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      Genetic and molecular characterization of a novel reassortant H3N2 influenza virus from a sick pig in Eastern China in 2019

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          Abstract

          Swine influenza viruses (SIVs) cause clinical respiratory symptoms associated with high mortality rates among pigs. Because pigs can be a “mixing vessel” for influenza viruses, the SIV might pose a serious threat to animal and human health. In this study, an H3N2 SIV [A/swine/Zhejiang/19/2019(H3N2) (ZJ-SW19)] was isolated from a sick pig in Eastern China in 2019, and its molecular genetics were characterized. Phylogenetic analysis demonstrated the hemagglutinin (HA) and neuraminidase (NA) segments of ZJ-SW19 are highly homologous with those of H3N2 SIVs, belonging to human-like lineages; in contrast, the remaining six SIV segments (PB2, PB1, PA, NP, M, and NS) demonstrate the highest similarity with H1N1 SIVs isolated in East Asia during 2014–2020. The in vitro analysis of the virus’s growth kinetics revealed that ZJ-SW19 can replicate efficiently in various mammalian and avian cell lines (including MDCK, A549, and DF-1). The receptor-binding analysis results indicated that ZJ-SW19 can bind to human-like receptors (α-2,6-linked sialic acid) and avian-like receptors (α-2,3-linked sialic acid). Moreover, ZJ-SW19 demonstrated significant differences compared with avian- and human-origin H3N2 influenza viruses in the antigenic analysis. Finally, in the pathogenicity test, ZJ-SW19 effectively replicated in the mouse lungs with moderate virulence. Therefore, continuous circulation of novel reassortant H3N2 SIVs indicates the need for long-term, close surveillance of influenza viruses in pig herds.

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          MEGA6: Molecular Evolutionary Genetics Analysis version 6.0.

          We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
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            Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals.

            Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.
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              Universal primer set for the full-length amplification of all influenza A viruses.

              To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A virus, we need reliable, simple methods that not only characterize partial sequences but analyze the entire influenza A genome. We designed primers based on the fact that the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved between all influenza A viruses and unique for each segment. The primers designed for each segment contain influenza virus specific nucleotides at their 3'-end and non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7, and N9 subtypes, the segment specific sequences of the neuraminidase genes are different. Therefore, we optimized the primer design to allow the amplification of those neuraminidase genes as well. The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.
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                Author and article information

                Contributors
                wuhaibo@zju.edu.cn
                Journal
                Vet Res
                Vet Res
                Veterinary Research
                BioMed Central (London )
                0928-4249
                1297-9716
                10 February 2025
                10 February 2025
                2025
                : 56
                : 38
                Affiliations
                [1 ]State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, and National Clinical Research Center for Infectious Diseases, School of Medicine, the First Affiliated Hospital, Zhejiang University, ( https://ror.org/05m1p5x56) Hangzhou, 310003 China
                [2 ]Animal Husbandry and Veterinary Institute, Zhejiang Academy of Agricultural Science, ( https://ror.org/02qbc3192) Hangzhou, 310021 China
                Author notes

                Handling editor: Marie Galloux.

                Author information
                http://orcid.org/0000-0001-5550-5186
                Article
                1462
                10.1186/s13567-025-01462-7
                11808988
                76251871-82f5-4199-a7a6-4df7ce975e51
                © The Author(s) 2025

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 5 September 2024
                : 12 November 2024
                Funding
                Funded by: National Science Foundation of the People’s Republic of China
                Award ID: 32273092
                Award Recipient :
                Funded by: Zhejiang Provincial Natural Science Foundation of China
                Award ID: LY24H190001
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100012226, Fundamental Research Funds for the Central Universities;
                Award ID: 2022ZFJH003
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © L’Institut National de Recherche en Agriculture, Alimentation et Environnement (INRAE) 2025

                Veterinary medicine
                swine influenza virus,h3n2,reassortant,eastern china
                Veterinary medicine
                swine influenza virus, h3n2, reassortant, eastern china

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