Case Descriptions

The index patient was a 54-year-old woman who had headache, myalgias, and abdominal pain on August 26, 1999, followed several days later by respiratory symptoms. She went to the hospital on August 31, where she was diagnosed with bilateral pneumonia and adult respiratory distress syndrome; she died on September 1. A serum sample obtained on August 31 was reactive for IgM antibodies. The patient’s 71-year-old brother had had a febrile illness on August 7, 1999, and was hospitalized 2 days later with a clinical diagnosis of acute abdominal pain, pyelonephritis, shock, and bilateral pulmonary infiltrates; he died on August 10. HPS was not suspected, and no serum or tissue was available for testing when HPS was diagnosed in the index patient.

The Ministry of Health initiated a routine evaluation of household and neighborhood contacts on September 13, 1999. Blood was obtained from 10 asymptomatic contacts, including the 10-year-old grandson of the index patient. On September 15, the grandson became febrile, and headache and vomiting developed. Two days later, he (patient 99-7913) was evaluated as an outpatient. His physical examination showed fever (38°C) and no respiratory symptoms. His leukocyte count was 13,000/µL, hematocrit 46.9%, hemoglobin 15.7 g/dL, and platelet count 125,000/µL. The plasma C reactive protein was 39 mg/L. Diffuse bilateral interstitial pulmonary infiltrates were detected on chest radiograms, and the patient was treated with a macrolide antibiotic for presumed pneumonia. He returned to the hospital the morning of September 18 without fever, with arterial pressure 110/60 mmHg, tachycardia (100 beats per minute), and weakness. Pneumonia, obstructive bronchial syndrome, and dehydration were diagnosed. He was treated with intravenous penicillin, hydration, and aerosolized salbutamol. He returned to the hospital again on the evening of September 18 with respiratory failure and shock and died on September 19 within hours of arrival. No additional serum or tissue samples were obtained at the outpatient visit or in the hospital.

Epidemiologic Studies

Routine epidemiologic evaluation of each confirmed HPS case in Chile includes rodent trapping around the patient’s household and evaluation of household and family contacts. The latter includes a clinical evaluation for history of recent fever or other symptoms and the administration of a questionnaire to assess risk factors for hantavirus infection. A serum sample is obtained from household and family contacts by venipuncture and transported to the Institute of Public Health in Santiago for determination of hantavirus antibodies.

Biosafety Procedures

We followed the recommendations of the Centers for Disease Control and Prevention (CDC) in all aspects of this work (10). Antibody studies were conducted at biosafety level 2 (BSL-2) facilities, but viral isolation attempts were conducted at BSL-3 laboratories in the Instituto de Salud Pública laboratory in Santiago.

Detection of Antibodies to SNV in Patient Serum

We detected antibodies from patient serum samples by enzyme-linked immunosorbent assay (ELISA) with nucleocapsid (N) antigens of SNV and Laguna Negra (formal name: Laguna Negra virus [LNV]) virus. These diagnostic tests were obtained from CDC in Atlanta, Georgia (11). SNV antigen was used in the solid phase for detecting IgG antibodies, and LNV antigen was used in an antigen-capture format for detecting specific IgM antibodies, as described (11).

Isolation of ANDV from Patient Serum

Virus was isolated by a conventional method, with three blind passages in monolayers of Vero E6 cells (12). We grew Vero E6 cells to confluence in T25 flasks with minimal essential media containing 10% fetal calf serum and antibiotics, and then replaced the media with 1.5 mL of fresh media containing 0.5 mL of serum from patient 99-7913. After 1 hour, we added 4.5 mL of fresh media and maintained the cells at 37°C in 5% CO2 for 26 days, refeeding twice per week (Tissue Culture 1 or TC-1). At 26 days, the cells were treated with trypsin and split 1:2 into fresh T25 flasks (TC-2). At 24 days postinoculation (dpi), we trypsinized the cells and replated them into fresh T25 flasks (TC-3). At 14 dpi, we trypsinized the cells and replated them into fresh T25 flasks (TC-4). At 13 dpi, we treated the cells with trypsin and replated them after removing 5 x10⁴ for indirect immunofluorescence assay (IFA) analysis.

IFA

Fifty thousand inoculated Vero E6 cells were washed twice with phosphate-buffered saline and dried on a microscope spot-slide for IFA testing. As a control, we used uninoculated Vero E6 cells that were processed in parallel. The cells were stained according to Gallo et al. (13), using a 1:1,000 dilution of rabbit polyclonal anti-Andes N. Cells were considered to be positive for hantavirus antigen if we observed punctate cytoplasmic and Golgi staining in the presence of anti-N antibody but not in the presence of preimmune rabbit serum or if the cells had not been injected with a source of ANDV.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

We conducted nested RT-PCR to detect a portion of the viral N gene by using generic primers for S segment as described (14). The coordinates of the primers, designated S1 and S2 (outer primers), were at 2 and 593, whereas the inner primers S3 and S4 were at coordinates 22 and 359. We determined that an amplification reaction was positive if we could visualize a 338-bp band after electrophoresis through agarose, as described (14).

Phylogenetic Studies

Nucleotide sequences examined corresponded to positions 22–359 of antigenome-sense sequence of nucleoprotein gene. Genetic relationships of the Chilean isolate with homologous sequences of previously characterized hantavirus were obtained by the maximun parsimony method with the Clustal W and PHYLIP packages (15).

Virus Isolation

Figure 1

Figure 1. Immunofluorescence assay (IFA) of Vero E6 cells infected with Chilean hantavirus CHI-7913 isolate. A, IFA with seropositive human sera from a Chilean HPS patient; arrow shows infected Vero E6 cells expressing...

Inoculated and noninoculated Vero E6 cells were cultivated for several weeks. After three blind passages in new monolayers of Vero E6 cells, the presence of hantaviral antigens was tested by IFA with serum samples from seropositive Chilean HPS patients. Only the cells derived from the patient serum-inoculated tissue culture expressed fluorescent inclusion bodies (Figure 1). Over 90% of the cells became fluorescent. The specificity of this reaction was further tested by ELISA with cellular lysates prepared from infected and noninfected cells and ANDV antibody-positive rabbit sera. The absorbance values obtained by using lysates of infected cells were higher than those from noninfected cells (data not shown). To further confirm the presence of hantavirus infection in the Vero E6 cells, we also used nested RT-PCR of the nucleoprotein gene to test tissue cultures inoculated with patient serum with generic primers for S segment (14). Only the RNA extract from patient sera-inoculated cells showed an amplified product with the expected 338-bp fragment. The amplified product was not detected in mock-infected cells (data not shown).

Figure 2

Figure 2. Maximum-parsimony tree analysis comparing S nucleotide sequence of CHI-7913 virus with homologous sequences of previously characterized hantaviruses. Nucleotide sequences examined correspond to positions 22–359 of antigenome-sense sequence of nucleoprotein (N) gene....

The amplified DNA product was sequenced and compared with the sequence of prototype strains (Figure 2). The Chilean hantavirus isolate, designated CHI-7913, showed 96.2% nucleotide identity with the prototypical ANDV sequence, but only 81.1%–82.5% identity with SNV from the southwestern United States (14).