Rodent Tissues

Five hundred sixty-six tissue specimens (74 spleen, 225 liver, and 267 kidney) from 425 woodrats were tested for infectious arenavirus. The specimens were from the Museum of Texas Tech University (Lubbock, TX) or Museum of Southwestern Biology (University of New Mexico, Albuquerque, NM). The specimens from the Museum of Southwestern Biology were chosen to represent localities in which antibody to an arenavirus was found in one or more Neotoma species in a previous study (13).

Virus Assay

Tissue specimens were tested for infectious arenavirus as described previously (12). Briefly, 0.2 mL of a 10% w/v crude tissue homogenate was inoculated onto a monolayer of Vero E6 cells in a 25-cm² plastic culture flask (Corning, Inc., Corning, NY). The inoculum was incubated on the cell monolayer at 37C for 60 minutes; then the monolayer was overlaid with 7.0 mL of a minimum essential medium containing Earle's salts, 1.5 mg/mL sodium bicarbonate, 2% v/v heat-inactivated (56ºC for 30 minutes) fetal bovine serum, 0.29 mg/mL L-glutamine, 100 U/mL penicillin G, 100 µg/mL streptomycin sulfate, and 100 U/mL nystatin. The cell culture was maintained at 37C in a humidified atmosphere of 5% CO₂ in air for 13 days. Half the culture medium was replaced with fresh maintenance medium on day 6 or 7 after inoculation. Cells were scraped from the monolayer on day 13 after inoculation and coated onto 12-well glass microscope slides (Cel-Line Associates, Inc., Newfield, NJ). The cell spots were air-dried, fixed in cold acetone, and then tested for arenaviral antigen by using an indirect fluorescent antibody test (12). In that test, cell spots were stained with a hyperimmune mouse ascitic fluid prepared against the WWA virus prototype strain AV 9310135, and mouse immunoglobulin G (IgG) bound to cell-associated arenaviral antigen was detected by using a goat anti-mouse IgG fluorescein isothiocyanate conjugate (Cappel Laboratories, West Chester, PA).

Genetic Characterization of Viral Isolates

The nucleotide sequence of a fragment of the N protein gene of each of 12 isolates was determined. Four of the 12 isolates were from the spleens and kidneys of two animals, rodents 62425 and 62439 (Table 1). Total RNA was extracted from monolayers of infected Vero E6 cells by using TRIzol Reagent (Life Technologies, Inc., Grand Island, NY). Reverse transcription of RNA from isolates AV 96010149, AV 96010151, AV 96010025, and AV 96010024 was carried out by using Superscript II RTase (Life Technologies) in conjunction with oligonucleotide ARE-3'END (14). This oligonucleotide apparently is complementary to the 19-nt fragment at the 3' terminus of the S genomic segment of all arenaviruses. Polymerase chain reaction (PCR) amplification of the first-strand cDNA was carried out by using Taq DNA polymerase (Promega Corp., Madison, WI) in conjunction with oligonucleotides 1010C and NW1696R (2-3), which flank a 616-nt region of the N protein gene of the WWA virus prototype strain AV 9310135 (12). Reverse transcription and PCR (RT-PCR) amplification of a fragment of the N protein gene of each of the eight other isolates was carried out by using the Access RT-PCR Kit (Promega Corp.) in conjunction with oligonucleotides AVNP1 (5'-CCCTTCTTYTTNYTCTTRATGACTA-3') and AVNP2 (5'-GGKAGRGCNTGGGAYAACAC-3'). AVNP1 and AVNP2 flank a 518-nt region in the fragment of the WWA virus N protein gene that is amplified by using oligonucleotides 1010C and NW1696R. They were designed based on N protein gene sequence data for the WWA virus prototype strain AV 9310135 (GenBank Accession No. U52180), WWA virus strains AV 96010149, AV 96010151, AV 96010025, and AV 96010024, TAM virus strain W-10777 (U43690), and PIC virus strain An 3739 (K02734). Size separation of PCR products was done by agarose gel electrophoresis; the products of the expected size were purified form gel slices by using QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, CA). One strand of each 1010C-NW1696R PCR product was sequenced directly by using the dye termination cycle sequencing technique (Applied Biosystems, Inc., Foster City, CA) in conjunction with oligonucleotide 1010C. The sequence of the other (i.e., complementary) strand of each of these products was determined by cloning the PCR product in the TA cloning vector PCRII (Invitrogen Corp., Carlsbad, CA) and then using a plasmid-specific oligonucleotide (M13) to initiate the cycle sequencing reaction. Both strands of the AVNP1-AVNP2 PCR products were sequenced directly by using the same oligonucleotides that were used to prime the RT-PCR, i.e., AVNP1 and AVNP2. The 12 nucleotide sequences generated in this study were deposited with the GenBank nucleotide sequence database under Accession Nos. AY012710 - AY012721.

Data Analysis

The analyses of nucleotide sequence data were restricted to the 518-nt fragment of the WWA virus N protein gene that is flanked by oligonucleotides AVNP1 and AVNP2. The GenBank database sequences included in the analyses were Accession Nos. U52180 (WWA virus, strain AV 9310135), U43690 (TAM, W-10777), K02734 (PIC, An 3739), U43689 (Parana, 12056), U43687 (Flexal, BeAn 293022), U62561 (Pirital, VAV-488), U43688 (Latino, 10924), U34248 (Oliveros, 3229-1), U70802 (Junin, XJ), X62616 (Machupo, AA288-77), U43686 (Guanarito, INH-95551), U41071 (Sabiá, SPH 114202), U43685 (Amapari, BeAn 70563), M20304 (Tacaribe, TRVL 11573), M20869 (LCM, Armstrong), and U80004 (Lassa, LP). The computer software package CLUSTAL W1.7 (15) was used to construct an alignment of the predicted amino acid sequences, and the computer program TransAlign (16) was used to generate a multiple nucleotide sequence alignment from the amino acid sequence alignment. Pairwise genetic distances were computed by using the p distance model as implemented in the computer program MEGA, version 1.02 (17). Percent sequence identities were calculated by subtracting the genetic distances from 1.0 and multiplying by 100. Phylogenetic analysis was carried out on the multiple amino acid sequence alignment by using the neighbor-joining method (gamma model, alpha = 2) as implemented in MEGA, version 1.02. Bootstrap support (18) for the results of the phylogenetic analysis was based on 500 pseudoreplicate datasets generated from the original multiple amino acid sequence alignment.

Viral Isolates

Twenty-three arenaviral isolates were recovered from tissues of 14 (3.3%) of 425 Neotoma rodents (Table 1). The 23 isolates included three WWA virus strains (AV 9310135, AV 9310041, and AV 9310040) that were reported previously (12).

Figure 1

Figure 1. . Locations of 14 arenavirus-positive Neotoma rodent collections. San Juan County, southeastern Utah = N. cinerea and N. mexicana (one virus-positive animal each species); Cimarron County, western Oklahoma = N. albigula...

The virus-positive animals included two N. albigula from McKinley County, northwestern New Mexico; two N. albigula from Cimarron County, western Oklahoma; three N. mexicana from Socorro County, central New Mexico; five N. micropus from the Chaparral Wildlife Management Area (Dimmit and La Salle counties), southern Texas; and one N. mexicana and one N. cinerea from San Juan County, southeastern Utah (Table 2, Figure 1). The virus-positive animals from McKinley County were two (50%) of four woodrats (all N. albigula) collected on July 15, 1993, from Whitewater Arroyo. The positive N. albigula from Cimarron County were two (22.2%) of nine woodrats (seven N. albigula and two N. mexicana) collected on October 12, 1985, from a site near Kenton. The positive N. mexicana from Socorro County were three (42.9%) of seven woodrats (all N. mexicana) collected on September 24, 1994, from a site in the Magdalena Mountains. The positive N. micropus from Dimmit County were 4 (13.8%) of 29 woodrats (all N. micropus) collected in a 3-day period (July 17 through July 19, 1999) from the western region of the Chaparral Wildlife Management Area. The positive N. micropus from La Salle County was one (25.0%) of four woodrats (all N. micropus) collected on July 20, 1999, from the eastern region of the Chaparral Wildlife Management Area. The positive N. mexicana and N. cinerea from San Juan County were 2 (12.5%) of 16 woodrats (11 N. mexicana, 2 N. cinerea, and 3 N. albigula) collected in an 8-day period (June 29 through July 6, 1994) from Natural Bridges National Monument. Information from the Museum of Southwestern Biology indicated that the positive N. mexicana and N. cinerea were collected from different sites in Natural Bridges National Monument.

The nucleotide sequences of the isolates from rodent 62425 (one isolate each from kidney and liver; strains AV 96010151 and AV 98360019, respectively) were identical. In contrast, the nucleotide sequences of the isolates from rodent 62439 (again, one isolate each from kidney and liver; strains AV 96010154 and AV 98360020, respectively) were 99.6% identical. Further study is needed to determine whether the differences between the isolates from rodent 62439 represent the coexistence of multiple virus genotypes (alleles) in the same rodent. An alternative explanation is that the sequence differences are the result of adaptation of the isolates to growth in cultured (Vero E6) cells or manipulation of viral nucleic acid extracted from cultured cells.

Nucleotide and amino acid sequence identities among WWA virus prototype strain AV 9310135 and 12 other isolates from Neotoma rodents ranged from 74.7% to 100.0% and 84.9% to 100.0%, respectively (Table 3). When compared with other arenaviruses, the isolates from the Neotoma rodents exhibited 69.9% to 73.7% nucleotide sequence identity with TAM virus, 61.0% to 63.3% identity with PIC virus, and less than 62.0% sequence identity with all other arenaviruses.

Figure 2

Figure 2. . Phylogeny of the North American arenaviruses based on a neighbor-joining analysis of nucleocapsid protein amino acid sequence data. Distances and groupings were determined by using the gamma distance algorithm (alpha...

Phylogenetic analysis of N protein amino acid sequence data indicated that isolates from Neotoma rodents represent a phylogenetic lineage (viral species) that is in a sister relationship to the lineage represented by TAM virus (Figure 2). We concluded that all isolates recovered from the Neotoma rodents were strains of WWA virus.