High rate of subclinical chikungunya virus infection and association of neutralizing antibody with protection in a prospective cohort in the Philippines

Abstract

Background: Chikungunya virus (CHIKV) is a globally re-emerging arbovirus for which previous studies have indicated the majority of infections result in symptomatic febrile illness. We sought to characterize the proportion of subclinical and symptomatic CHIKV infections in a prospective cohort study in a country with known CHIKV circulation.

Methods/findings: A prospective longitudinal cohort of subjects ≥6 months old underwent community-based active surveillance for acute febrile illness in Cebu City, Philippines from 2012-13. Subjects with fever history were clinically evaluated at acute, 2, 5, and 8 day visits, and at a 3-week convalescent visit. Blood was collected at the acute and 3-week convalescent visits. Symptomatic CHIKV infections were identified by positive CHIKV PCR in acute blood samples and/or CHIKV IgM/IgG ELISA seroconversion in paired acute/convalescent samples. Enrollment and 12-month blood samples underwent plaque reduction neutralization test (PRNT) using CHIKV attenuated strain 181/clone25. Subclinical CHIKV infections were identified by ≥8-fold rise from a baseline enrollment PRNT titer <10 without symptomatic infection detected during the intervening surveillance period. Selected CHIKV PCR-positive samples underwent viral isolation and envelope protein-1 gene sequencing. Of 853 subjects who completed all study procedures at 12 months, 19 symptomatic infections (2.19 per 100 person-years) and 87 subclinical infections (10.03 per 100 person-years) occurred. The ratio of subclinical-to-symptomatic infections was 4.6:1 varying with age from 2:1 in 6 month-5 year olds to 12:1 in those >50 years old. Baseline CHIKV PRNT titer ≥10 was associated with 100% (95%CI: 46.1, 100.0) protection from symptomatic CHIKV infection. Phylogenetic analysis demonstrated Asian genotype closely related to strains from Asia and the Caribbean.

Conclusions: Subclinical infections accounted for a majority of total CHIKV infections. A positive baseline CHIKV PRNT titer was associated with protection from symptomatic CHIKV infection. These findings have implications for assessing disease burden, understanding virus transmission, and supporting vaccine development.

Fig 1. Study flow chart.

Fig 2. Dot plot of chikungunya virus (CHIKV) plaque reduction neutralization test (PRNT) titers at enrollment and 12 months in per-protocol subjects.

All 19 symptomatic and 87 subclinical CHIKV infections had enrollment PRNT titer <10 and 12-month titers ranging from 229 to 2,030 in symptomatic infections and 64 to 3,347 in subclinical infections; 239 subjects had positive titers at enrollment ranging from 10 to 1,785; and 505 subjects had negative titers at both enrollment and 12 months.

Fig 3. Phylogenetic tree showing five chikungunya viruses (CHIKVs) characterized in the study.

The tree was constructed by neighbor-joining methods (1,000 bootstrap replications) using envelope protein-1 (E1) nucleotide sequences (1,320 bp) of 46 CHIKV strains; the five from the study are designated in bold. Bootstrap support values are shown for major nodes. Scale bar indicates nucleotide substitutions per site. Genotypes are indicated on the right. The sequences were named according to virus/country/strain/year of collection or isolation. GenBank accession numbers are shown in parentheses.