W Rutvisuttinunt1, P Chinnawirotpisan2, B Thaisomboonsuk3, P Rodpradit4, C Ajariyakhajorn5, W Manasatienkij6, S Simasathien7, S K Shrestha8, I K Yoon9, C Klungthong10, S Fernandez11. 1. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: Wiriyar@afrims.org. 2. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: PiyawanC@afrims.org. 3. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: ButsayaT@afrims.org. 4. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: PrinyadaR@afrims.org. 5. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: ChuanpisA@afrims.org. 6. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: WudtichaiM@afrims.org. 7. Phramongkutklao Hospital, Bangkok, Thailand. Electronic address: ssriluck@hotmail.com. 8. Walter Reed/ AFRIMS Research Unit Nepal, Kathmandu, Nepal. Electronic address: ShresthaSK@afrims.org. 9. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: YoonI@afrims.org. 10. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: ChontichaK@afrims.org. 11. Department of Virology, Armed Forces Research Institute of Medical Sciences (AFRIMS), 315/6 Rajavithi Road, 10400 Bangkok, Thailand. Electronic address: Fernandezs@afrims.org.
Abstract
BACKGROUND: Influenza virus (IFV) isolates obtained from mammalian cell cultures are valuable reagents used for vaccine production, antigenic characterization, laboratory assays, and epidemiological and evolutionary studies. Complete genomic comparison of IFV isolates with their original clinical specimens provides insight into cell culture-driven genomic changes which may sequentially alter the virus phenotype. OBJECTIVES: The genome of the viral isolates and of the viruses in the clinical specimens was examined by deep sequencing in order to determine nucleotide heterogeneity (measured number of variances or numbers of mixed bases) as a marker for IFV population diversity. STUDY DESIGN: Clinical respiratory specimens were collected between July and October 2012 and identified by RT-PCR as positive for influenza A H3N2 or H1N1, or influenza B. The viruses in the clinical specimens were amplified using mammalian cell culture. Next generation sequencing (NGS) was used to investigate genomic differences between IFV isolates and their corresponding clinical specimens. RESULTS: There was less nucleotide heterogeneity in 5 of 6 viral isolates compared to the corresponding clinical specimens, especially for influenza B. A phylogenetic analysis of the hemagglutinin (HA) gene consensus sequences obtained from deep and Sanger sequencing showed that the viral isolates and their corresponding clinical specimens contained the same IFV strains with less than 5% pair-wise genetic distance. CONCLUSION: The IFV sequence data analysis detected a substantial decrease in nucleotide heterogeneity from clinical specimens to viral cultures in 5 out of 6 investigated cases.
BACKGROUND:Influenza virus (IFV) isolates obtained from mammalian cell cultures are valuable reagents used for vaccine production, antigenic characterization, laboratory assays, and epidemiological and evolutionary studies. Complete genomic comparison of IFV isolates with their original clinical specimens provides insight into cell culture-driven genomic changes which may sequentially alter the virus phenotype. OBJECTIVES: The genome of the viral isolates and of the viruses in the clinical specimens was examined by deep sequencing in order to determine nucleotide heterogeneity (measured number of variances or numbers of mixed bases) as a marker for IFV population diversity. STUDY DESIGN: Clinical respiratory specimens were collected between July and October 2012 and identified by RT-PCR as positive for influenzaA H3N2 or H1N1, or influenza B. The viruses in the clinical specimens were amplified using mammalian cell culture. Next generation sequencing (NGS) was used to investigate genomic differences between IFV isolates and their corresponding clinical specimens. RESULTS: There was less nucleotide heterogeneity in 5 of 6 viral isolates compared to the corresponding clinical specimens, especially for influenza B. A phylogenetic analysis of the hemagglutinin (HA) gene consensus sequences obtained from deep and Sanger sequencing showed that the viral isolates and their corresponding clinical specimens contained the same IFV strains with less than 5% pair-wise genetic distance. CONCLUSION: The IFV sequence data analysis detected a substantial decrease in nucleotide heterogeneity from clinical specimens to viral cultures in 5 out of 6 investigated cases.
Authors: Wiriya Rutvisuttinunt; Chonticha Klungthong; Butsaya Thaisomboonsuk; Piyawan Chinnawirotpisan; Chuanpis Ajariyakhajorn; Wudtichai Manasatienkij; Thipwipha Phonpakobsin; Chanthap Lon; David Saunders; Sonam Wangchuk; Sanjaya K Shrestha; John Mark S Velasco; Maria Theresa P Alera; Sriluck Simasathien; Darunee Buddhari; Richard G Jarman; Louis R Macareo; In-Kyu Yoon; Stefan Fernandez Journal: J Clin Virol Date: 2017-07-14 Impact factor: 3.168
Authors: Wan Beom Park; Nak Jung Kwon; Su Jin Choi; Chang Kyung Kang; Pyoeng Gyun Choe; Jin Yong Kim; Jiyoung Yun; Gir Won Lee; Moon Woo Seong; Nam Joong Kim; Jeong Sun Seo; Myoung Don Oh Journal: J Korean Med Sci Date: 2020-02-24 Impact factor: 2.153