Serum samples were obtained from 823 febrile patients at the Secretaria de Salud de Yucatán and other health institutions in Merida during January–October 2007. The patients resided in all 3 states of the Yucatan Peninsula of Mexico: Yucatan (n = 809), Quintana Roo (n = 8) and Campeche (n = 6). The study was approved by the Institutional Biosafety Committees at Iowa State University (Ames, IA, USA) and the Universidad Autónoma de Yucatán (Mérida, Mexico).

All serum samples were examined at a dilution of 1:20 by plaque reduction neutralization test (PRNT) by using CVV (strain CVV-478), and PRNTs were performed as described (7). A subset of serum samples with antibodies that neutralized CVV were titrated and further analyzed by PRNT by using CVV, CHLV (strain CHLV-Mex07), KRIV (strain KRIV-Mex07), SOURV (strain NJO-94f), Maguari virus (strain BeAr7272), and Wyeomyia virus (strain prototype). All of these viruses belong to the Bunyamwera (BUN) serogroup except SOURV, which belongs to the California (CAL) serogroup.

Titers were expressed as the reciprocal of highest serum dilutions yielding >90% reduction in the number of plaques (PRNT₉₀). For etiologic diagnosis, the PRNT₉₀ antibody titer for each virus was required to be >4-fold greater than that to the other viruses tested.

Antibodies that neutralized CVV were detected in 146 (18%) of 823 study participants. The mean ages of patients with and without antibodies that neutralized CVV were 32.0 and 22.3 years, respectively. Logistic regression analysis showed that the risk for infection increased significantly with age (p = 0.0001).

Serum samples from 50 seropositive patients were titrated and analyzed by comparative PRNT to identify the orthobunyaviruses responsible for these infections. Six persons were seropositive for CVV, 5 for CHLV, and 1 for SOURV or a SOURV-like virus; 38 had antibodies to an undetermined orthobunyavirus (Table). Because SOURV was the only CAL serogroup virus used in this study, and another CAL serogroup virus may have been responsible for the infection, the person who had a SOURV PRNT₉₀ titer >4-fold than that to the other viruses tested received a conservative PRNT diagnosis of seropositive for SOURV or a SOURV-like virus. Because interserogroup crossreactivity of neutralizing antibodies to viruses in the BUN and CAL serogroups has not been seen, the 17 persons with antibodies that neutralized SOURV and >1 of the BUN serogroup viruses might have been exposed to >1 viruses from each serogroup.

CHLV and POTV share the same medium RNA segment, so antibodies for these viruses cannot be differentiated by PRNT. Furthermore, antibodies to CHLV and POTV cannot be differentiated by complement fixation test (4). Thus, we cannot dismiss POTV as a possible cause of infection in some or all of the study participants who were seropositive for CHLV. However, it appears more likely that these persons had been infected with CHLV because this virus has been isolated in the Yucatan Peninsula, whereas no direct evidence has been found for POTV in this region.

As already noted, serum samples from 38 (76%) of the study participants analyzed by comparative PRNT had antibodies to an undetermined orthobunyavirus. Most of these persons had low PRNT₉₀ titers; the highest PRNT₉₀ titer for 29 of these persons did not exceed 40. Because neutralizing antibody levels decline over time, these findings may indicate that many of these infections occurred years ago, and the trace amounts of neutralizing antibodies that remained were insufficient to yield a >4-fold difference between the titers of the virus responsible for the infection and the other viruses used in the PRNTs. Another explanation is that some of these persons had been infected with an orthobunyavirus not included in the PRNTs, although all orthobunyaviruses known to occur in the Yucatan Peninsula were represented.

We found 18% of the 823 Yucatan residents participating in our study had evidence of orthobunyavirus exposure. This number is presumably an underestimate; additional seropositive persons might have been identified if the initial PRNTs had not been restricted to CVV. In particular, additional seropositive persons likely would have been detected if SOURV was used in the initial PRNTs, because a screening algorithm that includes only a BUN serogroup virus would likely miss many CAL serogroup virus infections. Nevertheless, we provide evidence that orthobunyaviruses commonly infect humans in the Yucatan Peninsula.

Previous serosurveys have provided information on the seroprevalence of orthobunyaviruses in humans in the United States. For example, antibodies that neutralized CVV were detected in 42/356 (12%) residents in Maryland and Virginia in 1961–1963 (8). Antibodies that neutralized Maguari virus or Tensaw virus were detected in 71/≈300 humans in Florida in the 1980s (9); as observed in our study, the highest PRNT titers for most of the seropositive persons in that study did not exceed 40.

All persons in our study cohort initially sought care for unspecified fever; however, we could not determine whether any of these febrile illnesses were a direct consequence of orthobunyavirus infection. The detection of acute orthobunyavirus infections is limited because no IgM-capture ELISA for orthobunyavirus diagnosis exists. PRNTs can be used to identify recent orthobunyavirus infections when paired acute and convalescent serum samples are available, but for our study, only single serum samples were available from each participant. Orthobunyavirus viremias in humans are transient and of low magnitude, which makes reverse transcription PCR ineffective for the detection of orthobunyavirus RNA in serum samples. However, a duplex reverse transcription PCR was recently developed for the detection of CVV RNA in human cerebrospinal fluid (10).

In conclusion, we provide evidence that orthobunyaviruses commonly infect humans in the Yucatan Peninsula. These viruses are also a common cause of infection of livestock in this region (4). Our findings underscore the need to determine whether orthobunyaviruses represent an unrecognized cause of illness in humans and vertebrate animals in Mexico.