Zika Virus looming epidemic in Pakistan: seroprevalence findings by plaque reduction neutralization test in the Sindh Region of Pakistan

Background Zika virus (ZIKV) has emerged as a cause of febrile illness in children and adults globally. West Asian and Middle Eastern countries have not yet experienced the widespread emergence of ZIKV. In Pakistan, detection of ZIKV antibodies have been reported. However, the validity of this data is questionable given the current understanding of flaviviral antigenic cross-reactivity. In order to determine if ZIKV is circulating in the Sindh region of Pakistan, patients presenting to healthcare centers with an acute febrile dengue-like illness were evaluated for ZIKV infection. Methods Dengue virus (DENV) screening in patients was performed using a commercial ELISA Rapid Test NS1 antigen capture test. All DENV negative samples were tested for Zika virus, using a commercial IgM capture ELISA kit. Additionally, a plaque reduction neutralization test (PRNT) was performed to measure neutralizing antibodies. Singleplex, two-step Real-time PCR using general primers and probes was performed for the detection of Zika virus nucleic acid. Results Patients with acute dengue-like illness (DLI) presenting at healthcare centers in different sites throughout the Sindh region of Pakistan were recruited. A total of 745 patient samples were tested for ZIKV via IgM ELISA and RT-PCR. Thirty-seven samples (4.9%) tested positive for ZIKV IgM without any cross-reactivity to other flaviviruses tested simultaneously. These were considered as presumptive positive for ZIKV, selected presumptive positive samples (n=20) were confirmed using PRNT50 using Vero cells. All 20 samples showed ZIKV neutralization at PRNT50. Conclusions Our study provides evidence that ZIKV is circulating in the Sindh region of Pakistan and is a probable cause of clinical dengue-like illness (DLI) cases that are seen seasonally in Pakistan. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the Defense Threat Reduction Agency, Basic Research Award # HDTRA1-14-1-0022, to the University of Florida ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol was approved by Ethics Review Committee, Aga Khan University, Karachi, Pakistan (#3183-PAT-ERC-14). All enrolled subjects gave written informed consent in accordance with the Declaration of Helsinki I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All the data is present within the manuscript