In collaboration with the Peruvian Ministry of Health, the Naval Medical Research Center Detachment, Lima, Peru, has established a febrile illness surveillance program designed to identify the causes of febrile diseases in the country. The program carries out passive surveillance in Ministry of Health posts, where febrile patients are identified, signs and symptoms are recorded, and acute- and convalescent-phase blood samples are collected for diagnostic studies. Tests routinely performed in support of the surveillance program include immunoglobulin (Ig) M ELISA for flaviviruses, alphaviruses, bunyaviruses, arenaviruses, and rickettsia, as well as virus isolation, reverse transcription–PCR (RT-PCR), and sequencing. The study protocol was approved by the Naval Medical Research Center Institutional Review Board (Protocol NMRCD.2000.0006, Department of Defense 31535), in compliance with all federal regulations governing the protection of human patients; the protocol also was reviewed by the Peruvian Ministry of Health.

Clinical diagnoses were considered confirmed if laboratory testing resulted in isolation of virus from the specimen, virus detection by RT-PCR, or a 4-fold or greater increase in IgM antibody titers. Diagnoses were considered presumptive if the IgM titer was elevated in the acute-phase sample only or a 4-fold or greater increase was discerned upon comparison of acute- and convalescent-phase titers. In the absence of laboratory evidence of a specific pathogen, cases were classified as negative.

Viruses were isolated by using a modification of a published protocol (23). Acute-phase serum samples, obtained no more than 5 days after disease onset, were transported on dry ice to the Naval Medical Research Center Detached laboratory in Lima and stored at –80°C. Serum samples were thawed and diluted 1:5 in minimum essential medium containing 2% heat-inactivated fetal bovine serum and antimicrobial agents. African green monkey (Vero) (37°C) and/or mosquito C6/36 (28°C) cell cultures were each injected with 200 μL of the diluted serum into 25-cm² flasks. Upon observation of viral cytopathic effect, or 10 days postinoculation (dpi) if no cytopathic effect was observed, cells were removed from the flasks and placed on 12-well glass spot-slides for examination by immunofluorescence assay using group- and virus-specific polyclonal and monoclonal antibodies. The antibodies used were reactive with dengue viruses 1–4, yellow fever virus, St. Louis encephalitis virus, Rocio virus, Ilheus virus, West Nile virus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, Mayaro virus, Trocara virus, Oropouche virus, Caraparu virus, Murtucu virus, Guaroa virus, hantavirus, Machupo virus, and Tacaribe virus; after the cardiovirus isolation, EMCV antibody also was used.

Upon receipt of the viruses at the Centers for Disease Control and Prevention (CDC; Atlanta, Georgia, USA), a master seed stock was prepared from each virus and working stocks were made by passage in Vero E6 cells. Cytopathic effect developed rapidly, and the cultures were harvested at 3–4 dpi; some infected cells were fixed in 2.5% glutaraldehyde for electron microscopy examination. For virus reisolation at CDC, acute-phase serum samples were injected into Vero E6 cells. Virus isolates were obtained in Vero E6 cells from acute-phase serum of both case-patients. The isolates, IQD6726 (Iquitos) and FSC575 (Cusco), were passaged in Vero E6 cells to obtain stocks for further analysis. Litters of suckling mice were inoculated intracerebrally with both strains of virus. Brain, spleen, and liver tissues were harvested and fixed in 2.5% glutaraldehyde for electron microscopic examination.

RNA was extracted from the virus isolates by using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA). cDNA reactions consisted of 2 μL of extracted viral RNA, 50 ng of random hexamers (Applied Biosystems, Foster City, CA, USA), 4 μL of 5× first strand buffer (Invitrogen, Carlsbad, CA, USA), 200 μmol/L of each dNTP (GE Healthcare, Piscataway, NJ, USA), 40 U of RNasin ribonuclease inhibitor (Promega, Madison, WI, USA), and 200 U of SuperScript II reverse transcriptase (Invitrogen) in a total volume of 20 μL. The cDNA reactions were incubated for 10 min at 25°C, followed by 45 min at 42°C and 4 min at 95°C. For the PCR assays, 3 genome regions were targeted, the 5′-NTR, VP1, and 3D regions (Table). Two sets of PCR primers targeting the viral protein 1 (VP1) gene were used, yielding overlapping PCR fragments. PCR reactions consisted of 2 μL of cDNA, 200 μmol/L of each dNTP (GE Healthcare), 5 μL of 10× buffer with MgCl₂ (Roche Molecular Biochemicals, Indianapolis, IN, USA), 2.5 U of Fast Start Taq DNA Polymerase (Roche), and forward and reverse primers (10 pmol of each of AN312 and AN315; 40 pmol each of AN283, AN285, AN393, and AN286; 20 pmol each of P1 and P2; and 60 pmol each of 1C340F and 2B188R), in a total volume of 50 μL. Cycling parameters were 42ºC for 50 min, 50°C for 10 min, and 95ºC for 5 min, followed by 45 cycles of 95ºC for 30 sec, 45ºC for 40 sec, and 60ºC for 1 min. All PCR products were analyzed by electrophoresis in 2% agarose gels stained with ethidium bromide.

Amplicons were purified from an agarose gel (QIAGEN gel extraction kit) before sequencing. Both strands were sequenced by using the PCR primers, a Prism BigDye Terminator version 1.1 or 3.1 Ready Reaction Cycle Sequencing kit (Applied Biosystems), and a model 3100 or 3130 Genetic Analyzer (Applied Biosystems). The sequences determined in this study were deposited in the GenBank sequence database, accession nos. EU979543–EU979548.

Sequences were identified in GenBank by using BLAST to compare sequences (www.ncbi.nlm.nih.gov/blast). For phylogenetic reconstruction, sequences were aligned with those of other cardioviruses by using PileUp (Wisconsin Sequence Package, version 11.1; Accelrys, San Diego, CA, USA), and trees were generated with the neighbor-joining method implemented in ClustalX version 1.83 (25). Genetic distances were estimated by the Kimura 2-parameter method. To assess the confidence of branching patterns of the neighbor-joining tree, 1,000 bootstrap replicates were performed.

Serum specimens were tested for neutralizing antibody against EMCV by a modified microneutralization assay (26). Approximately 80–100 median 50% cell culture infectious dose (CCID₅₀) of isolate FSC575 and serial dilutions of serum (starting at 1:10 and ending at 1:1,280) were incubated together at 37°C for 2.5 h before Vero E6 cells were added to the wells. After incubation for 5 days at 37°C, each plate was stained with 0.05% crystal violet in 25% ethyl alcohol and, after drying, the optical density in each well was measured at 570 nm. Each specimen was run in triplicate; the final titer was estimated by use of the Spearman-Karber method (27). Positive-control anti-EMCV serum was generously provided by Ann Palmenberg.

Pathogen-specific IgM titers were determined by using an adapted IgM-capture ELISA (28). Briefly, 96-well plates were coated with antihuman IgM antibody to capture patient IgM molecules. Virus-specific IgM was detected by adding viral antigen, followed by virus-specific mouse hyperimmune ascitic fluid and horseradish peroxidase-labeled antimouse IgG. After the addition of colorimetric substrate, absorbance was read at 490 nm. All acute- and convalescent-phase samples were initially screened at 1:100. Samples exceeding the reference cutoff value, calculated as the median of 7 antibody-negative samples plus 3 standard deviations, were considered IgM positive. Positive samples were subsequently retested by using 4-fold serial dilutions to determine end-point titers. Viruses included in the ELISAs included dengue viruses 1–4, yellow fever virus, St. Louis encephalitis virus, Rocio virus, Ilheus virus, West Nile virus, Venezuelan equine encephalitis virus, eastern equine encephalitis virus, Mayaro virus, Trocara virus, Oropouche virus, Caraparu virus, Murtucu virus, Guaroa virus, hantavirus, Machupo virus, and Tacaribe virus; after the cardiovirus isolation, EMCV was also used.

The first patient was identified in the Amazonian city of Iquitos, Loreto Department, Peru. Iquitos (3°44.69′S, 73°15.25′W) is situated at an elevation of 120 m and has a population of ≈350,000. The city is surrounded by the Nanay, Itaya, and Amazon rivers and is accessible only by air or river. The climate is tropical, mean temperature is 25°C, and annual rainfall is 2.7 m. Rodents are common in urban and rural areas; swine and primates are abundant in rural zones.

Case-patient 1 was a 59-year-old housewife from Iquitos. She sought treatment at Hospital Apoyo Iquitos after 3 days of fever, pallor, poor appetite, malaise, nausea, and headache. She recovered completely from fever after 6 days of treatment as an outpatient. Acute-phase (3 days postonset), convalescent-phase (15 days postonset), and late convalescent–phase (8 months later) blood samples were obtained. She had no identifiable focus of infection, negative thick and thin smears for malaria, and a negative tourniquet test. She reported no yellow fever or hepatitis B vaccination and no travel outside of Iquitos within 2 weeks before onset. The patient was interviewed 8 months after seeking treatment when virus isolation results were completed. She reported only limited contact with rats and cats in her house and a neighbor’s house where she played bingo frequently. After 7 days and nights of trapping in both houses and in a sewer adjacent to the neighbor’s house, no rodents were collected.

The second case-patient was identified in the Cusco Regional Hospital but had traveled from Quebrada, a small rural village on the Yanatile River, about 8 hours travel time from the city of Cusco. Quebrada (approximately 13°55′S, 71°40′W) is located in Yanatile district, Calca Province, in the Department of Cusco. The weather in Quebrada is warmer than that in Cusco (temperate climate) at an altitude of 2,926 m. Malaria and leishmaniasis are endemic to the area. The city is accessible by air and land from Cusco.

Case-patient 2 was a 39-year-old male farmer from Cusco-Acomayo/Quebrada Calca. He sought treatment at the local hospital after 7 days of fever. Additional symptoms were headache, malaise, retro-ocular pain, sweats, weight loss, arthralgia, photophobia, poor appetite, myalgia, chills, pallor, nausea, vomiting, and abdominal pain. He was hospitalized with a diagnosis of febrile syndrome and urinary tract infection (UTI). Urinalysis showed 20–25 leukocytes per high-power field. The patient had elevated values for alkaline phosphatase (680 U/L; reference range 68–240 U/L), total bilirubin (1.68 mg/dL; reference value <1.0 mg/dL), and direct bilirubin (1.06 mg/dL; reference value <0.2 mg/dL). Thick and thin smears for malaria and Bartonella spp. were negative, as were urine culture and a tourniquet test for dengue hemorrhagic fever. The patient was treated with oral ciprofloxacin (750 mg 2×/d for 7 days), oral paracetamol (acetaminophen; 500 mg as needed for fever >38.5ºC), and intravenous fluids. He recovered completely after 7 days as an inpatient. At the time of his convalescent-phase sample (15 days postonset), his physical examination was within normal limits. He reported tuberculosis in a family member contact (treated for 5 months), yellow fever vaccination in 1994, and no vaccination against hepatitis B. No case investigation was carried out for this patient to determine risk factors for disease. However, the typically rural populations near Cusco have contact with a variety of animal species such as mules, dogs, cats, swine, rodents, rabbits, llamas, alpacas, and vicuñas.

Virus isolates were obtained from acute-phase serum specimens of both patients by inoculation of Vero E6 cell cultures. Electron microscopic studies of infected Vero E6 cells and mouse tissues demonstrated cytoplasmic accumulations of particles consistent with the features of picornaviruses. Virions averaged 24 nm in diameter and were occasionally found in paracrystalline arrays (Figure 1, panel A). The infected cells were notable for areas of vesiculation and membrane proliferation (Figure 1, panel C), consistent with the replication complexes, which have been described for picornavirus-infected cells (29). Newborn mice inoculated intracerebrally all died at 3 dpi. Rapid death in neonatal mice, coupled with ultrastructural evidence of picornavirus infection, was consistent with the presence of a cardiovirus.

To confirm the diagnosis, we used cardiovirus-specific RT-PCR assays targeting the 5′-nontranslated region and the VP1 and 3D coding regions (Table). Each of the cardiovirus-specific primer sets amplified a specific DNA product of the expected size; in each instance, the identity of the amplicon was confirmed by sequencing. In a phylogenetic reconstruction done on the basis of sequences from the VP1 capsid region, the Peru viruses clustered with sequences of EMCV strains derived from pigs from Belgium, Cyprus, and Italy; the EMCV reference strains and field strains from pigs and orangutans formed separate subgroups within the EMCV species group (Figure 2, panel A). The VP1 nucleotide sequences of the Peru strains were 91%–100% identical to those of other strains within their phylogenetic subgroup. VP1 nucleotide identities of the Peru cardioviruses to the other EMCV subgroups ranged from 71% to 80% and from 47% to 51% to the rodent and human viruses in the Theilovirus genus, respectively. The Peru strains were also most closely related to the European pig strains in the 5′-NTR and 3D regions (Figure 2, panels B, C). Within the larger EMCV 5′-NTR group, containing the Peru cardioviruses, pairwise nucleotide identities ranged from 95.8% to 100.0% (Figure 2, panel B). Relatedness of the Peru cardioviruses’ 5′-NTR to the EMCV and Theiler’s viruses outside of this group ranged from 30.3% to 58.4%. Within the large 3D clade containing the Peru cardioviruses and European pig strains (Figure 2, panel C), the nucleotide identities ranged from 95.2% to 100.0%. 3D nucleotide identities of the Peru cardioviruses to the EMCVs outside the Peru clade ranged from 80.0% to 87.6%. Relatedness of the Peru cardioviruses’ 3D region to the Theiler’s viruses ranged from 56.7% to 61.4%.

Serum from case-patient 1 was seropositive for cardiovirus by IgM monoclonal antibody-capture ELISA; titer was high. Seroconversion was documented by an increase in titer from <8 in the acute-phase sample (day 3) to >1,024 in the convalescent-phase sample (day 15). The neutralization assay yielded similar results; acute-phase titer was <10 and a convalescent-phase titer was >1,280. Serum samples from case-patient 2, collected on days 7 and 15, were negative in both assays (titers <8 and <10, respectively), despite the presence of virus in the day 7 serum sample.