Molecular Epidemiology of Emerging Arboviruses in Pakistan

Abstract

In Pakistan, the burden of arboviruses was largely unknown, as no formal surveillance system was in place. Three types of arboviruses studied are dengue, chikungunya, and Zika viruses. Dengue fever (DENV) has been prevalent in Pakistan from years, and chikungunya virus (CHIKV) recently (2016) emerged in Pakistan. Zika virus has recently drawn the interest of community. Since the Arboviruses share similar clinical presentation, so the differential diagnosis may be difficult. This study objective was to describe the molecular epidemiology of emerging arboviruses, viral genotypes of dengue viruses and phylogenetic relationships of Chikungunya viruses in Pakistan. Blood samples were collected from suspected Arboviruses; clinical and epidemiological characteristics like onset of fever >38.5°C for 7 days and with one or more symptoms such as nausea, vomiting, diarrhea, eye pain, hemorrhagic features, rashes, joint pain, joint swelling, arthritis. Samples were collected from different tertiary care hospitals and clinics in numerous geographic regions in the country across Pakistan. A predesigned questionnaire (patient medical record) Performa including epidemiological information (demographic, clinical characteristics, travel history and risk factors associated with mosquito bites) was filled with sample collection. During 2017-2019, A total of 2612 samples were processed at Virology Department, National Institute of Health for Arboviruses were screened for three viruses i.e., Dengue virus, Chikungunya virus, Zika virus. Out of which 1365 (52%) were found positive by real-time PCR: Among them 774 (56.7%) were found positive for CHIKV, whereas DENV was detected in 591 (43.2%) cases. Out of analyzed data DENV-2 was the dominant serotype 425 (71%) followed by DENV-1: 166 (28%) and DENV-3: 7 (1%). No sample came positive for Zika virus out of processed samples. Sample positivity for CHIKV and DENV ranged from 0.5% in 2016, 65.7% in 2017, 0.5% in 2018 and 33.5% in 2019 respectively. CHIKV and DENV cases were identified, with approximately half of the males (764, 56%), falling into the 15-49 years age group (529, 49%) followed by 5-14 years olds (171, 16.0%). Geographic distribution of 774 CHIKV positive cases was observed across country: Sindh: 472 (61%); Khyber Pakhtunkhwa (KPK): 188 (24%), Punjab: 54 (7%), Baluchistan: 32 (4%) and Federal capital: 28 (4%). Out of 591 DENV Sindh: 26 (4%); Khyber Pakhtunkhwa (KPK): 171 (29%), Punjab: 284 (48%), Baluchistan: 35 (6%) and Federal capital: 75 (13%). Clinical Characteristics of chikungunya positive patients showed children ≤10 years were more often presented with rashes (42%) than the adult population. However, joint pain and joint swelling were commonly found 91% and 40% in 11-20 years of age followed by 21-30 age groups respectively. Patients with DENV1 infections were less likely to report dengue-related symptoms than patients with any other serotype, according to clinical signs and symptoms in Dengue case data. These patients were also less likely to have a temperature above 38oC (P= 0.03). Arthralgia was found to be 4.0 times more common in DENV-2 infected patients than in other serotypes. Arthralgia and other DENV related symptoms such as headache, rash, or nausea were less common in patients with DENV-3 infections. Dengue virus partial E-NS1 gene genetic characterization revealed DENV genotypes within serotypes such as DENV-2 (Cosmopolitan), DENV-3 (III) and DENV-1 (IV) found in Pakistani strains which showed close relationship at nucleotide and amino acid level with the dengue strains previously reported from Pakistan. Genetic characterization of emerging chikungunya virus discovered that the Pakistani CHIKVs strains clustered with Indian Ocean lineage that is further divided into three clades, Analysis showed that Pakistan viruses belongs to clade C2.3a which are closely related to 2016 strains of India, Bangladesh and Thailand. The phylogenetic analysis of chikungunya virus showed close genetic relation with the global strains. Our findings confirm the emerging and reemerging trend of dengue and chikungunya virus in Pakistan. The present study provides information on chikungunya virus emerged in the country for the first time: its early identification of variants of increased virulence and vector borne diseases preventive measures in the endemic areas. Thus, it is imperative to implement preventive and control strategies in high-risk areas. Our findings further underscore the strengthening of laboratory systems in provincial and at-risk regions to obtain accurate data for patient treatment and resource allocation