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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: J Med Virol. 2010 Sep;82(9):1586–1593. doi: 10.1002/jmv.21864

Confirmation of Choclo Virus as the Cause of Hantavirus Cardiopulmonary Syndrome and high serum antibody prevalence in Panama

Randin Nelson 1,*, Raul Cañate 2,*, Juan Miguel Pascale 2, Jerry W Dragoo 3, Blas Armien 2, Anibal G Armien 4, Frederick Koster 1,5,†
PMCID: PMC2927102  NIHMSID: NIHMS217434  PMID: 20648614

Abstract

Choclo virus (CHOV) was described in sigmodontine rodents, Oligoryzomys fulvescens, and humans during an outbreak of hantavirus cardiopulmonary syndrome (HCPS) in 1999 to 2000 in western Panama. Although HCPS is rare, hantavirus-specific serum antibody prevalence among the general population is high suggesting that CHOV may cause many mild or asymptomatic infections. The goals of this study were to confirm the role of CHOV in HCPS and in the frequently detected serum antibody and to established the phylogenetic relationship with other New World hantaviruses. CHOV was cultured to facilitate the sequencing of the small (S) and medium (M) segments and to perform CHOV-specific serum neutralization antibody assays.

Sequences of the S and M segments found a close relationship to other Oligoryzomys-borne hantaviruses in the Americas, highly conserved terminal nucleotides, and no evidence for recombination events. The maximum likelihood and maximum parsimony analyses of complete M segment nucleotide sequences indicate a close relationship to Maporal and Laguna Negra viruses, found at the base of the South American clade. In a focus neutralization assay acute and convalescent sera from 6 Panamanian HCPS patients neutralized CHOV in dilutions from 1:200 to 1:6400. In a sample of antibody-positive adults without a history of HCPS, 9 of 10 sera neutralized CHOV in dilutions ranging from 1:100 to 1:6400. Although cross-neutralization with other sympatric hantaviruses not yet associated with human disease is possible, CHOV appears to be the causal agent for most of the mild or asymptomatic hantavirus infections, as well as HCPS, in Panama.

Keywords: hantavirus, phylogeny, neutralizing antibody, Bunyaviridae

INTRODUCTION

Hantavirus cardiopulmonary syndrome (HCPS) is a severe and often fatal infection of the lung and other tissues, caused by one of at least 16 hantaviruses (Family Bunyaviridae, Genus Hantavirus) distributed throughout most of the Americas [Schmaljohn and Hjelle, 1997] [Koster and Hjelle, 2004]. Hantaviruses are negative-strand RNA viruses containing three segments, a small (S), medium (M), and large (L) segments encoding nucleocapsid (N), glycoproteins GPC, and RNA-dependent RNA polymerase, respectively. Most hantaviruses in the Americas, including the Sin Nombre virus (SNV) in North America and the Andes virus (ANDV) in South America, are associated with mortality rates of 20 to 35% and are contrasted with low levels of seroprevalence in endemic regions, usually below 2%. One exception is the Laguna Negra virus (LNV) in the Gran Chaco of western Paraguay, where indigenous peoples uncommonly experience symptomatic infection and mortality yet seroprevalence to hantavirus infection exceeds 20% in many communities [Ferrer et al., 1998; Williams et al., 1997].

The other exception is Choclo virus (CHOV), the only virus ascribed to human disease in Panama [Bayard et al., 2004; Vincent et al., 2000]. CHOV was identified by RT-PCR from samples taken from the host Oligoryzomys fulvescens, a common peridomestic sigmodontine rodent in Panama. More than 140 cases of HCPS, including 28 fatalities, have been recorded in Panama since 2000. All hospitalized patients tested by RT-PCR and amplimer sequencing were shown to have CHOV infections (Pascale et al., unpublished data). HIgh serum antibody prevalence [Armien et al., 2004] may be due to infection exclusively with CHOV or may be due in part to infections with other hantaviruses. Hantaviruses in Panama not associated with human disease include Calabazo virus (CALV) [Hjelle et al., 1995; Salazar-Bravo et al., 2004; Vincent et al., 2000] and an unnamed hantavirus in Sigmodon hirsutus [Armien et al., 2009]. Extensive cross-reactivity to nucleoproteins of the American hantaviruses prevents the use of EIA or Western blot technologies when assigning an infecting strain [Hjelle et al., 1997]. Studies utilizing neutralizing antibody specificities, on the other hand, may permit tentative assignment [Chu et al., 1994]

This study was undertaken to culture CHOV, obtain a complete sequence of the viral S and M segments, identify phylogenetic relationships, and develop a focus neutralization assay in order to implicate CHOV in the high antibody prevalence among Panamanians.

METHODS

Viral Isolation

Virus was obtained from the spleen of a rodent, O. fulvescens (specimen voucher no. NK101588, UNM MSB 96073), captured on 6 March 2000 in Las Tablas, Los Santos Province, Panama. The virus is named for a cantina ‘El Choclo’ of interesting reputation in the neighborhood Barriada 8 Noviembre near Las Tablas. One-hundred mg of tissue was homogenized by a bead beater using 2.5-mm zirconium/silica beads in phosphate buffered saline (PBS) and diluted 1:50, 1:200, and 1:1000 in 1.0 ml complete Vero media (Eagle’s minimal essential medium [EMEM] containing 10% fetal bovine serum (FBS), gentamicin (50 μg/mL), and 20 mM glutamine). Vero E6 cells (Vero C1008, ATCC CRL 1586, passage 8) were grown to confluency in 25-cm2 flasks in Vero complete media. Media was removed from the monolayer and the diluted homogenates were added, incubated on a slow plate rocker at room temperature for 2 h, then the tissue homogenate was removed. Fresh media with 2.5% FBS was added and the monolayers were incubated at 36°C in a 5% CO2 atmosphere, with media changed twice weekly. Passage of the monolayer to fresh flasks after trypsinization (0.5% trypsin/5 mM EDTA) was accomplished after 4 weeks (first passage) and thereafter every 2 to 2.5 weeks. All experiments involving infectious viruses were performed in a biosafety level 3 laboratory.

RT-PCR

A nested reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect viral RNA in culture supernatants from each passage. Typically 170-μL aliquots (approximately 2 μg total RNA) of supernatant media were extracted using the QIAampViral RNA kit (Qiagen Inc, Valencia, CA) according to the manufacturer’s directions. RT-PCR was initiated using AMVrt and Amplitaq LD with the outer antisense primer for 1 h at 42°C. Subsequent reaction conditions were 94°C for 5 min, followed by 8 cycles consisting of 10 s at 94°C, 20 s at 50°C, and 60 s at 72°C, and finally by 28 cycles with the annealing temperature of 55°C. The outer primers at the 5′ end of the segment were 5′-ACTGCACGGCAAAAGCTTAAA-3′ (58F) and 5′-GGATATAAGCACCAATTGACCT-3′ (379R) producing a 320-bp amplimer. The inner pair was 5′-GGACCCGGATGAAGTTAACAA-3′ (102F) and 5′-AATTTTTGAGCTGCCACCAA-3′ (222R) producing a 120 bp amplimer. The products were visualized on agarose gel, purified and sequenced to confirm specificity to CHOV.

Focus and Neutralization Assays

Replicating virus was titered using a focus assay as published [Bharadwaj et al., 2000]. Vero E6 cells were seeded onto 48-well plates and incubated until confluent. Ten-fold dilutions (1:10 through 1:107) of virus-containing culture supernatant were added to the monolayers in a 200-μL volume of viral culture medium (EMEM, HEPES buffer, 2.5% FBS, and 50 mg/mL gentamicin) and incubated for 2 h at 37°C. After adsorption, the supernatant was aspirated and 1 mL/well of viral overlay media (VCM and 1.2% methylcellulose) was added and incubated for 7 days. After 7 days the overlay media was removed; the monolayer was washed once with PBS. Ice cold methanol containing 0.5% H2O2 was added and incubated at room temperature for 30 min; fixative then was aspirated and PBS added for storage until immunoperoxidase assay.

For the immunoperoxidase assay, fixed cell monolayers were washed twice with PBS, and 200 μL/well of 1:1000-diluted rabbit anti-SN virus serum was incubated for 1 h at 37°C. After aspiration and washing twice with PBS, 200 μL of peroxidase-conjugated AffinPure Goat anti-rabbit IgG (H+L) (Pierce Immunochemicals, Rockford, IL) diluted 1:1000 in PBS was added and incubated for 1 h at 37°C. Following aspiration and washing, 200 μL of DAB/metal concentrate diluted with 1x peroxidase substrate buffer was added to the monolayers and incubated until brown foci appeared, usually 15 to 30 min. Foci/well were enumerated with a 20x inverted scope ocular.

Neutralizing antibody was measured in human serum using diluted test sera in the focus titration assay. Serum samples containing anti-hantavirus antibody were collected from family members and neighbors of HCPS patients in the community of San Jose, Los Santos Province from 2001 to 2003. Serum was diluted (1:20, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800 dilutions) was incubated for 2 h at 37°C with stock virus diluted to a concentration of 35 to 50 focus-forming units (FFU)/well prior to incubation on Vero cell monolayers. Neutralizing antibody titers were expressed as the reciprocal of the highest serum dilution that results in an 80% reduction in the number of foci compared to virus controls. Discrepant results were resolved by two additional focus neutralization assays. Stock CHOV used was E6 passage 3, with a titer of 4.3 log10 FFU/mL. Stock SNV and ANDV demonstrated approximately the same titer in the focus assay titration as was documented by the originating laboratory.

Viral Sequencing

To sequence complete S and M segments, nested primers were designed from consensus sequences of ANDV and LNV viruses in GenBank. To sequence the 5′ and 3′ termini of each segment, dephosphorylated terminal nucleotides were ligated with T4 ligase and appropriately sized amplimers were cloned into pCRII vector (Invitrogen, Carlsbad, CA). The resulting clones were sequenced by the dideoxy method. Sequences were acceptable only if forward and reverse sequencing results agreed. Multiple overlapping amplimers were sequenced to achieve an 80% duplication of the entire genome.

Phylogenetic Analysis

Sequences of the N protein gene of four strains of ANDV and one strain each of 20 other hantaviruses were compared to the sequence of the CHOV. The sequence of the GPC gene of the CHOV was compared to sequences of 4 strains of ANDV and 20 other hantaviruses (Table 1). The accession numbers of closely related viruses were Maporal virus (MAPV) (N, AY267347; GPC, AY363170) and LNV (N, AF005727; GPC, AF005728). Sequences were aligned using ClustalX [Thompson et al., 1994] followed by visual inspection using MacClade 4 [Maddison and Maddison, 2003]. The N protein and GPC gene sequences were 1302 (with stop codon) and 3465 characters in length, respectively.

Table 1.

Virus sequences used in this study. Accessions numbers are for N protein (top) and GPC gene (bottom of the pair – when present)

Virus Genbank No. General locality Host
S segment M segment
CHOV DQ285046 DQ285047 Panama Oligoryzomys fulvescens
ANDV AF324902 AF324901 Argentina Homo sapiens
ANDV AF291702 AF291703 Chile Oligoryzomys longicaudatus
ANDV AF325966 Argentina Oligoryzomys chacoensis
ANDV AF482712 Argentina Homo sapiens
BAYV L36929 L36930 USA Homo sapiens
BCCV L39949 L39950 USA Sigmodon hispidus
BMJV AF482713 Argentina Oligoryzomys chacoensis
ELMCV U11427 U26828 USA Reithrodontomys megalotis
HNTV M14626 M14627 Korea Apodemus agrarius
LECV AF482714 AF028022 Argentina Oligoryzomys flavescens
LNV AF005727 AF005728 Paraguay Calomys laucha
MACV AF482716 Argentina Necromys benefactor
MAPV AY267347 AY363179 Venezuela Oligoryzomys fulvescens
MONV U32591 USA Peromyscus maniculatus
MULEV U54575 USA Sigmodon hispidus
NYV U09488 U36801 USA Peromyscus leucopus
ORNV AF482715 AF028024 Argentina Oligoryzomys longicaudatus
PHV M34011 X55129 USA Microtus pennsylvanicus
PUUV M32750 M29979 Russia Clethrionomys glareolus
PRGV AF482717 Argentina Akodon azarae
RIOMV U52136 Bolivia Oligoryzomys microtis
RIOSV U18100 Costa Rica Reithrodontomys mexicanus
SEOV M34881 M34882 Japan Rattus norvegicus
SNV L37904 L37903 USA Peromyscus maniculatus
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Phylogenetic analyses of DNA sequences were conducted using Maximum Parsimony (MP) and Maximum Likelihood (ML) in the software package PAUP* 4.0b10 [Swofford, 2002]. Fulhorst et al. (2004) excluded third base positions from their MP and ML analyses. Third base positions were included but only for transversion changes. Transversion changes continued to increase as genetic distance increased, suggesting that a phylogenetic signal may still be present for these changes at the third position, yet transition changes plateau, suggesting saturation of substitutions has occurred for transitions at the third base position. In the MP analysis all characters were weighted based on their rescaled consistency index (RCI). A heuristic search option with 100 replications of random addition of taxa and TBR branch swapping was used to generate parsimony trees. Bootstrap support [Felsenstein, 1985] for results of MP analyses were based on 1000 repetitions of resampling data using the heuristic search option, with 10 replications of random addition of taxa.

Modeltest (v 3.06) [Posada and Crandall, 1998] was used to calculate the appropriate model for ML analyses. The GTR + G + I (0.6787 and 0.3440 for G and I, respectively) was used to construct phylogenies for the S segment and the GTR + G (0.3439) model was used to construct the M segment phylogeny based on the results of the Modeltest analysis.

RESULTS

Isolation of the Virus

RT-PCR of serial supernatants from blind passages identified the appearance of detectable viral RNA from animal #588 by the fourth blind passage in both tissue inocula diluted 1:50 and 1:1000. Virus was first detected by focus titration assay from the third passage at <100 focus forming units (FFU)/mL, and the highest level of virus was found in the sixth passage with a titer of 5 × 104 FFU/mL. Viral RNA was extracted from sixth passage supernatants, or third passage of virus-positive supernatants, for subsequent genomic sequencing.

Nucleotide Sequence Analysis

The S segment of CHOV isolate 588 (Accession No. DQ285046) was 1516 nt in length, of which 1302 bases encode a predicted nucleocapsid protein 434 amino acids in length, beginning at nt 43. The S segment also contains a potential second ORF (open reading frame) beginning at nt 122, with a predicted protein of 63 amino acids in length. Terminal nucleotides were conserved as seen in other hantaviruses [Chizhikov et al., 1995] with the 32- and 28-nt equivalent to the 5′ and 3′ ends, respectively, being identical to those of ANDV and LNV. The 20-nt differences between the CHOV and SNV in the S segment coding region included 16 nonsynonymous changes.

Comparison of the CHOV S segment sequences with those of other hantaviruses indicates the highest degree of identity with South American viruses (78.3 to 79.8% nt; 89.4 to 91.2% aa) (Table 2). Nucleotide similarity between CHOV and the North American viruses was close to that of the South American viruses and ranged from 75.9 to 79.3 % (83.2 to 89.2% aa). Comparison of deduced N protein amino acid sequences shows an 89.9% identity to MAPV, an 88.5% identity to SNV, and a 90% identity to ANDV.

Table 2.

Nucleotide differences (below diagonal) and amino acid differences (above diagonal) between pairs of hantaviruses for the S segment

ANDV1 ANDV2 ANDV3 ANDV4 BMJV LECV ORNV MACV PRGV MAPV CHOV RIOM LNV ELMC RIOS SNV MONV NYV MULE BAYV BCCV SEOV HTNV PUUV PHV
ANDV1 0 0 15 1 14 15 14 26 21 36 41 38 41 71 76 59 52 52 59 49 57 157 151 123 114
ANDV2 74 0 15 1 14 15 14 26 21 36 41 38 41 71 76 59 52 52 59 49 57 157 151 123 114
ANDV3 209 210 0 16 6 7 1 24 18 40 43 41 43 72 78 56 53 53 62 47 58 162 151 124 115
AND4 79 9 213 0 15 16 15 26 22 35 41 39 42 72 77 60 53 52 60 50 56 157 151 123 115
BMJV 203 205 163 209 0 1 5 24 18 37 38 41 43 71 75 53 50 52 62 47 58 161 149 123 113
LECV 197 202 166 202 110 0 6 25 19 36 39 40 44 72 74 54 51 53 63 48 59 161 149 123 112
ORNV 207 206 8 209 164 165 0 23 17 39 42 40 42 71 77 55 52 52 61 46 57 161 150 123 114
MACV 241 241 227 240 231 228 227 0 17 42 46 45 50 70 77 62 57 54 60 52 57 159 155 124 115
PRGV 237 237 216 239 231 216 216 218 0 40 44 43 44 71 79 59 53 52 59 48 59 159 152 127 114
MAPV 257 254 260 252 279 262 256 285 265 0 44 36 45 67 69 59 57 55 59 50 57 155 151 121 111
CHOV 267 279 263 283 274 274 263 281 275 281 0 47 51 68 73 50 47 47 65 49 62 165 155 123 108
RIOM 258 245 244 246 259 251 244 276 264 234 280 0 29 72 77 65 58 56 58 50 56 161 156 124 116
LNV 267 266 272 269 266 259 267 284 276 281 270 226 0 74 81 62 58 54 61 54 61 163 156 124 117
ELMC 300 301 285 303 292 294 283 313 309 301 314 297 321 0 38 64 65 65 73 65 70 163 164 127 116
RIOS 328 326 322 328 321 320 317 329 323 308 320 324 316 268 0 70 73 71 80 69 76 167 164 123 119
SNV 305 300 294 305 288 288 292 285 305 314 304 303 301 297 311 0 27 27 72 54 67 167 162 129 114
MONV 276 281 283 282 289 291 279 307 291 294 294 295 315 298 324 217 0 16 68 48 58 166 159 128 109
NYV 294 284 295 283 302 292 293 285 294 289 299 292 299 294 332 213 198 0 68 51 60 162 158 128 110
MULE 293 307 301 307 299 291 303 307 310 296 310 290 305 312 351 314 314 304 0 30 41 168 160 124 115
BAYV 296 295 274 297 293 292 274 304 297 279 305 286 285 303 322 294 292 286 248 0 33 162 155 121 107
BCCV 301 313 301 313 273 277 301 296 307 285 301 291 294 317 342 317 311 311 251 248 0 162 156 126 112
SEOV 480 480 476 477 478 475 474 476 487 487 490 503 489 491 506 494 504 484 508 512 481 0 75 161 160
HTNV 469 475 473 475 479 481 472 468 476 465 484 478 476 486 509 484 491 497 482 461 474 343 0 166 163
PUUV 409 421 427 427 427 424 427 418 442 418 433 424 416 417 416 417 432 425 415 425 408 475 510 0 86
PHV 395 391 400 398 399 402 398 415 418 390 394 405 396 405 414 396 395 394 408 398 403 484 486 347 0
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Sequence Analysis of the M Segment

The complete M segment (Accession No. DQ285047) is 3465 nt in length, encoding a glycoprotein precursor of 1155 amino acids. Again, CHOV is more similar to the South American viruses (73.4 to 74.9% and 82.6 to 83.8% nt and aa, respectively) (Table 3). CHOV and North American viruses had nucleotide similarity ranging from 70.8 to 72.3% and amino acid similarity from 76.1 to 79.6%. Comparison of amino acid sequences shows that MAPV has an 83.6% identity to, a 79.6% identity to SNV, and a 83.7% identity to ANDV.

Table 3.

Nucleotide differences (below diagonal) and amino acid differences (above diagonal) between pairs of hantaviruses for the M segment

ANDV1 ANDV2 ORNV LECV MAPV LNV CHOV SNV NYV BAYV BCCV ELMC SEOV HTNV PHV PUUV
ANDV1 0 9 85 72 168 142 188 238 257 262 267 291 512 506 476 524
ANDV2 184 0 81 71 166 146 187 242 259 267 272 295 511 507 478 524
ORNV 711 708 0 51 174 150 194 246 256 269 267 299 507 505 486 533
LECV 713 718 629 0 161 146 187 238 252 269 266 290 505 497 483 527
MAPV 835 841 842 827 0 205 189 257 267 278 283 302 521 500 490 534
LNV 820 832 807 812 876 0 201 257 267 269 268 288 512 508 485 535
CHOV 870 874 879 895 889 922 0 236 258 245 260 276 522 512 484 532
SNV 953 960 968 947 970 970 961 0 58 212 222 226 521 505 462 512
NYV 965 974 986 999 965 955 987 647 0 234 237 240 523 508 471 512
BAYV 971 971 998 995 970 1003 964 911 947 0 130 259 509 503 477 526
BCCV 959 961 995 1007 988 965 975 893 915 752 0 264 517 510 488 527
ELMC 1044 1050 1035 1057 1061 1019 1013 939 960 986 963 0 509 487 475 522
SEOV 1418 1410 1410 1413 1454 1413 1421 1414 1410 1371 1355 1358 0 258 594 621
HTNV 1400 1402 1432 1417 1392 1400 1407 1401 1376 1374 1382 1377 937 0 587 619
PHV 1375 1385 1397 1392 1365 1360 1396 1327 1325 1375 1378 1333 1545 1512 0 314
PUUV 1452 1471 1487 1469 1462 1454 1477 1422 1452 1463 1451 1454 1600 1590 1061 0
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The tree topologies for the MP and ML analyses were similar. CHOV was basal to the South American viruses in the ML analyses for the S segment, whereas it formed a polytomy with MAPV and the remaining South American viruses in the MP analyses (Figure 1a and 1b). The relationship of CHOV to the South American viruses also was found in both the ML and MP analyses using the M segment (Figure 1c and 1d). However, the bootstrap support for this relationship was stronger than in the analysis of the S segment. The differences in tree topologies between this study and that of Fulhorst et al. (2004) can be explained by low bootstrap support of two nodes (compare Figure 1 to their Fig. 1 [p. 141]). Because bootstrap support is low in analyses from both studies, it is difficult to reject either hypothesis. In both studies the North American viruses are paraphyletic with respect to the South American viruses.

Figure 1.

Figure 1

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A. Maximum likelihood analysis of 1302 nt of the S segment of 25 hantaviruses. Tree based on the GTR + G (0.6787) + I(0.3440) model of nucleotide evolution. Numbers associated with node are Bayesian posterior probabilities. Model for Bayesian analyses is the same used for Maximum Likelihood (topologies were identical). B. Single most parsimonious tree based on 675 informative of 1302 total base pairs of the N protein. Characters were weighted based on a rescaled consistency index. Third base positions were analyzed using transversions only. Tree length = 587.794; RCI = 0.5148. Numbers above branches represent percentage of 1000 bootstrap replications supporting each node. Nodes with less than 50% support are collapsed. C. Maximum likelihood analysis of 3465 nt of the M segment of 16 hantaviruses. Tree based on the GTR + G(0.3439) model of nucleotide evolution. Numbers associated with node are Bayesian posterior probabilities. Model for Bayesian analyses is the same used for Maximum Likelihood (topologies were identical). D. Single most parsimonious tree based on 2077 informative of 3465 total base pairs of the GPC gene. Tree length = 6307; CI = 0.4635. Third base positions were analyzed using transversions only.

Numbers above branches represent percentage of 1000 bootstrap replications supporting each node. Nodes with less than 50% support are collapsed.

Neutralization of Choclo Virus by Human Sera

The presence of neutralizing antibody was detected by the focus reduction assay in sera from all 6 individuals with RT-PCR-confirmed CHOV infection and typical HCPS. Three adults with 3 to 5 days of symptomatic acute disease and who required intubation had lower serum titers (1:200 to 1:1600) than the 3 individuals with milder disease who did not require intubation and who were tested 30 to 60 days after hospitalization (Table 4). Among 10 individuals with IgG antibody, as detected by EIA and strip immunoblot, and who denied previous hospitalization for respiratory infection, 9 had significant neutralizing antibody to CHOV, with titers ranging from 1:100 to 1:6400. One individual in this group did not have a detectable CHOV-neutralizing antibody, as determined in four independent assays. Seronegative controls from Panama did not have neutralizing antibody to CHOV, and no subject from Panama had neutralizing antibody to either SNV or ANDV. Two patients with mild HCPS in New Mexico had neutralizing antibody to SNV isolate 777 but not to CHOV. The hantaviruses identified by RT-PCR in Z. brevicauda (CALV) and in S. hirsutus (unnamed virus) could not be grown in Vero cell culture despite multiple blind passages in two different laboratories, and therefore neutralizing antibody to these viruses is not reported.

Table 4. Neutralization titers of Panamanian sera to CHOV isolate 588.

Clinical Group Pt # Clin. Severity Neut. CHOV Neut. SNV Neut. ANDV
Acute HCPS 15355 fatal 1:400 <1:20 <1:20
 (day 5–8 of sxa) 16617 moderate 1:200 <1:20 <1:20
15467 moderate 1:1600 <1:20 <1:20
Convalescent HCPS 18483 mild 1:3200 <1:20 <1:20
 (day 15–20 of sxa) 15586 mild 1:6400 <1:20 <1:20
17644 mild 1:3200 <1:20 <1:20
Seropositive/no resp hxb 384 No resp hx 1:100 <1:20 <1:20
390 No resp hx 1:1600 <1:20 <1:20
401 No resp hx 1:200 <1:20 <1:20
407 No resp hx 1:400 <1:20 <1:20
409 No resp hx 1:400 <1:20 <1:20
412 No resp hx 1:1600 <1:20 <1:20
703 No resp hx 1:6400 <1:20 <1:20
705 No resp hx <1:20 <1:20 <1:20
717 No resp hx 1:1600 <1:20 <1:20
733 No resp hx 1:3200 <1:20 <1:20
Seronegative/neg hx 7 sera No resp hx <1:20 <1:20 <1:20
Acute HCPS, USA NM 1 Mild <1:20 1:800 <1:20
NM 2 mild <1:20 1:1600 <1:20
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a

interval between onset of symptoms and diagnosis of HCPS.

b

seropositive and no history of acute respiratory infection requiring hospitalization.

DISCUSSION

The first goal of the study was to culture the virus and facilitate accurate identification of phylogenetic relationships. The complete genomic sequence of the S and M segments permitted the conclusion that CHOV is distinct from all other hantaviruses, according to established criteria [Elliott et al., 2000]. Even though CHOV and MAPV share the same host, O. fulvescens, these two strains are approximately 10% distinct at the amino acid level of the N protein. This distinction is likely due to independent evolution permitted by the extensive ecologic and physical separation between western Panama and western Venezuela. Rodents in the sigmodontine genus Oligoryzomys are natural hosts for at least 6 hantaviruses [Bharadwaj et al., 1997; Bohlman et al., 2002; Gonzalez Della Valle et al., 2002; Medina et al., 2009; Meissner et al., 2002; Powers et al., 1999]. The oryzomine rodent-borne viruses in the Argentina/Chile clade of hantaviruses show little amino acid diversity ([Bohlman et al., 2002; Medina et al., 2009], suggesting recent divergence. The greater diversity of CHOV from other oryzomine hantaviruses suggests its more remote divergence from the common ancestor.

The second goal of the study was to use the specificity of the neutralization assay to indicate whether the high serum antibody prevalence was due to previous CHOV infection. The focus neutralization assay of sera from CHOV-positive PCR-confirmed HCPS patients demonstrated the presence of neutralizing antibody titers of levels similar to other published reports [Bharadwaj et al., 2000]. As expected, there was no cross-neutralization between CHOV, ANDV, and SNV, but cross-neutralization studies with more closely related viruses, including CALV [Vincent et al., 2000] and MAPV, a hantavirus isolated from O. fulvescens in Venezuela [Fulhorst et al., 2004; Milazzo et al., 2002], is needed for definitive specificity.

In five communities in the Azuero peninsula investigators found antibody prevalence ranging from 3% to 45% [Armien et al., 2004]. Antibody was detected in samples from children as young as 4 years of age and increases steadily to highest prevalence in the 40- to 50-year-old cohort, suggesting that steady exposure and infection occurs throughout life in rural areas. From this cohort data it is estimated that among the 250,000 people of the Azuero peninsula there may be more than 10,000 individuals with serum anti-hantavirus antibody. Questionnaires indicated that none of the 275 antibody-positive individuals tested had given a history compatible with an HCPS-like illness, although histories of febrile respiratory illnesses not requiring hospitalization were common [Armien et al., 2004]. An ongoing clinical trial in four clinics during two years of observation has identified 110 individuals with fever and anti-hantavirus antibody detected by IgM-ELISA. CHOV RNA was detected in acute blood samples by RT-PCR, yet no progression to symptomatic pulmonary disease was found (B. Armien, unpublished data).

If the majority of antibody positive individuals were infected with CHOV, as suggested by neutralization assays, and if the reported 140 cases of HCPS is a good estimation of total cases, the ratio of mild or asymptomatic CHOV infection to HCPS may be as high as 50:1 in Panama. LNV may also be associated with a high ratio of mild to severe infection, although strain-specific neutralization titers have not been published [Chu et al., 2006]. In regions where multiple hantaviruses co-circulate in rodent populations, such as Panama, Paraguay [Chu et al., 2006] and Brazil [Figueiredo et al., 2009; Raboni et al., 2009], neutralization assays studies can identify the dominant hantaviruses that infect humans. Since each rodent host has habitat preferences [Armien et al., 2009; Chu et al., 2003], the identification of hantavirus species causing human disease can have useful public health implications.

ACKNOWLEDGEMENTS

We acknowledge the support of Karl M. Johnson, and sequencing support from the Center for Genomic Medicine at the University of New Mexico. We thank Brian Hjelle for providing the virus stocks of SNV and ANDV.

Funding for this work was obtained from a Gorgas Memorial Institute Research Award to R.C.; an Opportunity Pool Award and Supplements to the ICIDR Program, NIH-U19 45452; the RCE program U54 AI057156; support from the Ministry of Health, Republic of Panama (B.A. and J.M.P); and a grant from DARPA under grant no. DARPA-N00014-1-0900 (J.W.D).

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