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Research Article Isolation of a Reassortant H1N2 Swine Flu Strain of Type (Swine-Human-Avian) and Its Genetic Variability Analysis

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Research Article Isolation of a Reassortant H1N2 Swine Flu Strain of Type (Swine-Human-Avian) and Its Genetic Variability Analysis Hindawi BioMed Research International Volume 2018, Article ID 1096079, 10 pages https://doi.org/10.1155/2018/1096079 Research Article Isolation of a Reassortant H1N2 Swine Flu Strain of Type (Swine-Human-Avian) and Its Genetic Variability Analysis Long-Bai Wang , Qiu-Yong Chen, Xue-Min Wu , Yong-Liang Che, Cheng-Yan Wang, Ru-Jing Chen, and Lun-Jiang Zhou Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fujian Animal Disease Control Technology Development Center, Fuzhou, Fujian 350013, China Correspondence should be addressed to Lun-Jiang Zhou; [email protected] Received 3 January 2018; Accepted 26 February 2018; Published 29 May 2018 Academic Editor: Jialiang Yang Copyright © 2018 Long-Bai Wang et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Weisolated an infuenza strain named A/Swine/Fujian/F1/2010 (H1N2) from a pig suspected to be infected with swine fu. Te results of electron microscopy, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, and whole genome sequencing analysis suggest that it was a reassortant virus of swine (H1N1 subtype), human (H3N2 subtype), and avian infuenza viruses. To further study the genetic evolution of A/Swine/Fujian/F1/2010 (H1N2), we cloned its whole genome fragments using RT-PCR and performed phylogenetic analysis on the eight genes. As a result, the nucleotide sequences of HA, NA, PB1, PA, PB2, NP, M, and NS gene are similar to those of A/Swine/Shanghai/1/2007(H1N2) with identity of 98.9%, 98.9%, 99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, respectively. Similar to A/Swine/Shanghai/1/2007(H1N2), we inferred that the HA, NP, M, and NS gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were derived from classical swine infuenza H3N2 subtype, NA and PB1 were derived from human swine infuenza H3N2 subtype, and PB2 and PA genes were derived from avian infuenza virus. Tis further validates the role of swine as a “mixer” for infuenza viruses. 1. Introduction isolatedintheUnitedStatesin1998isan“avian-swine- human” reassortant strain [6]. In addition, the pandemic Swine infuenza is an acute and highly contagious fu caused H1N1 infuenza virus originated in April 2009 is a “swine- by swine infuenza virus (SIV), which infects human and pigs. human-avian” reassortant virus [7]. In fact, two out of Its clinical symptoms in swine include high fever, decreased the four most serious infuenza pandemics in the history appetite, lethargy, sneezing, coughing, and difculty in breath are caused by reassortment of gene segments from avian [1]. In virus classifcation, swine infuenza viruses are single- infuenza viruses [8, 9]. stranded negative-sense RNA viruses belonging to the family At present, there are three subtypes of swine infuenza Orthomyxoviridae. Te genome of swine infuenza virus is in the world, namely, H1N1, H3N2, and H1N2, which can composed of 8 gene fragments with diferent sizes, namely, be further divided into classic swine H1N1, avian H1N1, PB2, PB1, PA, HA, NP, NA, M, and NS, respectively [2]. human-like H3N2, reassortant H3N2, and reassortant H1N2 Te 8 genomic segments of diferent infuenza viruses can [10, 11]. In addition to these three subtypes, strains of subtypes be randomly shufed to generate new reassortant viruses H5N1,H9N2,andH3N1havealsobeenisolatedinswine [3]. As pigs possess both NeuAc-2,3Gal and NeuAc-2,6Gal populations; however they are not prevalent enough in swine receptors, they can infect both human and avian infuenza populations to establish stable lineages [12–14]. Te H3N2 viruses[3].Asaresult,pigsbecomea“mixer”forreassort- swine infuenza subtype was frst reported in Taiwan in 1969, ment of infuenza viruses and an “incubator” for emergence andtheH3N2swineinfuenzaviruseswereisolatedfrom of new infuenza strains [4, 5]. Pigs also play an important pigs in Hong Kong in 1970 [15]. Afer that, they have become role in the ecological distribution and genetic evolution of widespread in swine worldwide. H1N2 infuenza subtype infuenza viruses. For example, the Sydney-like H3N2 mutant was frst isolated from Japanese swine herds in 1978 and 2 BioMed Research International subsequently widespread in other countries such as France, were abandoned, whereas those died between 24 and 96 ∘ UK, and USA. Te H1N2 swine infuenza viruses are diverse hours were stored overnight at 4 C. Allantoic fuid was in gene origin, which results in their diferent levels of collected and the hemagglutination activity was detected. If prevalence and danger. In China, H1N2 swine infuenza virus the hemagglutination result is positive, we preserved chicken wasfrstisolatedinZhejiangin2004andthenisolatedin embryo allantoic fuid; otherwise the allantoic fuid were Guangxi, Shanghai, Guangdong, and other places from 2005 blindly passed for three generations and discarded if the result to 2010. was still negative. From 2009 to 2013, our laboratory carried out epidemi- ological study of swine fu in Fujian Province. An infuenza 2.3. Virus Identifcation strain was isolated from 13 pigs suspected to be infected. We then performed electron microscopy, hemagglutination (HA) Swine Infuenza RT-PCR. A pair of primers was designed assay, hemagglutination inhibition (HI) assay, and whole according to the M gene conserved region of swine fu reg- � genome sequencing analysis to infer its biological character- istered in GenBank, i.e., SIV-F: 5 -CAAGACCAATCCTGT- � � istics and evolutionary history. As a result, we confrmed that CACCTC-3 ,andSIV-R:5-AAGACGATCAAGAATCCAC � the virus was a “swine-human-avian” reassortant virus. AA-3 . It was then amplifed into a fragment of 684 bp by synthetic company Dalian Biological Engineering Technol- 2. Materials and Methods ogy. Te viral RNA was extracted by conventional methods. Te 10.0 �L RT reaction system consists of DEPC water (1.25 � × � � 2.1. Materials L), 5 AMV Bufer (2.0 L), 25 mg MgCl2 (1.0 L), dNTP mixture (1.0 �L), RNase inhibitor (0.25 �L) of AMV (0.5 �L), Materials and Reagents. Te lungs and lymph nodes used random primer (1.0 �L), and RNA sample template (3.0 �l). ∘ ∘ in the experiment were collected from a large-scale swine Te RT reaction conditions were 30 Cfor10min,42Cfor ∘ farm suspected to be infected with swine infuenza in Fujian 1h,and99 Cfor5min.Te25.0�LPCRreactionsystem Province, China. EDS-76 and ND were positive sera provided consists of Nuclease-Free Water (10.0 �L), GoTaq Green by Fujian Institute of Animal Husbandry and Veterinary Master Mix (10.0 �L), upstream primer (0.5 �L), downstream Medicine; the standard antigens and its antisera of H1, H3, primer (0.5 �L), and RT reaction product (4.0 �L). Te PCR ∘ ∘ H5, H7, and H9 subtypes and antisera of N1, N2, and N9 reaction conditions were 95 Cfor5min,94Cfor1min, ∘ ∘ ∘ subtypes were purchased from Harbin Veterinary Research 54 Cfor1min,72C for 1 min with 35 cycles, and then 72 C ∘ Institute; RNA extraction kit Trizol, plasmid extraction kit, for 10 min and 4 C for preservation. Afer the reaction, LATaq DNA polymerase, RNasin Inhibition, M-MLV, dNTP, the PCR products were electrophoresed on 1% agarose gel, BamHI, HindIII, and PMD18-T vector were purchased from and the positive product was sequenced by Bioengineering Dalian Bao Biological Company; Escherichia coli engineering (Shanghai) Co., Ltd. strain DH5a was preserved by our laboratory. Electron Microscopy. Afer allantoic fuid was centrifuged by Test Animals. BALB/c mice were collected from Fujian diferential and density gradient method, the morphology Medical Laboratory Animal Center; 5-week-old piglets were and size of the virus were observed by transmission electron purchased from a large-scale pig farm in Fuzhou, which were microscopy. confrmed to be negative in serological antibody examina- tion; SPF eggs were purchased from Fuzhou Dabei Agricul- Agar Difusion Test. Te isolated chicken embryo allantoic tural Biotechnology Co. and incubated to 9 to 11 days of age. fuid was purifed, which can be used as antigen afer adding SDS. Afer that, it was under agar difusion test with standard serum. Te results were collected at 24 h, 48 h, and 72 h, 2.2. Virus Isolation respectively. Acquisition and Processing of Disease Materials.Telungand lymphnodetissuesofpigssuspectedtobeinfectedwithswine 2.4. Identifcation of Serum Subtypes. Te HA subtype of infuenza were cut into pieces and ground into homogenate swine infuenza virus was identifed by comparing the iso- under sterile conditions. Hank’s solution was added to it to lated virus with the standard positive sera of swine infuenza make 10% suspension solution, which was then undergone by hemagglutination assay (HA) and hemagglutination inhi- frozen and thawed for three times, centrifuged at 12000 rpm bition assay (HI). Neuraminidase inhibition assay (NI) was for 10 min. Te supernatant was fltered and sterilized, and used to identify NA subtypes. ∘ placed at −70 C freezer for further use. Hemagglutination Assay (HA). We added 50 �lofvirus Virus Isolation and Culture.Tesupernatantwasinoculated antigeninthefrstwellofeachrowandusedapipetteto into allantoic cavity of 9 to 11-day-old SPF chicken embryos mix well; we then added 50 �l of the mixed solution to the with 0.1 mL/embryo, and each sample was inoculated into second well, diluted consecutively until the 11th well. Te 12th 4 embryos. Te inoculated embryos were incubated in an hole without antigen was taken as a control. Afer that, we ∘ incubator at 37 Candarelativehumidityof55%.Teeggs added 50 �l 1% pigeon erythrocyte suspension to each well, were candled twice a day to inspect the vitality of chicken oscillated using microoscillator for 1 min and recorded the embryos for 6 days. Te embryos died within 24 hours results afer 20 ∼ 30minquiescenceinroomtemperature. BioMed Research International 3 Hemagglutination Inhibition Assay (HI). According to the normal saline. Te body temperature of all piglets was hemagglutination test, we made 8-unit and 4-unit antigen by measured every day afer inoculation, and the diet and mental adding physiological saline.
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