Detection and phylogenetic characterization of arbovirus dual-infections among persons during a chikungunya fever outbreak, Haiti 2014

Abstract

In the context of recent arbovirus epidemics, questions about the frequency of simultaneous infection of patients with different arbovirus species have been raised. In 2014, a major Chikungunya virus (CHIKV) epidemic impacted the Caribbean and South America. As part of ongoing screening of schoolchildren presenting with acute undifferentiated febrile illness in rural Haiti, we used RT-PCR to identify CHIKV infections in 82 of 100 children with this diagnosis during May-August 2014. Among these, eight were infected with a second arbovirus: six with Zika virus (ZIKV), one with Dengue virus serotype 2, and one with Mayaro virus (MAYV). These dual infections were only detected following culture of the specimen, suggesting low viral loads of the co-infecting species. Phylogenetic analyses indicated that the ZIKV and MAYV strains differ from those detected later in 2014 and 2015, respectively. Moreover, CHIKV and ZIKV strains from co-infected patients clustered monophyletically in their respective phylogeny, and clock calibration traced back the common ancestor of each clade to an overlapping timeframe of introduction of these arboviruses onto the island.

Fig 1. Chikungunya virus-induced CPE in Vero E6 cells with filtered plasma specimens.

(A) Mock-infected Vero E6 cells, and (B) and (C), CHIKV-induced CPE in Vero E6 cells inoculated with CHIKV-positive plasma samples. All images at 200X original magnification.

Fig 2. Mixed CPE in Vero E6 cells inoculated with filtered plasma specimens.

Panels (A) and (B), mock-infected Vero E6 cells. Panels (C) and (D), Vero E6 cells inoculated with virus-containing plasma. Original image magnifications are indicated beneath the panel identification letters.

Fig 3. Time-scaled Bayesian phylogenetic maximum clade credibility (MCC) trees of arbovirus strains.

MCC trees for (A) CHIKV, (B) ZIKV, (C) DENV-2 and (D) MAYV were inferred from full genome sequences using BEAST v1.8.4. Gray arrows indicate strains from CHIKV and ZIKV co-infected patients, orange from CHIKV and DENV-2, black from CHIKV and MAYV (sequence for CHIKV strain from the co-infected patient was not determined). Letters next to arrows indicate ID of co-infected patients. Tips are colored by sampling location as indicated in the legends to the left of each tree; diamonds at each node indicate strong statistical support along branches defined as posterior probability > 0.9. For MAYV (D), insert box shows relationship of recent Haitian and Brazilian strains.