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Etiologic studies of acute febrile disease were conducted in sites across South America, including Cusco and Iquitos, Peru. Patients' clinical signs and symptoms were recorded, and acute- and convalescent-phase serum samples were obtained for serologic examination and virus isolation in Vero E6 and C6/36 cells. Virus isolated in Vero E6 cells was identified as encephalomyocarditis virus (EMCV) by electron microscopy and by subsequent molecular diagnostic testing of samples from 2 febrile patients with nausea, headache, and dyspnea. The virus was recovered from acute-phase serum samples from both case-patients and identified with cardiovirus-specific reverse transcription-PCR and sequencing. Serum samples from case-patient 1 showed cardiovirus antibody by immunoglobulin M ELISA (acute phase <8, convalescent phase >1,024) and by neutralization assay (acute phase <10, convalescent phase >1,280). Serum samples from case-patient 2 did not contain antibodies detectable by either assay. Detection of virus in serum strongly supports a role for EMCV in human infection and febrile illness.

Venezuelan equine encephalitis (VEE) virus was isolated in 1993, 1994, and 1995 from human cases of acute, undifferentiated, febrile illness in the Peruvian Amazon Basin. Two virus isolates were recovered in 1994 from Peruvian soldiers at a jungle outpost near Pantoja in northern Peru, and 10 isolates were obtained from military personnel and civilians in 1993-1995 in Iquitos, an urban center in northeastern Peru. The genetic relationship of these isolates to other VEE virus strains was determined by sequencing 856-867 nucleotide reverse transcription-polymerase chain reaction fragments derived from the PE2 glycoprotein gene. The sequences were compared with those of other VEE virus strains, including representatives of the IAB, IC, ID, IE, II, and IIIC subtypes. The two Pantoja isolates were most closely related to subtype IC and ID viruses previously isolated in Colombia and Venezuela, and to the ID viruses isolated during the 1970s in Iquitos. All of the recent Iquitos isolates were similar to one another, but they were more closely related to Panamanian ID strains than to isolates previously obtained in Iquitos, Peru, or in Colombia and Venezuela. The recent Iquitos VEE viral isolates were the first Panama-genotype VEE ID virus strains identified outside of the Republic of Panama.

A survey was conducted from October 1, 1993 to June 30, 1995 to determine the arboviral etiologies of febrile illnesses in the city of Iquitos in the Amazon River Basin of Peru. The study subjects were patients who were enrolled at medical care clinics or in their homes by Peruvian Ministry of Health (MOH) workers as part of the passive and active disease surveillance program of the MOH. The clinical criterion for enrollment was the diagnosis of a suspected viral-associated, acute, undifferentiated febrile illness of < or = 5 days duration. A total of 598 patients were enrolled in the study. Demographic information, medical history, clinical data, and blood samples were obtained from each patient. The more common clinical features were fever, headache, myalgia, arthralgia, retro-ocular pain, and chills. Sera were tested for virus by the newborn mouse and cell culture assays. Viral isolates were identified initially by immunofluorescence using polyclonal antibody. An ELISA using viral-specific monoclonal antibodies and nucleotide sequence analysis were used to determine the specific variety of the viruses. In addition, thin and thick blood smears were observed for malaria parasites. Venezuelan equine encephalitis (VEE) virus subtype I, variety ID virus was isolated from 10 cases, including three cases in October, November, and December 1993, five cases in January and February 1994, and two cases in June 1995. The ELISA for IgM and IgG antibody indicated that VEE virus was the cause of an additional four confirmed and four presumptive cases, including five from January through March 1994 and three in August 1994. Sixteen cases were positive for malaria. The 18 cases of VEE occurred among military recruits (n = 7), agriculture workers (n = 3), students (n = 3), and general laborers (n = 5). These data indicated that an enzootic strain of VEE virus was the cause of at least 3% (18 of 598) of the cases of febrile illnesses studied in the city of Iquitos in the Amazon Basin region of Peru.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.