Evidence of the presence of the Zika virus in Mexico since early 2015
To assess the possible circulation of Zika virus (ZIKV) prior to the first documented case in Mexico, we reanalyzed the stored samples from the states of Veracruz and Yucatán, which were originally collected to test for dengue (DENV) and chikungunya (CHIKV) but were negative for these viruses despite the symptomatology. The samples were originally collected between the 30 and 46 epidemiological weeks (EW) when the ZIKV was not yet declared as a Public Health Emergency of International Concern (PHEIC). From the total 4016 negative samples, a total of one hundred samples, 50 from Veracruz (CHIK− DENV−) and 50 from Yucatán (4 CHIK− DENV− and 46 CHIK− or DENV−), were tested for Zika virus by using RT-PCR. Results showed that in Veracruz and Yucatán, 20 % (10/50) and 70 % (35/50) were, respectively, ZIKV positive, indicating unequivocally the presence of ZIKV at least since July 2015. We also tested non-confirmed suspect measles cases from early 2015 for ZIKV by RT-PCR. Remarkably in 11 Mexican states, 86 % (18/21) were positive with the earlier symptoms onset as early as May 2015. Finally, RT-PCR analyses on RNA extracted from Aedes aegypti mosquitoes captured from January to March 2015 showed the presence of ZIKV, strongly suggesting that the vector was already carrying the virus at the start of 2015.
KeywordsZika virus Mexico RT-PCR Retrospective analysis
Given the overlapping symptomatology of Zika virus disease and measles , we also tested non-confirmed suspect measles cases from early 2015 (stored at −20 C) for Zika virus by RT-PCR. Remarkably in 11 Mexican states, 86 % (18/21) were positive for ZIKV with the earlier symptoms onset as early as May 2015 (EW 19; Fig. 1a), with sixteen samples corresponding to 1- to 7-year-old children from ten Mexican states. These results indicate that syndromic classification of exanthematous diseases and Zika fever should be revised.
Moreover, as part of an active entomo-virological surveillance for dengue through the RNLSP, RT-PCR analyses of RNA extracted from Aedes aegypti mosquitoes (macerated groups of no more than 25 specimens) collected at the southern state of Guerrero were performed. The capture of mosquitoes was carried out since January 2015. Briefly, adult female mosquitoes were grouped in pools of 4 up to 25 (mean = 9.3) specimens. Each pool was homogenized with a tissue disruptor (Qiagen) and centrifuged at 2000 rpm for 5 min at 4 °C. Viral RNA was extracted from the supernatant using QIAamp Viral RNA minikit (QIAGEN, Inc., Valencia, CA) and stored at −20 °C until further use. Each pool was processed for the presence of ZIKV using the above-described RT-PCR protocol. To validate the test results, a positive control (RNA synthesized from a reference strain provided by InDRE) and a negative control (without template) were included in each RT-PCR run. The threshold (CT) was determined based on the positive and negative controls, with a detection limit of ≤39 cycles.
It is noteworthy that ZIKV positive results were found from the first tested sample, strongly suggesting that the vector was already carrying the virus at the very beginning of 2015 (Fig. 1b). Interestingly, despite the fact that the Mexican Epidemiological Surveillance System has implemented protocols for microcephaly surveillance, no cases have been reported in these states yet, probably due to the fact that we have not reached a considerable burden of disease as previously reported by Brasil et al. . In addition, no increase in Guillain–Barré syndrome or other child born defects linked to the ZIKV have been described.
This study highlights firstly the usefulness of retrospective analyses of available samples to establish the scope of the Zika virus epidemic, and secondly the potential need to revise and update the diagnostic algorithms of flavivirus and viral exanthems in endemic areas with state-of-the-art available technology.
We are grateful to all the staff of the Department of Virology InDRE and to the Red Nacional de Laboratorios de Salud Pública (RNLSP) for sending the samples used in this study.
José Alberto Díaz-Quiñonez designed the experimental strategies; reviewed the results, figures, and text; contributed to fruitful discussions; and wrote the manuscript. Irma López-Martínez designed the experimental strategies, reviewed the results, and contributed to fruitful discussions. Belem Torres-Longoria designed the experiments, reviewed the results, and contributed to discussions. Mauricio Vázquez-Pichardo designed and performed the experiments, and constructed the figure. Edith Cruz-Ramírez reviewed the results and contributed to discussions. José Ernesto Ramírez-González carried out sequencing of PCR fragments and DNA data analyses. Cuitláhuac Ruiz-Matus designed the experimental strategies, reviewed the results, and contributed to fruitful discussions. Pablo Kuri-Morales designed the experimental strategies, reviewed the results, and contributed to fruitful discussions.
This study was partially funded by CONACyT (Consejo Nacional de Ciencia y Tecnología, Mexico) Grant No. 210399.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest whatsoever.
This article uses human samples as part of the Mexican Epidemiological Surveillance System, according to the Mexican law (NOM-017-SSA2-2012 para la vigilancia epidemiológica).