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      Molecular characterization of human group A rotavirus genotypes circulating in Rawalpindi, Islamabad, Pakistan during 2015-2016

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          Abstract

          Group A rotaviruses (RVA) are one of the major causes of acute gastroenteritis (AGE) in young children worldwide. Owing to lack of proper surveillance programs and health facilities, developing countries of Asia and Africa carry a disproportionately heavy share of the RVA disease burden. The aim of this hospital-based study was to investigate the circulation of RVA genotypes in Rawalpindi and Islamabad, Pakistan in 2015 and 2016, prior to the implementation of RVA vaccine. 639 faecal samples collected from children under 10 years of age hospitalized with AGE were tested for RVA antigen by ELISA. Among 171 ELISA positive samples, 143 were successfully screened for RT-PCR and sequencing. The prevalence of RVA was found to be 26.8% with the highest frequency (34.9%) found among children of age group 6–11 months. The most predominant circulating genotypes were G3P[8] (22.4%) followed by G12P[6] (20.3%), G2P[4] (12.6%), G1P[8] (11.9%), G9P[6] (11.9%), G3P[4] (9.1%), G1P[6] (4.2%), G9P[8] (4.2%), and G3P[6] (0.7%). A single mixed genotype G1G3P[8] was also detected. The findings of this study provide baseline data, that will help to assess if future vaccination campaigns using currently available RVA vaccine will reduce RVA disease burden and instigate evolutionary changes in the overall RVA biology. The high prevalence of RVA infections in Pakistan require to improve and strengthen the surveillance and monitoring system for RVA. This will provide useful information for health authorities in planning public health care strategies to mitigate the disease burden caused by RVA.

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          Global Illness and Deaths Caused by Rotavirus Disease in Children

          To estimate the global illness and deaths caused by rotavirus disease, we reviewed studies published from 1986 to 2000 on deaths caused by diarrhea and on rotavirus infections in children. We assessed rotavirus-associated illness in three clinical settings (mild cases requiring home care alone, moderate cases requiring a clinic visit, and severe cases requiring hospitalization) and death rates in countries in different World Bank income groups. Each year, rotavirus causes approximately 111 million episodes of gastroenteritis requiring only home care, 25 million clinic visits, 2 million hospitalizations, and 352,000–592,000 deaths (median, 440,000 deaths) in children <5 years of age. By age 5, nearly every child will have an episode of rotavirus gastroenteritis, 1 in 5 will visit a clinic, 1 in 60 will be hospitalized, and approximately 1 in 293 will die. Children in the poorest countries account for 82% of rotavirus deaths. The tremendous incidence of rotavirus disease underscores the urgent need for interventions, such as vaccines, to prevent childhood deaths in developing nations.
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            Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains.

            Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.
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              Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.

              The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.
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                Author and article information

                Contributors
                Role: Data curationRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – original draft
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Writing – review & editing
                Role: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – review & editing
                Role: SoftwareRole: SupervisionRole: Writing – review & editing
                Role: Formal analysis
                Role: Formal analysis
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                30 July 2019
                2019
                : 14
                : 7
                : e0220387
                Affiliations
                [1 ] Department of Biosciences, COMSATS University (CUI), Tarlai Kalan, Chak Shahzad, Islamabad, Pakistan
                [2 ] KU Leuven-University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Viral Metagenomics, Leuven, Belgium
                Defense Threat Reduction Agency, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: Laboratory of Virology, Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institute of Health, Hamilton, MT, United States of America

                Author information
                http://orcid.org/0000-0002-9924-6355
                Article
                PONE-D-18-35563
                10.1371/journal.pone.0220387
                6667158
                31361761
                5b1e27fb-f793-42bb-8e2f-8ec9ec817880
                © 2019 Sadiq et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 12 December 2018
                : 15 July 2019
                Page count
                Figures: 10, Tables: 3, Pages: 28
                Funding
                This research was funded by ASMA SADIQ, 2BM1-196, the Higher Education Comission of Pakistan (HEC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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