoographic characteristics

As indicated previously, the statistical tests of the associations between antibody status, antibody titer, success of virus isolation, and gender and size were restricted to localities 3 through 5 and 7 through 11 (Table 1).

Antibody to WWAV strain AV 9310135 was found in 106 (26.9%) of the 393 woodrats captured at these 8 localities. Forty-nine (46.3%) of the 106 antibody-positive woodrats and 145 (50.1%) of the 287 antibody-negative woodrats were male. There was no association between antibody status and gender in the group of 393 woodrats (χ2 test, 3 df).

The prevalence of antibody by size class in the male woodrats and in the female woodrats ranged from 13.3% to 29.2% and from 13.6% to 46.7%, respectively (Table 6). There was no association between antibody status and size class in the group of 179 male woodrats in size classes II, Ill, and IV (χ2 test, 5 df). Likewise, there was no association between antibody status and size class in the group of 178 female woodrats in size classes II, Ill, and IV (χ2 test, 5 df).

Table 6.
Prevalence of Antibody (IgG) to WWAV Strain AV 9310135 in 393 White-Throated Woodrats Captured at 8 Localities in Arizona, by Gender and Size Class

The antibody titers in the antibody-positive male woodrats and in the antibody-positive female woodrats ranged from 320 through 1,310,720 (Table 7). There was no obvious association between antibody titer and size class in the group of 106 antibody-positive woodrats, regardless of gender.

Table 7.
Antibody Titers to WWAV Strain AV 9310135 in Male White-Throated Woodrats and Female White-Throated Woodrats Captured at 8 Localities in Arizona, by Size Class

Arenavirus was isolated from 1 (10.0%) of the 10 antibody-positive animals in size class i, 5 (25.0%) of the 20 antibody-positive animals in size class II, 11 (16.9%) of the 65 antibody-positive animals in size class iii, and none of the 11 antibody-positive animals in size class IV. The virus-positive animal in size class I was TK114586. There was no association between the success of virus isolation and gender in the group of 106 antibody-positive woodrats (χ2 test, 3 df). There also was no association between the success of virus isolation and size class in the group of 106 antibody-positive woodrats (χ2 test, 7 df).

Arenavirus was isolated from the samples of kidney from 16 woodrats and from the samples of spleen from 9 (56.3%) of these 16 woodrats (Table 3). The antibody titers in the 9 woodrats for which arenavirus was isolated from kidney and spleen ranged from 1280 to 327,680 (median: 5120), whereas the antibody titers in the 7 woodrats for which arenavirus was isolated from kidney but not spleen ranged from 20,480 to greater than 1,310,720 (median: 327,680) (Table 3), suggesting that the success of virus isolation from spleen was negatively associated with antibody titer.

Arenavirus was isolated from 14 (58.3%) of the 24 antibody-positive animals captured at locality 10, none of the 13 antibody-positive animals captured at locality 4, and none of the 9 antibody-positive animals captured at locality 7 (Table 1). The male-to-female ratio in the 24 antibody-positive woodrats captured at locality 10 was not significantly different from the male-to-female ratio in the group of 22 antibody-positive woodrats captured at localities 4 and 7 (χ2 test, 3 df). There was no significant difference between the mean of the nose-to-rump lengths of the 7 antibody-positive male woodrats captured at locality 10 and the mean of the nose-to-rump lengths of the 11 antibody-positive male woodrats captured at localities 4 and 7 (Student's 2-tailed t-test, 16 df). There also was no significant difference between the mean of the nose-to-rump lengths of the 17 antibody-positive female woodrats captured at locality 10 and the mean of the nose-to-rump lengths of the 11 antibody-positive female woodrats captured at localities 4 and 7 (Student's 2-tailed t-test, 26 df).

Discussion

The principal host relationships of some South American arenaviruses appear to represent a long-term shared evolutionary relationship between the Arenaviridae and the Cricetidae, subfamily Sigmodontinae (Bowen et al. 1997). Evidence for this long-standing relationship includes the present-day association of phylogenetically closely related arenaviruses with phylogenetically closely related sigmodontine rodents, e.g., JUNV with the drylands vesper mouse (C. musculinus) in Argentina (Mills et al. 1992) and MACV with a vesper mouse (Calomys species) in Bolivia (Johnson et al. 1966, Salazar-Bravo et al. 2002).

Arenaviruses phylogenetically closely related to WWAV strain AV 9310135 and CTNV strains AV A0400135 and AV A0400212 have been isolated from a white-throated woodrat (N. albigula) captured in western Oklahoma, a bushy-tailed woodrat (N. cinerea) and Mexican woodrat (N. mexicana) captured in southern Utah, and Mexican woodrats (N. mexicana) captured in central New Mexico (Fulhorst et al. 2001b). Further, strains of BCNV have been isolated from big-eared woodrats (N. macrotis) captured in southern California (Cajimat et al. 2007b). Collectively, N. albigula, N. cinerea, N. macrotis, N. mexicana, and N. micropus represent 3 of the 4 major phylogenetic subdivisions in the genus Neotoma (Edwards and Bradley 2002). The broad geographical distribution of phylogenetically closely related arenaviruses in association with phylogenetically distantly related woodrat species suggests that the present-day association between the Arenaviridae and the Cricetidae, subfamily Neotominae (specifically, the genus Neotoma), was established long ago. This relationship may have been established as early as the divergence of the neotomine rodents from the sigmodontine rodents, which has been dated to 18.1 to 16.8 million years ago (Steppan et al. 1994).

The Eighth Report of the International Committee on Taxonomy of Viruses (Salvato et al. 2005) indicated that strains of different arenavirus species should exhibit significant differences in pairwise comparisons of amino acid sequences or significant differences in 2-way serological tests. in this study, nonidentities between the amino acid sequences of the GP-C and between the amino acid sequences of the N proteins of different South American arenavirus species were as low as 15.8% (ALLV strain CLHP-2472 and FLEV strain BeAn 293022) and 11.9% (JUNV strain XJ13 and MACV strain 9530537), respectively. The level of sequence divergence between BBTV strains AV D0390174 and AV D0390324, TTCV strains AV D0390060 and AV D0150144, and WWAV strain AV 9310135 in this study is comparable to the level of sequence divergence between strains of phylogenetically closely related South American species.

The results of the Bayesian analyses of the GP-C and N protein gene sequences indicated that BBTV and TTCV are phylogenetically more closely related to WWAV and CTNV than to BCNV or TAMV, but did not solve the phylogenetic relationships among BBTV, CTNV, TTCV, and WWAV. Accordingly, BBTV, CTNV, TTCV, and WWAV could be grouped together in a species complex. other members of the WWAV species complex may include the arenaviruses associated with the white-throated woodrat in western oklahoma, the bushy-tailed woodrat in southern Utah, the Mexican woodrat in southern Utah, and the Mexican woodrat in central New Mexico (Ful-horst et al. 2001b).

The rodent fauna of McKinley County in northwestern New Mexico includes N. albigula, N. cinerea, N. mexicana, and N. stephensi (Find-ley et al. 1975) and the rodent fauna of Gila and Graham Counties in Arizona includes N. albigula, N. mexicana, and N. stephensi (Hoffmeister 1986). Our knowledge of the natural host relationships of WWAV is limited to the isolation of strain AV 9310135 and 2 other WWAV strains from 2 (12.5%) of 16 white-throated woodrats captured in 1993 in McKinley County (Fulhorst et al. 1996). Hypothetically, the WWAV infections in the white-throated woodrats captured in McKinley County were acquired from bushy-tailed woodrats, Mexican woodrats, southern plains woodrats, Stephen's woodrats (N. stephensi), or rodents other than woodrats. Similarly, the virus-positive white-throated woodrats captured in Gila County and in Graham County in this study could have acquired their infections from rodents other than white-throated woodrats. Future research on the ecology of the arenaviruses naturally associated with white-throated woodrats in Arizona and New Mexico should include field and laboratory studies to define better the principal host relationships of these viruses.

The results of the analyses of cytochrome b gene sequence data in this study indicated that the woodrats captured in Gila County, the woodrats captured in Graham County, and white-throated woodrat NK50020 from McKinley County, New Mexico, are conspecific. The high level of genetic divergence among BBTV, TTCV, and WWAV suggests that the present-day association of 1 or more of these virus species with N. albigula does not represent a long-standing shared evolutionary relationship between the Arenaviridae and N. albigula. Alternatively, the high level of genetic divergence among BBTV, TTCV, and WWAV could be interpreted as evidence that the evolution of these species in association with N. albigula has significantly outpaced the evolution of the cytochrome b gene of the white-throated woodrat (N. albigula).

Chronic infections in individual rodents appear to be critical to long-term maintenance of arenaviruses in nature (Childs and Peters 1993). The results of a laboratory study on the biology of WWAV strain AV 9310135 in white-throated woodrats captured in southeastern New Mexico (Fulhorst et al. 2001a) indicated that the duration of infection is dependent upon the age of the woodrat at the onset of infection. inoculation of newborn animals resulted in chronic infections whereas inoculation of juvenile and adult animals resulted in transient infections. These findings led to the hypothesis that vertical (dam-to-progeny) virus transmission in the white-throated woodrat (N. albigula) is critical to the long-term maintenance of WWAV in nature.

The isolation of arenavirus from the brain of TK114586 (size class I) strongly indicates that some white-throated woodrats in nature are infected with BBTV at a very young age. The failure to isolate arenavirus from the 9 other antibody-positive woodrats in size class I (Table 7) suggests that anti-arenavirus antibody in other young white-throated woodrats is a consequence of mother-to-offspring antibody transfer rather than a humoral antibody response to infection.

The failure to isolate arenavirus from 26 antibody-negative animals captured at locality 4 and 18 antibody-negative animals captured at locality 7 (Table 1) suggests that naturally infected white-throated woodrats usually develop a measurable antibody response soon after the onset of infection. Collectively, the isolation of arenavirus from TK114586, the lack of an association between antibody status and size class in the group of 179 male woodrats in size classes II, III, and IV, and the lack of an association between antibody status and size class in the group of 178 female woodrats in size classes II, III, and IV indicate that vertical virus transmission is an important mode of arenavirus transmission in the white-throated woodrat (N. albigula) in nature.

In the laboratory study mentioned previously (Fulhorst et al. 2001a), the infections in the white-throated woodrats inoculated with WWAV strain AV 9310135 at birth persisted in brain and kidney through 164 days of age, the infections in the white-throated woodrats inoculated with WWAV strain AV 9310135 at 4 months of age or older were transient, and the clearance of arenavirus from blood and spleen in the animals inoculated at birth and the sterilization of the infections in the animals inoculated at 4 months of age or older coincided with the advent of a vigorous humoral antibody response to strain AV 9310135. The results of this study (specifically, the failure to isolate arenavirus from the spleens of 7 of the 16 virus-positive animals in size classes II, III, and IV, and the negative association between success of virus isolation from spleen and antibody titer in the 16 virus-positive animals in size classes II, III, and IV) suggest that arenavirus infections in naturally infected white-throated woodrats are systemic only during the acute phase of infection.

The isolation of arenavirus from the kidneys of all the virus-positive animals in size classes II, III, and IV suggests that arenavirus persists in the kidneys of some naturally infected white-throated woodrats. The isolation of arenavirus from the samples of urine from only 4 of 7 virus-positive animals suggests that virus shedding in some naturally infected white-throated woodrats is either transient or intermittent.

The failure to isolate arenavirus from the 22 antibody-positive woodrats captured at localities 4 and 7 was unexpected because arenavirus was isolated from 13 (54.1%) of the 24 antibody-positive woodrats captured at locality 10. The difference in the success of virus isolation between localities did not appear to be related to a difference in gender or size (age) between the antibody-positive woodrats captured at localities 4 and 7 and the antibody-positive woodrats captured at locality 10. Hypothetically, arenavirus was not isolated from the antibody-positive woodrats captured at localities 4 and 7 because the arenaviruses associated with these woodrats do not adapt to growth in cultured Vero E6 cells as readily as AV D0390324 and the other strains of BBTV isolated from the woodrats captured at locality 10.

Humans usually become infected with arenaviruses by inhalation of infectious virus in aerosolized droplets of urine or secretions from rodents. The isolation of arenavirus from the samples of urine from 4 woodrats captured at locality 10 suggests that some naturally infected white-throated woodrats are infectious to humans.

Six members of the Arenaviridae have been causally associated with severe febrile disease in humans: LASV, LCMV, GTOV, JUNV, MACV, and SABV (Peters 2002). The human health significance of arenaviruses associated with woodrats and other cricetid rodents indigenous to the western United States is the subject of ongoing research supported by the U.S. Public Health Service, National Institutes of Health. A specific objective of this research is to develop assays for detection of arenavirus-specific RNA in acute-phase biological specimens from febrile patients infected with arenaviruses naturally associated with woodrats, cotton rats, and other cricetid rodents indigenous to North America. Knowledge of the nucleotide sequences of the arenaviruses isolated from the woodrats in this study could prove very useful in efforts to design accurate nucleotide sequence-based assays for arenavirus-specific RNA in acute-phase biological specimens from febrile patients infected with Tacaribe serocomplex viruses naturally associated with woodrats and other cricetid rodents indigenous to the western United States.

Acknowledgments

Mary Louise Milazzo, Maria N.B. Cajimat, and Michelle L. Haynie contributed equally to this study. John R. Suchecki, B. Dnate’ Baxter, Serena A. Reeder, and Nevin N. Durish (Texas Tech University, Lubbock) and Sammie Gardner and Jason E. Comer (University of Texas Medical Branch, Galveston) assisted with dissection of the woodrat carcasses. Sammie Gardner and Jason E. Comer also assisted with the assays for antibody in the blood samples and the assays for arenavirus in tissues collected from the carcasses. The sample of liver from rodent NK50020 was obtained from the Museum of Southwestern Biology, University of New Mexico, Albuquerque. Francisca M. Mendez-Harclerode (University of Texas Health Science Center, San Antonio) determined the nucleotide sequence of the cytochrome b gene of rodent NK50020. Teresa N. Quitugua (University of Texas Health Science Center, San Antonio) and Robert Fleischer (Smithsonian Institution, National Zoological Park, National Museum of Natural History, Center for Conservation and Evolutionary Genetics, Washington, DC) provided the laboratory materials and space for the genetic characterization of rodent NK50020 and rodents TK93637, TK113981, TK114581, and TK114533, respectively. National Institutes of Health grant AI-41435 (“Ecology of emerging arenaviruses in the southwestern United States”) provided the financial support for this study. Patricia Repik (National Institute of Allergy and Infectious Diseases) facilitated the grant support for this study.

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