Black Creek Canal Virus Research Articles

A lethal disease model for New World hantaviruses using immunosuppressed Syrian hamsters.

BackgroundHantavirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaviruses are found on the American continent where the most prevalent viruses like Andes virus and Sin Nombre virus are known to cause hantavirus pulmonary syndrome. New World hantavirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes virus and Maporal virus; the only hantaviruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantavirus pulmonary syndrome in humans.Methodology/Principal findingsHamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantavirus strains (Bayou virus, Black Creek Canal virus, Caño Delgadito virus, Choclo virus, Laguna Negra virus, and Maporal virus). In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantavirus results in an acute disease that precisely mimics both hantavirus disease in humans and Andes virus infection of hamsters.Conclusions/ SignificanceInfected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaviruses reaching a lethal outcome with signs of disease mimicking human disease.

Muleshoe Virus and Other Hantaviruses Associated with Neotomine or Sigmodontine Rodents in Texas.

The broad objective of this study was to increase our knowledge of Muleshoe virus and other hantaviruses associated with cricetid rodents in Texas. Anti-hantavirus antibody was found in 38 (3.2%) of 1171 neotomine rodents and 6 (1.8%) of 332 sigmodontine rodents from 10 Texas counties; hantaviral RNA was detected in 23 (71.9%) of 32 antibody-positive rodents. Analyses of nucleocapsid protein gene sequences indicated Muleshoe virus infection in four hispid cotton rats (Sigmodon hispidus) from northern Texas; Bayou virus, three Texas marsh oryzomys (Oryzomys texensis) from the Gulf Coast; Limestone Canyon virus, five brush mice (Peromyscus boylii) from western Texas; and Sin Nombre virus-five Texas mice (P. attwateri), one Lacey's white-ankled deer mouse (P. laceianus), four white-footed mice (P. leucopus), and one fulvous harvest mouse (Reithrodontomys fulvescens) from northern, central, or southern Texas. The results of this study together with the results of a previous study revealed that Muleshoe virus, perhaps in association with S. hispidus, is distributed across northern Texas. Finally, the results of Bayesian analyses of glycoprotein precursor (GPC) gene sequences and pairwise comparisons of complete GPC (amino acid) sequences strengthened support for the notion that Muleshoe virus is distinct from Black Creek Canal virus, Bayou virus, and all other species included in the Bunyaviridae, genus Hantavirus.

Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection

New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV), Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3, 4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents.

Pathology of Black Creek Canal Virus Infection in Juvenile Hispid Cotton Rats (Sigmodon hispidus)

The purpose of this study was to assess the effect of inoculum dose on the pathogenesis of Black Creek Canal virus (BCCV) infection in the hispid cotton rat (Sigmodon hispidus), the principal host of BCCV. No sign of illness was observed in any of the 52 juvenile hispid cotton rats inoculated with 3.1, 1.1, -0.9, or -2.9 log(10) median infectious doses(VeroE6) (ID(50-VeroE6)) of BCCV and euthanized on day 9, 18, 27, or 54 postinoculation (PI). Analysis of virus assay and serological data indicated that inoculum dose could significantly affect the pathogenesis of BCCV infection in juvenile hispid cotton rats. For example, the six animals inoculated with 3.1 or 1.1 log(10) ID(50-VeroE6) and euthanized on day 54 PI were virus positive and antibody positive, whereas the six animals inoculated with -0.9 or -2.9 log(10) ID(50-VeroE6) and euthanized on day 54 PI were virus positive but antibody negative. Microscopic examination of tissues from the animals inoculated with 3.1 or 1.1 log(10) ID(50-VeroE6) revealed diffuse, subacute pneumonitis in the lungs of all the animals euthanized on day 18 PI or thereafter, and indicated that the severity of pneumonitis was dependent upon inoculum dose as well as duration of infection (i.e., amount of time elapsed since inoculation).

New and Old World hantaviruses differentially utilize host cytoskeletal components during their life cycles

Recently we reported that the N protein of the Old World hantavirus, Hantaan (HTNV), traffics on microtubules to the ER-Golgi intermediate compartment (ERGIC) prior to its movement to the Golgi and requires an intact ERGIC for viral replication. We have extended these studies to the New World hantaviruses, Andes virus (ANDV) and Black Creek Canal virus (BCCV), and an additional Old World hantavirus, Seoul virus (SEOV). These studies support microtubule-dependent trafficking of the N protein to ERGIC within the perinuclear region for the New and Old World hantaviruses. However, we observed that early entry events were distinct for HTNV and ANDV with respect to the pathway for entry and the dependence on an intact actin (ANDV) versus microtubule (HTNV) cytoskeleton for viral replication. These studies show for the first time that while Old and New World hantaviruses share common features in their pathways, they have evolved differences in their interaction with host cell machinery.

Detecting multiple levels of effect during survey sampling using a Bayesian approach: Point prevalence estimates of a hantavirus in hispid cotton rats ( Sigmodon hispidus)

Interpreting the results of survey samples of animals for zoonotic agents can be confounded by factors acting at various levels of scale. It is difficult to control for the numbers or characteristics of individuals surveyed even with standardized sampling. The survey results at any site may reflect the impact of individual level (e.g. age, gender) factors, local environmental conditions, and landscape structure. Incorporating these different scales to characterize more accurately prevalence estimates from survey results is problematic. We propose an empirical Bayesian format to deal with these factors and demonstrate the approach by modeling estimates of Black Creek Canal Virus infection prevalence (as determined by prevalence of antibodies) among local populations of hispid cotton rats ( Sigmodon hispidus) trapped in Dade County, Florida. Trapping was conducted at 110 sites for three nights using a standard protocol. A total of 1042 hispid cotton rats were captured (range 0–51 per site). A succession of Bayesian models was fit to identify variables that improved estimates of site-specific prevalences. At the individual level, both weight and sex were significant predictors of infection. Features of the local landscape, inferred from a LANDSAT image of the region, such as vegetation type and thermal indices, were also associated with increased likelihood of infection. Isolation of a trap site from suitable hispid cotton rat habitat was correlated with decreased prevalence. Finally, a spatially distributed intercept term showed regions of higher or lower than normal risk not explained by other variables. This suggests the rat populations near those sites may have been geographically connected to each other and/or disconnected from other sites.

334. Micro-Environmental Effects on Gene Transfer by Pseudotyped Lentiviral Vectors

Efforts to target lentivectors to specific tissues in vivo have primarily focused on vector pseudotyping with heterologous envelopes. One barrier to effective gene delivery into the different target organs following intravenous administration of lentivectors may be to find conditions that can bypass the endothelial barrier, which is likely the problem with the use of the conventional vesicular stomatitis virus G protein envelope (VSV-G). Towards this end, we conjectured that micro-environmental factors within specific tissues may also affect lentivector transduction efficiency. In this study, we examined the effect of micro-environmental conditions on lentivectors pseudotyped with two Bunyaviral envelopes. First, the Hantaan (HTNV) envelope was derived from a strain associated with hemorrhagic fever resulting in renal syndrome, and the second, Black Creek Canal Virus (BCCV) was derived from a strain associated with hemorrhagic fever with pulmonary syndrome. These viral envelopes were selected because, although closely related, they display distinct tissue tropisms, i.e., the HTNV strain preferentially infects the kidney, which is our target organ of interest. Preliminary evidence indicates that transduction of HeLa cells by HTNV, but not BCCV, pseudotypes can be significantly improved under acidic conditions and in a dose-dependent fashion in the presence of NaCl (3.6 ± 1.8 × 105 T.U/mL; n = 3) or urea (3.7 ± 2.9 × 105 T.U/mL; n = 3) compared to control conditions (9.3 ± 3.7 × 104 T.U./mL; n = 3). Moreover, decreasing the pH to 6.0 increased viral transduction with HTNV-pseudotyped lentivectors to 1.9 ± 1.6 × 105 T.U./mL from 9.3 × 104 T.U./mL. These conditions are important since they mimic those found within the kidney in vivo. The control VSV-G pseudotypes behaved similarly to BCCV pseudotypes. Enhanced transduction of cells by HTNV pseudotypes under these conditions was shown to occur in Huh7 hepatoma, C166 endothelial and RAG-1 renal carcinoma lines. To investigate the method of viral entry into the cells, we pre-treated the cells with 10 mM NH4Cl to block endosomal acidification. Transduction of cells by HTNV pseudotypes was inhibited by >90% in the presence of NH4Cl indicating that vector entry occurred via an endosomal pathway similar to that of native HTNV. Since we know that electrostatic interactions can affect the binding of many viruses to cell surface heparin sulfate (HS) proteoglycans, and we have investigated some of the conditions that could influence those interactions, we further examined the effect of HS and related molecules on HTNV and BCCV pseudotypes. Transduction by HTNV pseudotypes was significantly inhibited (>50%) in media supplemented with heparin, indicating this particular pseudotype requires HS proteoglycans to establish infection. Conversely, transduction was enhanced by 1–2 orders of magnitude in the presence of dextran sulfate (n = 3). Our findings indicate that micro-environmental conditions mimicking those within the kidney can significantly enhance transduction by HTNV pseudotypes and thus, improve our understanding of basic viral biology and their applications for effective vector design. Funded by NIDDK 69620–01.

Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region.

Black Creek Canal virus (BCCV) is a New World hantavirus which is associated with hantavirus pulmonary syndrome. We have examined the site of expression of the BCCV nucleocapsid protein (NBCCV) in the absence of BCCV glycoproteins and found that the majority of the protein is localized to the Golgi region. Immunofluorescence analysis of BHK21 cells expressing the NBCCV and La Crosse virus nucleocapsid protein (NLACV) showed different intracellular localization patterns of these proteins within the same cell: NLACV is cytoplasmic, whereas NBCCV is perinuclear. NBCCV was found to be colocalized with alpha-mannosidase II, a marker for the Golgi complex. Also, NBCCV was found to be associated with microsomal membranes following cell fractionation. Sedimentation analysis in density gradients revealed that the membrane association of NBCCV is sensitive to treatments with high-salt and high-pH solutions, which indicates that NBCCV is a peripheral membrane protein. Analysis of NBCCV truncation mutants revealed that the 141-amino-acid C-terminal portion of this protein was capable of targeting green fluorescent protein to the perinuclear region. The difference in the intracellular localization between the NBCCV and NLACV proteins suggests that the mechanisms involved in the morphogenesis of New World hantaviruses are distinct from that documented for other members of the Bunyaviridae family.

Transmission of Black Creek Canal virus between cotton rats.

Black Creek Canal (BCC) virus is a hantavirus associated with hantavirus pulmonary syndrome in southeastern North America. The virus was isolated from the spleen of a cotton rat (Sigmodon hispidus) trapped in southern Florida. Our previous studies have shown that we could consistently infect male cotton rats with BCC virus in the laboratory. These animals became persistently infected and virus could be detected in salivary glands, urine, and feces. In this report we show: (1) female and male cotton rats are equally susceptible to BCC virus infection, (2) susceptibility to infection was not influenced by age, (3) all inoculated rats transmitted the infection to uninoculated cage mates, and (4) offspring of infected rats became infected despite the presence of high maternal antibodies. The course of BCC virus infection, as determined by antibody response and the ability to isolate or detect virus, appeared to be similar regardless of whether the rats obtained their infection by inoculation or contact with inoculated rats. J. Med. Virol. 60:70-76, 2000. Published 2000 Wiley-Liss, Inc.

Black Creek Canal Virus infection in Sigmodon hispidus in southern Florida.

A total of 1,500 small mammals were collected and tested for antibodies cross-reactive to Sin Nombre virus (Hantavirus: Bunyaviridae) at 89 sites in a 1,600 km2 study area of southern Florida. More than 95% of the 123 seropositive animals were cotton rats (Sigmodon hispidus), suggesting infection by Black Creek Canal Virus, although seroreactive Rattus rattus (5 of 294) and Peromyscus gossypinus (1 of 39) also were captured. Crude seroprevalence in S. hispidus was 11%. Seroprevalence increased with body size and was more common in male (18%; n=451) than in female (6%; n=593) cotton rats. Infection within S. hispidus populations was widespread throughout the study area. Prevalence ranged from 0% to 60% at sites where more than five cotton rats were sampled but was not only a function of sample size. Sites with seropositive cotton rats were geographically clustered compared with sites with no seropositive cotton rats. Clustering was not due to the spatial distribution of sites with few animals, season of collection, or sex bias of animals captured at these sites. However, sites with no seropositive animals had an excess of animals in the intermediate size class (60-99 g) and a deficit of the largest and smallest animals. These data suggest that population structure within the habitat mosaic may play a significant role in the spatial distribution of hantavirus infection in local populations of reservoir species.

Pathogenesis of a North American hantavirus, Black Creek Canal virus, in experimentally infected Sigmodon hispidus.

This report describes the first detailed analysis of the replication, persistence, and excretion of a North American hantavirus in its natural rodent reservoir. Black Creek Canal virus was isolated from Sigmodon hispidus (cotton rat) shortly after the identification of a hantavirus pulmonary syndrome (HPS) case occurring in southern Florida. Six-week-old male cotton rats were inoculated subcutaneously with 1,000 tissue culture infectious doses. Viral complementary RNA (vcRNA) was quantified as a means of determining the site(s) of viral activity (transcription and replication). In the first few weeks post inoculation (pi), vcRNA was detectable in every tissue examined except blood. The quantities of vcRNA decreased over time, and by five months pi it could be detected only in the brain. In addition to using a quantitative polymerase chain reaction (QPCR) as a means of measuring viral replication/transcription, attempts were made to reisolate virus from all tissue samples taken. Virus could be isolated from every solid tissue examined, and the titers appeared to decrease over time, similar to the QPCR results. However, in contrast to the QPCR results, infectious virus was still routinely detectable at low levels in adrenal gland, liver, kidney, and testicle 150 days pi. Although results of testing for vcRNA in the blood were uniformly negative, infectious virus was detected at one week pi, reached highest titers at two weeks, and decreased dramatically by three weeks. After three weeks pi, infectious virus could only be detected sporadically in blood. Virus was isolated from urine collected during the first 70 days pi and throughout the entire study period in feces and wet bedding. These data indicate that the viral infection can be separated into an acute phase associated with high virus titers, and a chronic or persistent phase associated with lower virus titers and continued shedding of virus in excreta.

Role of actin microfilaments in Black Creek Canal virus morphogenesis.

We have investigated the involvement of cytoskeletal proteins in the morphogenesis of Black Creek Canal virus (BCCV), a New World hantavirus. Immunofluorescent staining of BCCV-infected cells revealed a filamentous pattern of virus antigen, the appearance of which was sensitive to treatment with cytochalasin D, an actin microfilament-depolymerizing drug. Double immunofluorescence staining of BCCV-infected Vero cells with anti-BCCV nucleocapsid (N) monoclonal antibody and phalloidin revealed a colocalization of the BCCV N protein with actin microfilaments. A similar, though less prominent, filamentous pattern was observed in BHK21 cells transiently expressing the BCCV N protein alone but not in cells expressing the BCCV G1 and G2 glycoproteins. Moreover, the association of the N protein with actin microfilaments was confirmed by coimmunoprecipitation with beta-actin-specific antibody. Treatment of the BCCV-infected Vero cells at 3 days postinfection with cytochalasin D decreased the yield of released BCCV by 94% relative to the yield from untreated cells. Pretreatment of Vero cells with cytochalasin D prior to and during BCCV adsorption and entry had no effect on the outcome of virus production. These results indicate that actin filaments may play an important role in hantavirus assembly and/or release.

Genetic Reassortment among Viruses Causing Hantavirus Pulmonary Syndrome

In order to determine the frequency and characteristics of reassortment among viruses causing hantavirus pulmonary syndrome (HPS), mixed infections were initiated in tissue culture by using two closely related strains of Sin Nombre virus, CC107 (from eastern California) and NMR11 (from New Mexico), which share the same species of rodent host in nature, the deer mouse (Peromyscus maniculatus). Potential reassortant virus plaques were screened by multiplex RT–PCR, using primers specific for individual genome segments of each strain. Reassortant viruses involving the M and S segments and, to a lesser extent, the L segment were detected in 8.5% of 294 progeny plaques tested. In addition, approximately 30% of the progeny virus plaques appeared to contain S or M segments originating from both parental virus strains, i.e., they were diploid. Most of these diploid virus genotypes were not stable, becoming either reassortant or parental virus strains upon plaque-to-plaque virus passage. In contrast to the results above, only one virus reassortant and four diploids were observed among 163 progeny virus plaques from mixed infections between Sin Nombre virus NMR11 and the genetically more distant Black Creek Canal virus, an HPS-causing virus from Florida, which has the cotton rat (Sigmodon hispidus) as its natural host.

Polarized entry and release in epithelial cells of Black Creek Canal virus, a New World hantavirus.

Black Creek Canal (BCC) virus is a newly identified hantavirus from Florida which is carried by the cotton rat (Sigmodon hispidus) and is associated with hantavirus pulmonary syndrome (HPS). We have investigated the interaction of BCC virus with polarized epithelial cells to examine whether entry and release of this virus occur at specific plasma membrane domains. The polarized Vero C1008 monkey kidney cell line was grown on permeable filters and infected with BCC virus either through the apical or basolateral surface. As shown by indirect immunofluorescence and radioimmunoprecipitation analysis, cells infected through the apical surface demonstrated a high level of susceptibility to BCC virus infection. In contrast, Vero C1008 cells infected basolaterally exhibited a barely detectable level of BCC virus-synthesized proteins. Titration of virus from apical and basolateral media of infected cells has demonstrated that virus titers released from the apical surface are about 1,200-fold greater than the titer of virus released into the basolateral media. The site of BCC virus release from polarized cells is, therefore, different from that previously described for release of other members of the family Bunyaviridae and may reflect one of the determinants of hantavirus pathogenesis. In addition, we have shown that BCC viral glycoproteins are expressed at the plasma membrane on the apical surface of polarized cells. Electron microscopy studies of the infected cells revealed evidence of BCC virus budding at the plasma membrane. This strongly indicates that, in contrast to most other members of the Bunyaviridae, BCC virus is assembled at the plasma membrane. Since the same site of virus assembly was recently described for Sin Nombre virus, it is likely that all of the new American hantaviruses associated with HPS utilize this same type of virus maturation.

Hantavirus pulmonary syndrome in Florida: Association with the newly identified Black Creek Canal virus

Hantavirus pulmonary syndrome (HPS) is a recently recognized viral zoonosis. The first recognized cases were caused by a newly described hantavirus, Sin Nombre virus (previously known as Muerto Canyon virus), isolated from Peromyscus maniculatus (deer mouse). We describe a 33-year-old Floridian man who resided outside the ecologic range of P maniculatus but was found to have serologic evidence of a hantavirus infection during evaluation of azotemia associated with adult respiratory distress syndrome. Small mammal trapping conducted around this patient's residence demonstrated the presence of antihantaviral antibodies in 13% of Sigmodon hispidus (cotton rat). Serologic testing using antigen derived from the Black Creek Canal hantavirus subsequently isolated from this rodent established that this patient was acutely infected with this new pathogenic American hantavirus. HPS is not confined to the geographical distribution of P maniculatus and should be suspected in individuals with febrile respiratory syndromes, perhaps associated with azotemia, throughout the continental United States.

Cross-Neutralization of Hantaviruses with Immune Sera from Experimentally Infected Animals and from Hemorrhagic Fever with Renal Syndrome and Hantavirus Pulmonary Syndrome Patients

Plaque-reduction neutralization tests were done with eight of nine known representative hantaviruses and immune sera from experimentally infected animals and from patients with hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS). Results obtained with animal sera demonstrated each virus to be antigenically unique. Neutralization with the HPS patient sera was highest with Sin Nombre (SN) virus and to a lesser extent with Black Creek Canal (BCC) virus. Sera from Korean HFRS patients reacted best with Hantaan virus, but cross-reactivity with all other viruses except Thottapalayam (TPM) virus was also observed. Sera from Swedish HFRS patients reacted best with Puumala virus but cross-reacted with Prospect Hill, SN, and BCC viruses and to a lesser extent with all of the other viruses except TPM virus.

Genetic and Serologic Analysis of Black Creek Canal Virus and Its Association with Human Disease and Sigmodon hispidus Infection

Black Creek Canal (BCC) virus is a newly identified virus associated with hantavirus pulmonary syndrome (HPS) in the southeastern United States. Nucleotide sequences were determined for the complete S and M and partial L genomic segments of a BCC virus isolate. Phylogenetic analysis indicates that each of the virus segments is unique, and there is no evidence of genetic reassortment having occurred between this and other previously characterized hantaviruses. All hantavirus-seropositive, wild-caught Sigmodon hispidus which were tested by polymerase chain reaction for the presence of BCC virus RNA were found to be positive, consistent with a chronic virus infection. This finding, together with serologic evidence, confirms S. hispidus as the primary rodent reservoir of this virus. Nucleotide sequence analysis of these PCR products indicates that BCC virus is genetically diverse and may pose a public health threat throughout much of the extensive range of this rodent species. Immunoprecipitation analysis demonstrated the presence of G1-, G2-, and N-specific antibodies in sera from BCC virus-infected patients with HPS and naturally infected S. hispidus. Comparison of the patterns of protein immunoprecipitation obtained with human acute- and convalescent-phase sera suggests that G1- and G2-specific antibodies increase relative to N-specific antibodies during the course of infection. The ratio of G1 to N protein immunoprecipitation by rodent or human acute-phase sera allowed differentiation of BCC and Sin Nombre virus infections.

Isolation of black creek canal virus, a new hantavirus from Sigmodon hispidus in Florida.

Numerous rodents were trapped for serologic and virologic studies following the identification of a hantavirus pulmonary syndrome (HPS) case in Dade County, Florida. Cotton rats (Sigmodon hispidus) were the most frequently capture rodent and displayed the highest seroprevalence to a variety of hantavirus antigens. Hantavirus genome RNA was detected in all the seropositive cotton rats tested, using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A virus was isolated from tissues of two seropositive cotton rats by cultivation of lung and spleen homogenates on Vero E6 cells. Nucleotide sequence information obtained by direct RT-PCR and the serologic relationships of this virus with the other hantaviruses indicate that this virus, Black Creek Canal virus, represents a new hantavirus distinct from the previously known serotypes.

Fatal Hantavirus Pulmonary Syndrome in an Adolescent

Hantavirus pulmonary syndrome (HPS) is a recently recognized viral zoonosis characterized by a febrile prodrome progressing to severe noncardiogenic pulmonary edema.1-4 This syndrome is caused by at least three newly described hantaviruses: the first, Sin Nombre virus, is the Southwestern hantavirus that caused an outbreak of respiratory failure during the summer of 1993; the second, Black Creek Canal virus, caused a case of HPS in Florida; and the third hantavirus was identified in lung tissue from a patient in Louisiana. Diagnosis is by enzyme-linked immunosorbent assay (ELISA) serology with elevated immunoglobulin M (IgM) titers against heterologous and homologous hantaviral antigens, positive immunohistochemistry on formalin-fixed tissue, or reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of hantaviral nucleotide sequence from frozen tissue.