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.

The virus family Arenaviridae comprises two serocomplexes. The lymphocytic choriomeningitis-Lassa (Old World) complex includes lymphocytic choriomeningitis (LCM), Lassa, Mopeia, Mobala, and Ippy viruses. The Tacaribe (New World) complex includes Tamiami (TAM), Whitewater Arroyo (WWA), Pichinde (PIC), Amapari, Flexal, Guanarito, Junin, Latino, Machupo, Oliveros, Parana, Pirital, Sabia, and Tacaribe viruses.

The arenaviruses have bipartite, single-stranded RNA genomes (1). The large (L) genomic segment encodes the viral RNA-dependent RNA polymerase and a zinc-binding protein. The small (S) genomic segment encodes the nucleocapsid (N) protein and glycoprotein precursor. The most comprehensive knowledge of the phylogeny of the family Arenaviridae is based on a fragment of the N protein gene (2-4).

Six arenaviruses are known to cause severe disease in humans. LCM virus is an agent of acute central nervous system disease (5) and congenital malformations (6). Lassa, Junin, Machupo, Guanarito, and Sabia viruses are etiologic agents of hemorrhagic fever in western Africa, Argentina, Bolivia, Venezuela, and Brazil, respectively (7).

The arenaviruses known to occur in North America are LCM, TAM, and WWA. LCM virus was introduced into the Americas along with its principal rodent host, Mus musculus (house mouse) (8). TAM virus is known only from Sigmodon hispidus (hispid cotton rat) in southern Florida (9-11). WWA virus was originally recovered from Neotoma albigula (white-throated woodrat) collected from northwestern New Mexico (12).

In a recent study (13), antibody to an arenavirus was found in five Neotoma species in the southwestern United States: N. albigula in Arizona, Colorado, and New Mexico; N. stephensi (Stephen's woodrat) in Arizona and New Mexico; N. mexicana (Mexican woodrat) in Arizona and Utah; and N. fuscipes (dusky-footed woodrat) and N. lepida (desert woodrat) in California. The purpose of the present study was to extend our knowledge of the geographic distribution and genetic diversity of the arenavirus(es) associated with Neotoma rodents in the southwestern United States.

Materials and Methods

All work with rodent tissues and infectious arenavirus was performed in a biosafety level 3 laboratory at the Centers for Disease Control and Prevention (Atlanta, GA) or University of Texas Medical Branch, Galveston.

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-[cm.sup.2] plastic culture flask (Corning, Inc., Corning, NY). The inoculum was incubated on the cell monolayer at 37 [degrees] C 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 (56 [degrees] C for 30 minutes) fetal bovine serum, 0.29 mg/mL L-glutamine, 100 U/mL penicillin G, 100 [micro]g/mL streptomycin sulfate, and 100 U/mL nystatin. The cell culture was maintained at 37 [degrees] C 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 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 518mt 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 from 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.

Table 1. Recovery of infectious arenavirus from tissues
of virus-positive woodrats (Neotoma species)

                                    Collected from
                         Date
Rodent     Species     collected    County    State

1627     N. albigula   07/15/93    McKinley    NM
1626     N. albigula   07/15/93    McKinley    NM
62415    N. mexicana   09/24/94    Socorro     NM
62425    N. mexicana   09/24/94    Socorro     NM
62439    N. mexicana   09/24/94    Socorro     NM
28731    N. albigula   10/12/85    Cimarron    OK
28742    N. albigula   10/12/85    Cimarron    OK
84648    N. micropus   07/18/99    Dimmit      TX
84703    N. micropus   07/19/99    Dimmit      TX
84708    N. micropus   07/19/99    Dimmit      TX
84761    N. micropus   07/18/99    Dimmit      TX
84816    N. micropus   07/20/99    La Salle    TX
36287    N. cinerea    07/06/94    San Juan    UT
36282    N. mexicana   07/05/94    San Juan    UT

                        Virus (strain) recovered from(a)

Rodent     Species       Spleen        Kidney         Liver

1627     N. albigula   AV 9310041    AV 9310135        nt
1626     N. albigula       nt        AV 9310040        nt
62415    N. mexicana       nt        AV 96010149       nt
62425    N. mexicana       nt        AV 96010151   AV 98360019
62439    N. mexicana       nt        AV 96010154   AV 98360020
28731    N. albigula       nt        AV 98490013    Negative
28742    N. albigula       nt        AV 97130039    TVP-6038
84648    N. micropus     Negative    AV A0400098       nt
84703    N. micropus   AV A0400337   AV A0400135       nt
84708    N. micropus       nt        AV A0400140       nt
84761    N. micropus   AV A0400373   AV A0400174       nt
84816    N. micropus   AV A0400412   AV A0400212       nt
36287    N. cinerea        nt        AV 96010025   AV 96010206
36282    N. mexicana       nt        AV 96010024   AV 96010205

(a) nt = not tested. The WWA virus prototype strain is bolded.
Isolates (strains) included in the (phylo-) genetic analyses
are underlined.

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 (Sabia, 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.

Results

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).

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.

[Illustration omitted]

Table 2. Results of virus isolation
attempts on tissues from 425 woodrats

                        Neotoma species(a)

County(b)          State   Nalb    Ncin   Nflo

Apache (1)          AZ     --      --     --
Cochise (2)         AZ     0/31    --     --
Coconino (2)        AZ     0/6     --     --
Maricopa (1)        AZ     0/23    --     --
Navajo (1)          AZ     0/29    0/1    --
Yavapai (2)         AZ     0/5     --     --
McKinley (3)        NM     2/16    --     --
Otero (9)           NM     0/33    --     --
Socorro (3)         NM     0/31    --     --
Cimarron (4)        OK     2/11    --     --
Major (4)           OK     --      --     0/45
McIntosh (2)        OK     --      --     0/12
Pottawatomie (2)    OK     --      --     0/7
Dimmit (1)          TX     --      --     --
La Salle (1)        TX     --      --     --
San Juan (8)        UT     0/3     1/2    --
Total                      4/188   1/3    0/64

                       Neotoma species(a)

County(b)          Nme    Nmic    Nste   Total

Apache (1)         0/6    --      0/5     0/11
Cochise (2)        --     --      --      0/31
Coconino (2)       --     --      0/3     0/9
Maricopa (1)       --     --      --      0/23
Navajo (1)         0/5    --      0/7     0/42
Yavapai (2)        0/2    --      --      0/7
McKinley (3)       --     --      --      2/16
Otero (9)          0/9    0/35    --      0/77
Socorro (3)        3/10   0/1     --      3/42
Cimarron (4)       0/5    --      --      2/16
Major (4)          --     0/38    --      0/83
McIntosh (2)       --     --      --      0/12
Pottawatomie (2)   --     --      --      0/7
Dimmit (1)         --     4/29    --      4/29
La Salle (1)       --     1/4     --      1/4
San Juan (8)       1/11   --      --      2/16
Total              4/48   5/107   0/15   14/425

(a) Nalb = Neotoma albigula, Ncin = N. cinerea, Nflo = N.
floridana, Nmex = N. mexicana, Nmic = N. micropus,
Nste = N. stephensi. Values are the number positive/number
tested; "-" = none tested.

(b) Number in parentheses indicates the number
of sites sampled in the county.

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.

Table 3. Nucleotide and amino acid sequence identities
among 13 arenavirus isolates recovered from
11 woodrats and Tamiami virus(a)

                                Virus or strain

                         AV 93   AV 93   AV 96   AV 96
Virus(b)     Strain      10135   10040   10149   10151

WWA        AV 9310135       --   100.0    86.5    86.5
WWA        AV 9310040    100.0      --    86.5    86.5
WWA        AV 96010149    95.3    95.3      --   100.0
WWA        AV 96010151    95.3    95.3   100.0      --
WWA        AV 98360019    95.3    95.3   100.0   100.0
WWA        AV 96010154    94.8    94.8    98.8    98.8
WWA        AV 98360020    95.3    95.3    99.4    99.4
WWA        AV 98490013    91.9    91.9    90.7    90.7
WWA        TVP-6038       91.9    91.9    90.7    90.7
WWA        AV A0400212    88.4    88.4    88.9    88.9
WWA        AV A0400174    88.9    88.9    89.5    89.5
WWA        AV 96010025    90.7    90.7    91.3    91.3
WWA        AV 96010024    93.0    93.0    94.2    94.2
TAM        W-10777        77.9    77.9    78.5    78.5

                        Virus or strain

           AV 98    AV 96   AV 98    AV 98    TVP-
Virus(b)   360019   10154   360020   490013   6038

WWA          86.5    85.3    85.3     82.3    82.6
WWA          86.5    85.3    85.3     82.3    82.6
WWA         100.0    98.1    98.5     80.1    80.3
WWA         100.0    98.1    98.5     80.1    80.3
WWA            --    98.1    98.5     80.1    80.3
WWA          98.8      --    99.6     81.3    81.5
WWA          99.4    99.4      --     81.3    81.5
WWA          90.7    90.7    91.3       --    99.4
WWA          90.7    90.7    91.3     98.8      --
WWA          88.9    88.9    89.5     86.6    86.6
WWA          89.5    89.5    88.9     85.5    85.5
WWA          91.3    91.3    91.3     87.8    87.8
WWA          94.2    94.2    94.2     88.4    88.4
TAM          78.5    78.5    78.5     78.5    78.5

                        Virus or strain

           AV A0    AV A0    AV 96   AV 96   TAM
Virus(b)   400212   400174   10025   10024

WWA         79.1     79.1     83.4    85.1   71.6
WWA         79.1     79.1     83.4    85.1   71.6
WWA         79.5     78.8     84.4    85.5   73.7
WWA         79.5     78.8     84.4    85.5   73.7
WWA         79.5     78.8     84.4    85.5   73.7
WWA         79.3     78.6     84.4    85.1   73.2
WWA         79.5     78.8     84.4    85.1   73.4
WWA         79.0     77.8     80.9    80.7   72.2
WWA         79.1     78.0     81.1    80.1   72.4
WWA           --     95.7     77.2    77.0   69.9
WWA         98.3       --     74.7    75.5   69.9
WWA         84.9     84.9       --    82.8   71.6
WWA         86.0     86.0     89.5      --   72.2
TAM         80.2     80.2     77.3    78.5     --

(a) Nucleotide and amino acid sequence identities
are listed above and below the dashes, respectively.

(b) WWA = Whitewater Arroyo, TAM = Tamiami.

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.

[Illustration omitted]

Discussion

Before the present study, WWA virus was known only from N. albigula in northwestern New Mexico (12). The present work provides unequivocal evidence that the virus also is naturally associated with N. cinerea, N. mexicana, and N. micropus, and that it occurs in Utah, central New Mexico, Oklahoma, and Texas. The recovery of WWA virus strains AV 98490013 and TVP-6083 from N. albigula is the first evidence that a Tacaribe complex virus occurs in Oklahoma. Likewise, the recovery of strains AV A0400174 and AV A0400212 from woodrats collected from southern Texas (Chaparral Wildlife Management Area) is the first evidence that N. micropus is naturally associated with a Tacaribe complex virus and that WWA virus occurs in Texas.

In a previous study (13), antibody to an arenavirus was found in N. fuscipes and N. lepida in southern California; N. albigula, N. mexicana, and N. stephensi in Arizona; and N. albigula in southwestern Colorado. Although the results of the present study indicate that WWA virus is geographically widely distributed in association with Neotoma rodents, further work is needed to determine whether the arenavirus associated with Neotoma rodents in California, Arizona, and Colorado is in fact WWA virus.

The results of the present study indicate that there can be substantial genetic heterogeneity among strains of WWA virus from different woodrat species from the same locality and among strains from conspecific woodrats collected from different localities. For example, nucleotide sequence identity between the strains recovered from N. mexicana and N. cinerea from Natural Bridges National Monument (San Juan County, Utah; strains AV 96010024 and AV 96010025, respectively) was 82.8%, and nucleotide sequence identity between strain AV 96010024 and the three strains recovered from N. mexicana collected from the Magdalena Mountains (Socorro County, New Mexico; strains AV 96010149, AV 96010151, and AV 96010154) was from 85.1% to 85.5%. In contrast, nucleotide sequence identity in strains recovered from conspecific rodents collected from the same locality (e.g., strains AV 9310135 and AV 9310040 from N. albigula from Whitewater Arroyo, and strains AV A0400174 and AV A0400212 from N. micropus from the Chaparral Wildlife Management Area) was [is greater than] 95.0%.

The results of previous studies (3,19,20) suggested that the present-day diversity of the arenaviruses is a product of long-term coevolution of the various viruses with their respective principal rodent hosts. In the present study, WWA viral strains AV 9310135 and AV 9310040 (both from N. albigula, northwestern New Mexico) appeared to be phylogenetically more closely related to strain AV 96010024 (N. mexicana, southeastern Utah) than to strains AV 98490013 and TVP-6038 (both from N. albigula, western Oklahoma). This situation suggests that the present-day association of WWA virus with N. albigula and N. mexicana does not represent a long-term shared evolutionary relationship between virus and rodent species. However, this conclusion assumes that recovery of WWA virus from a rodent represents a principal virus-host relationship. Perhaps some of the virus-positive rodents in the present study were infected by contact with other Neotoma species or even non-Neotoma rodent species.

The geographic range of the genus Neotoma extends from western Canada south to Guatemala, Honduras, and Nicaragua, and includes 33 states in the contiguous United States and 26 of the 32 states in Mexico (21). Thus, if the present-day association of WWA virus with the genus Neotoma represents a long-term shared evolutionary relationship between virus and rodent host, the geographic range of the virus may extend far beyond the southwestern United States. WWA virus recently was associated with several human deaths in California (22). Further study is needed to assess the human health significance of this virus in the southwestern United States and other regions in North America in which woodrats are indigenous.

Acknowledgments

Robert Baker and Terry Yates provided the tissue specimens that were tested for infectious arenavirus; Wen Li Kang amplified and cloned the PCR products generated from isolates AV 96010149, AV 96010151, AV 96010025, and AV 96010024.

This research was supported by the National Institutes of Health grant AI-41435 ("Ecology of emerging arenaviruses in the southwestern United States").

Dr. Fulhorst is assistant professor and member of the World Health Organization Collaborating Center for Tropical Diseases, University of Texas Medical Branch, Galveston. His research interests include the epidemiology of the arenaviruses, hantaviruses, and other viral zoonoses.

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Charles F. Fulhorst,(*) Remi N. Charrel,(*)([dagger]) Scott C. Weaver,(*) Thomas G. Ksiazek,([double dagger]) Robert D. Bradley,([sections]) Mary L. Milazzo,(*) Robert B. Tesh,(*) and Michael D. Bowen([double dagger])

(*) University of Texas Medical Branch, Galveston, Texas, USA; ([dagger]) Faculte de Medecine, Marseille, France; Centers for Disease Control and Prevention, Atlanta, Georgia, USA; and ([sections]) Texas Tech University, Lubbock, Texas, USA

Address for correspondence: Charles F. Fulhorst, Department of Pathology, Route 0609, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA; fax: [phone]; e-mail: [e-mail]