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- Title
Molecular detection and phylogenetic analysis of Astrovirus and Noroviruses from sewage water in Diyala-Iraq.
- Authors
Hamad, Abdulqader Y.; Mohammed, Nadira S.; Hasan, Abdulrazak S. H.
- Abstract
Background: Enteric viruses (EVs) are a diverse group of human pathogens primarily transmitted feco-orally, are a major cause of diarrheal disease in both developed and developing countries. Human Astroviruses (HAVs) and human noroviruses (HNVs) are shed in high numbers by infected individuals and persist for a long time in wastewater posing a serious threat to human health globally. Objectives: Molecular detection and genotyping of HAVs and HNVs from wastewater samples collected from sewage water plants (SWPs) and draining canals (DCs) in Diyala province-Iraq. Specimens and methods: This study was conducted in Diyala province-Iraq for the period from November 2019 to October 2021. A total of 100 water samples were collected from SWPs and DCs. Immunochromatographic assay (ICA) technique (Biozek, Netherlands) was used for direct detection of Rotaviruses, Noroviruses, and Astroviruses antigens. While the Hepatitis A virus antigen was detected by CerTest BioTEC (Spain). For molecular detection, the water samples were firstly ultra-centrifuged and the EV RNAs concentration were measured. Then viral nucleic acids (NA) were extracted and submitted for real time-Polymerase Chain Reaction (RT-PCR). Positive samples with high CT were further summited for Conventional PCR (PCR-Thermofisher (U.S.A.). For sequencing, PCR products were sent for Sanger sequencing (Macrogen Corporation - Korea). The sequencing results of the targeted samples were edited, aligned, and analyzed as long as with the respective sequences in the reference database using BioEdit Sequence Alignment Editor Software Version 7.1 (DNASTAR, Madison, WI, USA). A specific comprehensive tree was constructed. The observed variants were compared with their neighbor homologous reference sequences using the NCBI-BLASTn serve. Statistical analysis was done using the Statistical Packages for Social Science - Version 27), and P value was considered significant whenever it is equal or less than 0.05. Results: Using the RT-PCR technique the detection rate of HAVs and HNVs in SWPs and DCs was 58% for each. The detection rates of HAVs and HNVs were insignificant associated with wastewater collection sites (P=0.390) as well as with source of wastewater (P=0.774). Furthermore, 82% of the wastewater samples had co-detection of more than one virus. The conventional PCR product of ORF2 gene of HAV electrophoretically yields a band of 343 bps, while that of the ORF1 and ORF2 gene of HNV GI yields a band of 329 bps and that of the GII yields a band of 446 bps. The phylogenetic tree analyses revealed that the local Iraqi NVGII strains 1,2,3,4 and 5 are belong to the genotype NVGII.4, while the local Iraqi NVGI strains 6 and 7 belong to NVGI. On the Astrovirus side, the Iraqi local isolates 1 and 2 are 100% identical to the reference strain MG970106.1 of the NCBI. Conclusion: RT-PCR assay found a high detection rates of HAVs and HNVs in wastewater samples collected from sewage water plants and draining canals in Diyala province-Iraq. Conventional PCR, sequencing and phylogenetic analyses genotyped the local Iraqi isolates of these viruses. The study suggest that documenting sewage virome using molecular methods provides information for molecular epidemiology and may be useful in developing strategies to prevent further spread of viruses.
- Subjects
DIYALA (Iraq); IRAQ; CANALS; NOROVIRUSES; SEWAGE; REVERSE transcriptase polymerase chain reaction; ENTEROVIRUSES; HEPATITIS A virus
- Publication
Journal of Pharmaceutical Negative Results, 2022, Vol 13, p339
- ISSN
0976-9234
- Publication type
Article
- DOI
10.47750/pnr.2022.13.S01.42