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. We then added 50 �l8-unitanti- status of piglets were observed continuously for 28 days. gen into the frst well and 50 �l 4-unit antigen into the 2nd, 3rd, ..., and 11th well and 50 �lnormalsalinetothe12thwell 2.8. Whole Gene Sequencing as negative control. Afer that, we took 50 �lstandardpositive � serum to the frst well, mixed them even, and took 50 lto Primer Synthesis. According to the H1N2 subtype of swine the second well. Te process was repeated until the 11th well. infuenza virus in GenBank, 12 pairs of primers containing � Afer 15 min at room temperature, we added 50 l1%pigeon 8 gene fragments were designed by oligo 6.0 sofware. Te erythrocyte suspension, oscillated using microoscillator for 1 primers were available upon request. min and recorded the results afer 20 min quiescence in room temperature. Te hemagglutination inhibition titer of the Virus RNA Extraction and RT-PCR Reaction.TotalRNAwas serum was the maximum dilution of sera with 50% inhibition extracted from the viral RNA extraction kit Trizol. Te RT of erythrocyte agglutination. Hemagglutination inhibition reaction conditions were DEPC water (1.25 �L), MgCl2 (1.0 titer ≥ 4log3 serum was determined as positive. �L), 5 × MMLV Bufer (2.0 �L), dNTP Mixture (1.2 �L), MMLV (0.5 �L), RNase Inhibition (0.25 �L), Uni-12 primer ∘ Neuraminidase Inhibition Assay (NI). Te allantoic fuid of the (1.0 �L), RNA (3.0 �L); the reaction conditions were 30 Cfor ∘ ∘ virus was tested against the standard antiserum of three NA 10 min, 42 Cfor60min,and99Cfor5minandpreserved ∘ subtypes. at 4 C. Te PCR reaction conditions were 10× PCR Bufer (2.0 �L), dNTP Mixture (1.2 �L), DNA polymerase (0.2 �L), 2.5. Half of the Chicken Embryo Infection Determination. Te Up Primer (0.4 �L), Down Primer (0.4 �L), and template virus liquid of each dilution was inoculated into 9-day-old (2.0 �L) and flled to 20 �L by adding H2O. Te reaction ∘ SPF chicken embryos with 4 pieces/group and 0.1 mL/piece. conditions were as follows: predenaturation at 95 Cfor5min, ∘ ∘ ∘ Te embryos were then sealed with wax and cultured at 37 C denaturation at 94 C for 1 min, annealing at 53.5-61.0 Cfor1 ∘ ∘ for 7 days. Te embryos which died within 24 hours were min, extension at 72 C for 1 min, and extension at 72 Cfor abandoned and allantoic fuid from embryos which died afer 10 min. Te PCR products were identifed by 1.0% agarose gel 24 hours were collected for hemagglutination assay and the electrophoresis EID50 was calculated by Karber’s method. Gene Cloning and PCR Identifcation.TePCRproductwas 2.6. Identifcation of Viral Physical and Chemical Characteris- ligated into PMD18-T vector and transferred into DH5� tics. Virus allantoic fuid was centrifuged at 3000 rpm for 15 strain. Te recombinant plasmid was identifed by 1.0% min, and the supernatant was distributed into several tubes agarose gel electrophoresis, and the positive plasmid was sent for further use. We then added SDS (1 mg/mL, pH 8.2 for ∘ to Bioengineering (Shanghai) Co., Ltd., for sequencing. 1 h), chloroform (fnal concentration 48 mL/L at 4 Cfor10 ∘ min), ether (fnal concentration 200 mL/L at 4 Cfor24h), 2.9. Comparison of Gene Sequence Analysis. Te sequences hydrochloric acid (fnal concentration 1 mL/L for 3 min), wereputintoGenBankforhomologysearchusingBLAST, 1% potassium permanganate (for 5 min), formaldehyde (fnal and the sequences of subtypes human infuenza H3N2, concentration 0.2%), and 75% ethanol (for 5 min) and treated ∘ swine fu H3N2, classical swine fu H1N1, and representative them with water at 56 C for 0,5, 10,15, 20, 30, 60, 90, 120, and swinefuH1N2weredownloadedandanalyzedbyDNAStar 180 mins overnight. Te treated virus was inoculated into 9- sofware. Te sequence alignments were done by MUS- day-old SPF chicken embryo with 0.1 mL/embryo. Allantoic CLE sofware in MEGA6 (http://www.megasofware.net/). fuid or the embryo died in 24 ∼ 96 h were collected for HA Te phylogenetic trees were generated using the Neighbor- assay. Joining method with 1000 bootstrap replicates.

2.7. Animal Vaccination Test 3. Results BALB/cMiceInfectionAssay. Te virus with the dose 2× 5 3.1. Virus Isolation. We collected allantoic fuid from inocu- 10 EID50 was inoculated intranasally into ten 6-week-old lated SPF chick embryos died between 24 to 72 hours, whose BALB/c mice and physiological saline was inoculated into 10 5 mice as controls. One BALB/c mouse was dissected on the 1st, hemagglutination titer was determined to be 2 .Byautopsy, 3rd, 5th, 7th, 9th, 11th, and 13th days afer inoculation. Te we found that there are serious embryo hemorrhage lesions. lungs were harvested for virus isolation. Te mental and diet statusofthemicewerealsoobservedfor2weeks. 3.2. Virus Identifcation. RT-PCR amplifcation products were detected by agarose gel electrophoresis (Figure 1). As can Infection Assay on Piglets. Twelve 5-week-old piglets, which be seen, the amplifed fragments are of sizes about 684 bp as were tested negative for swine infuenza virus, were divided expected. Sequence analysis showed that the gene fragment into experiment and control group evenly. Piglets in exper- was derived from swine fu. By visualizing allantoic fuid 6 iment were inoculated intranasally with 2×10EID50 dose afer centrifugation using electron microscope, one can fnd virusesandthoseincontrolgroupwereinoculatedwith infuenza virus particles of diameter 80 ∼ 120 nm. Tey are 4 BioMed Research International

12345 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

2000 bp 1000 bp 2000 750 bp 500 bp 1000 250 bp 100 bp 750 500 250 Figure 3: RT-PCR electrophoresis of A/Swine/Fujian/F1/10 (H1N2) 100 strain.M:marker2000;1and2:PB2;3and4:PB1;5and6:PA;7and 8:NP;9:NS;10:M;11and12:HA;13:NA;14:negativecontrol. Figure1:RT-PCRresultsofMgene.1:marker2000;2,3,and4:M; 5: negative control.

3.3. Animal Vaccination. We frst performed BALB/c mouse infection assay. Te mice in the experiment group showed symptoms of mental exhaustion, coarse coat, and dyspnea symptoms 3 days afer inoculation, but no mice died. Te control group was normal. Infuenza virus was isolated from the lungs of mice from 1 to 5 days afer inoculation, and no virus could be isolated afer 7 days afer inoculation. We then infected virus with 6-weed-old piglets. Similar to mice, piglets in experiment group developed symptoms of high body temperature, loss of appetite, cough, and runny nose afer 4 days of inoculation, and the symptoms were relieved afer 7 days until all the piglets were recovery. No piglets died in the process. In contrast, piglets in control group were normal.

Figure 2: Electron specular photograph of allantoic fuid afer 3.4. Whole Genome Sequencing and Genetic Variation Anal- centrifugation. ysis. Te PB2, PB1, PA, HA, NP, NA, M, and NS genes of A/Swine/Fujian/F1/2010 (H1N2) strain were amplifed by RT-PCR, respectively. Afer sequencing, we obtained 2341 bp, 2302 bp, 2190 bp, 1709 bp, 1543 bp, 1443 bp, 991 bp, in spherical, horseshoe-shaped, and flamentous enveloped and 882 bp gene fragments (Figure 3), which were uploaded shapes (see Figure 2). to NCBI with accession numbers KM186312, KM186311, Tere are white precipitate line identifed in the agar KM186310, KM186305, KM186308, KM186307, KM186306, difusion assay between tested antigen and standard infuenza and KM186309, respectively. A serum, indicating that the isolated virus was an infuenza We then performed nucleotide homology and genetic A virus. We then performed HI assay between allantoic phylogenetic analysis using BLAST (https://blast.ncbi.nlm fuid and standard positive sera of EDS-76 and ND. Te .nih.gov/Blast.cgi), DNAStar (https://www.dnastar.com/), results showed that the isolated strain cannot be inhibited and MEGA6 using Neighbor-Joining method with 1000 by both EDS-76 and ND, indicating that it is not an EDS bootstrap replicates. We used K80 model to estimate the orNDVvirus.WealsoperformedHAandHIassayagainst distances between nucleotides. Specifcally, the eight gene the standard serum of H1 subtype and showed that it can fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were be inhibited by H1 serum with HI value 8 log2, suggesting compared with human infuenza, avian infuenza, and swine that the hemagglutinin of the virus is of H1 subtype. In infuenza virus deposited in NCBI fu database. addition, the NI assay suggested that the neuraminidase of Te nucleotide sequences of HA, NA, PB1, PA, PB2, the virus is of subtype N2. As a result, we named the virus NP, M, and NS genes all had the highest homology with A/Swine/Fujian/F1/2010 (H1N2). Finally, the EID50 value of A/Swine/Shanghai/1/2007 (H1N2) with identity 98.9%, the fourth-generation allantoic fuid isolated from culture 98.9%,99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, −4.6 was 10 /0.1 mL. Te virus lacks resistance to SDS, chloro- respectively. According to the phylogenetic tree, the form, ether, hydrochloric acid, 1% potassium permanganate, HA,NA,PB1,PA,PB2,NP,andMgenefragmentsof formaldehyde, and 75% ethanol and can easily be inactivated. A/Swine/Fujian/F1/2010 (H1N2) is genetically close to those ∘ Te titer of hemagglutination decreased by 2 titers at 56 Cfor of A/Swine/Shanghai/1/2007(H1N2), while its NS fragment 5 min. According to the method of determining the thermal is close to A/Swine/Guangxi/13/2006 (H1N2). We illustrated stability, it can be determined that the virus strain is heat- thephylogenetictreesofHA,NA,PB1,PA,andPB2in labile. Figures 4–8, respectively. BioMed Research International 5

34 A swine Iowa 17672 1988(H1N1) 44 A swine Wisconsin 1915 1988(H1N1) 94 A swine Kansas 3228 1987(H1N1) A Maryland 12 1991(H1N1) 98 100 A MD 12 1991(H1N1) A turkey IA 21089-3 1992(H1N1)

84 A swine California T9001707 1991(H1N1) 60 58 A swine Memphis 1 1990(H1N1) 90 A Swine Korea CY02 02(H1N2) 91 A swine Minnesota 55551 00 (H1N2) A swine North Carolina 98225 01(H1N2) A swine Guangxi 13 2006(H1N2) 68 100 100 A swine Hong Kong NS30 2004(H1N2) 100 A swine Indiana 9K035 99 (H1N2)

91 A swine Illinois 100084 01 (H1N2) 59 A swine Ohio 891 01(H1N2) A swine Guangdong 1 2010(H1N2)) A swine Shanghai 1 2007(H1N2) 100 50 A swine Fujian F1 2010(H1N2) 43 A swine Hubei 04 2009(H1N2)

80 A Florida 2 1993(H1N1) A New York 656 1995(H1N1) 100 A Finland 75 1988(H1N1)

70 A Taiwan 1132 1991(H1N1) 83 A Texas 36 91(H1N1)

100 A chicken Hong Kong 14 1976(H1N1) A duck Miyagi 66 1977(H1N1)

100 A swine England 101692 1997(H1N1) A turkey Germany 3 91(H1N1) 99 A swine France 3614 1984(H1N1) 69 53 A swine Belgium WVL2 1983(H1N1) 85 A swine Germany 2 1981(H1N1)

0.05 Figure 4: Phylogenetic tree based on HA nucleotide sequence.

3.5. Analysis on the Sources of Gene Fragments. Te analy- 4. Discussion sis of 8 gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain showed that the HA, NP, M, and NS gene fragments Swine as a host of infuenza virus can simultaneously infect were originated from classical pig infuenza H3N2 subtype, diferent types and subtypes of infuenza viruses. Terefore, NA and PB1 were derived from human swine infuenza most scholars believe that swine plays an important role in H3N2 subtype, and PB2 and PA gene were from avian infuenza pandemic as a “mixer” of virus reassortment [2]. infuenza virus, as shown in Figure 9. Te results indi- When pigs infect both human and avian infuenza viruses cated that the strain was a “swine-human-avian” reassortant atthesametime,newstrainsmaybeproduceddueto virus. gene reassortment, causing epidemics. Te southern part 6 BioMed Research International

99 A swine Hubei 04 2009(H1N2) 99 A swine Shanghai 1 2007(H1N2) 74 A swine Fujian F1 2010(H1N2) 99 A swine Guangxi 13 2006(H1N2) 54 A swine Hong Kong NS30 2004(H1N2)

82 A Turkey MO 24093 99(H1N2) 100 A Turkey MO 24093 99(H1N2)(2) 26 A swine Korea CY05 2007(H3N2) A Swine North Carolina 98225 01(H1N2)

3848 A Swine Indiana 9K035 99 (H1N2) 95 A Swine Minnesota 55551 00 (H1N2) A Swine Minnesota 593 99(H3N2) 40 92 A Swine Nebraska 209 98(H3N2)

93 A mallard South Dakota Sg-00127 2007(H3N2) A Swine Texas 4199-2 98 (H3N2) 100 85 A Swine Korea CY02 02(H1N2)

99 A Swine Illinois 100084 01 (H1N2) 37 98 A Swine Ohio 891 01(H1N2) A Swine Oklahoma 18717 99(H3N2) 100 A New York 570 1996(H3N2

75 A Buenos Aires 4459 96(H3N2) 53 A New York 516 1997(H3N2) 100 A Miyagi 29 95(H3N2) 96 A Siena 3 1995(H3N2) A Nanchang 12 93(H3N2) A duck Hong Kong 319 1978(H2N2) A duck Hong Kong 24 1976(H3N2)

63 A duck Hong Kong 245 1977(H3N2)) 99 A Duck Hong Kong 289 78(H9N2)

0.02 Figure 5: Phylogenetic tree based on NA nucleotide sequence.

of China is considered to be the source of the world’s fu viruses; and PB2 and PA were originated from H9N2 avian outbreak due to its unique ecological, geographical, and infuenza viruses. In 2007, Yao et al. reported a two-source climatic conditions. Fujian locates in southern China, next to H3N2 reassortant swine infuenza virus [16]. Te M and Guangdong, one of the most frequent infuenza out-breaking NS fragments of the strain were derived from the H1N1 places in the world. Tere are a lot of pig farms and waterfowl swine infuenza virus, whereas HA and NA were derived habitats in Fujian, greatly increasing the chance of infuenza from H3N2 human infuenza virus. In 1994, Brown et al. virus reassortment. obtained a swine-human-avian three-source recombinant Currently, there are a lot of reports about swine infuenza H1N2 swine infuenza virus [17]. Te HA gene of the strain virus reassortment all over the world. For example, the was derived from human infuenza H1N1. Te NA gene was H3N2 reassortant virus is isolated from North Carolina in derived from the porcine H3N2 subtype, while the other 1998,whoseHA,NA,andPB1fragmentsarederivedfrom six genes were originated from the H1N1 fu virus of the human infuenza H3N2 subtype virus; NP, NS, M, PB2, avian species. Karasin et al. ([6]; Karasin et al. 2002) also and PA were derived from classical swine infuenza H1N1 isolated the “swine-human-avian” triple-reassortant H1N2 BioMed Research International 7

40 A Nanchang 58 93(H3N2) 33 A Netherlands 179 1993(H3N2) 77 A New York 516 1997(H3N2) A Miyagi 29 95(H3N2) 33 89 A Siena 3 1995(H3N2) A Swine Indiana 9K035 99 (H1N2) 3340 A Swine Minnesota 55551 00 (H1N2) A Wisconsin 10 98 (H1N1)

80 A Swine Illinois 100084 01 (H1N2) 99 25 A Swine Korea CY02 02(H1N2) 33 A Swine Ohio 891 01(H1N2) 27 A Swine Texas 4199-2 98 (H3N2) A Swine Minnesota 593 99(H3N2) 7138 99 A Swine Nebraska 209 98(H3N2) A swine Korea CY05 2007(H3N2) A swine Korea CAN01 2004(H1N1) 100 A swine Minnesota SG1138 2002(H1N1)

99 A swine Guangdong 120 10(H1N1) A swine Guangxi 13 2006(H1N2)

51 A swine Hong Kong NS30 2004(H1N2) A swine Fujian F1 2010(H1N2) 58 100 A swine Hubei 04 2009(H1N2) 90 A swine Shanghai 1 2007(H1N2)

65 A swine Memphis 1 1990(H1N1) 72 A turkey IA 21089-3 1992(H1N1) A Maryland 12 1991(H1N1) 100 A swine Kansas 3228 1987(H1N1)

93 A swine Iowa 17672 1988(H1N1) 83 A swine Iowa 31483 1988(H1N1)

0.02 Figure 6: Phylogenetic tree based on PB1 nucleotide sequence.

swine infuenza virus in the United States from 1999 to 2001. (H1N2). Based on the role of pigs as intermediate hosts in Te HA, M, NP, and NS were originated from the classical the SIV interspecies transmission chain and the complexity H1N1 swine fu viruses, the NA and PB1 genes were from the of infuenza ecology in China, it is of great importance to human H3N2 infuenza viruses, and the PB2 and PA genes understand the prevalence of infuenza viruses circulating are from the avian fu viruses. in swine herds. Although EID50 of the isolated strains −4.6 In this study, a virus was isolated from the lymph nodes was 10 /0.1 mL, it could infect mice and piglets without and lungs of pigs suspected to be infected with swine killing them, of which the isolated strains were less patho- fu viruses. Our results showed that the virus had strong genic. pathogenicity to chick embryo and could kill the chicken Based on the whole genome sequencing analyses, embryo in 48 hours. Te allantoic fuid had hemagglutination A/Swine/Fujian/F1/2010 (H1N2) was a “swine-human-avian” activity. It is confrmed to be a H1N2 subtype virus by reassortant virus, highly similar to A/Swine/shanghai/1/2007 HA, HI, and NA assays, electron microscopy, and whole (H1N2), another reassortant virus isolated in Shanghai, genome sequencing and thus named A/swine/Fujian/F1/2010 China. We inferred that the HA, NP, M, and NS genes were 8 BioMed Research International

97 A swine Hubei 04 2009(H1N2) 97 A swine Shanghai 1 2007(H1N2) 78 A swine Fujian F1 2010(H1N2) 60 A swine Hong Kong NS30 2004(H1N2) A swine Guangdong 120 10(H1N1) 54 93 A swine Guangxi 13 2006(H1N2) 32 A Swine Korea CY02 02(H1N2) 27 A Wisconsin 10 98 (H1N1) A Swine Texas 4199-2 98 (H3N2)

2541 A Swine Minnesota 593 99(H3N2) 94 A Swine Nebraska 209 98(H3N2) 34 A Iowa CEID23 2005(H1N1)

100 100 A swine Korea CAN01 2004(H1N1) A Swine Indiana 9K035 99 (H1N2) 100 A Swine Ohio 891 01(H1N2) A duck Hokkaido 9 99(H9N2)

99 A Duck Germany 113 95(H9N2) 58 A duck Nanchang 1749 1992(H11N2)

96 A Miyagi 29 95(H3N2) A Siena 3 1995(H3N2) 100 A Nanchang 12 93(H3N2) 91 A Netherlands 179 1993(H3N2) A Maryland 12 1991(H1N1) A swine Memphis 1 1990(H1N1) 100 A turkey IA 21089-3 1992(H1N1) 53 A swine Iowa 17672 1988(H1N1) 33 34 A swine Iowa 31483 1988(H1N1) 41 A swine Kansas 3228 1987(H1N1)

0.02 Figure 7: Phylogenetic tree based on PA nucleotide sequence.

derived from the swine classical H1N1 infuenza virus. Te Authors’ Contributions NA and PB1 genes were derived from human H3N2 subtype infuenza viruses and PA and PB2 genes were from the avian Lun-Jiang Zhou conceived and designed the experiments. infuenza virus. Te genetic characteristics of swine infuenza Long-Bai Wang performed the experiments and analyzed the virus isolation show that swine does play an important role data. Long-Bai Wang and Lun-Jiang Zhou wrote the paper. Cheng-Yan Wang, Xue-Min Wu, Yong-Liang Che, Ru-Jing as a “mixer” during the spread of infuenza virus. Studies Chen,andLun-JiangZhoucontributedtothediscussionand on the molecular and genetic evolution of swine fu are helped to revise the paper. All authors have reviewed and of great importance for timely detection of swine fu. Te approved the fnal manuscript. surveillance of the variation of swine infuenza is critical for prevention and control of fu outbreaks. Acknowledgments Tis work was partially supported by the Public Welfare Sci- Conflicts of Interest entifc Research Projects of Fujian Province (no. 2009K10025- 5) and the Modern Agricultural Pig Industry Technology Teauthorshavedeclarednoconfictsofinterest. SystemProjectofFujianProvince(2014-2017). BioMed Research International 9

71 A Swine Minnesota 593 99(H3N2) A Swine Nebraska 209 98(H3N2) 71 A Swine Texas 4199-2 98 (H3N2) A Wisconsin 10 98 (H1N1) 51 A duck NC 91347 01(H1N2) 29 A Swine Oklahoma 18717 99(H3N2) A Swine Ohio 891 01(H1N2) 78 A Swine Korea CY02 02(H1N2) 70 62 A turkey Illinois 2004(H3N2) 72 98 A turkey Ohio 313053 2004(H3N2) A Swine Minnesota 55551 00 (H1N2) 76 A swine Korea CY05 2007(H3N2) A swine Hong Kong 1578 2003(H1N2) 49 54 A swine Hong Kong NS30 2004(H1N2) A swine Shanghai 1 2007(H1N2) 100 98 A swine Fujian F1 2010(H1N2) 65 A swine Hubei 04 2009(H1N2) A swine Guangdong 120 10(H1N1) A New York 516 1997(H3N2) A Nanchang 12 93(H3N2) 100 54 A Miyagi 29 95(H3N2) 95 A Siena 3 1995(H3N2) 74 A swine Iowa 31483 1988(H1N1) A swine Kansas 3228 1987(H1N1)

100 A turkey IA 21089-3 1992(H1N1) A Maryland 12 1991(H1N1) 96 A swine California T9001707 1991(H1N1) 90 A swine Memphis 1 1990(H1N1)

0.02 Figure 8: Phylogenetic tree based on PB2 nucleotide sequence.

References

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