Detection and subtyping of swine influenza H1N1, H1N2 and H3N2 viruses in clinical samples using two

Journal of Virological Methods 102(2002)53–59Detection and subtyping of swine influenza H1N1,H1N2and H3N2viruses in clinical samples using two multiplexRT-PCR assaysY.K.Choi a ,S.M.Goyal b ,S.W.Kang c ,M.W.Farnham a ,H.S.Joo a,*aDepartment of Clinical and Population Sciences ,Uni 6ersity of Minnesota ,385Animal Sci /Vet Medicine ,1988Fitch A 6enue ,St .Paul ,MN 55108,USAbDepartment of Veterinary Diagnostic Medicine ,Uni 6ersity of Minnesota ,St .Paul ,MN 55108,USAcDepartment of Animal Science ,Uni 6ersity of Minnesota ,St .Paul ,MN 55108,USAReceived 6September 2001;received in revised form 4December 2001;accepted 4December 2001AbstractA total of 360type A swine influenza virus-positive samples including cell culture isolates,nasal swabs or lung tissues along with 30virus-negative samples were tested for the detection and subtyping of H1N1,H1N2or H3N2by two multiplex reverse transcription (RT)-PCR assays.The positive samples had been collected between 1999and 2001from pigs with respiratory diseases,and type A influenza virus was isolated and subtyped by hemagglutination inhibition (HI)test at the Minnesota Veterinary Diagnostic Laboratory (MVDL).Two multiplex RT-PCR assays specific for H1and H3,and N1and N2were developed.RT-PCR products with unique sizes characteristic of each subtype of influenza A virus were sequenced,and the sequences were demonstrated to be specific for H1N1,H1N2or H3N2.Genomic RNAs or DNAs from 12common swine pathogens other than type A influenza viruses were not amplified when the PCR assays were performed with these primer sets.Positive amplification reaction could be visualized with RNA extracted from up to 10−5dilution of each reference virus with original infectivity titer of 105TCID 50/ml.Of the 360samples tested,swine influenza virus H1N1,H1N2and H3N2were identified in 200,13and 139samples,respectively.The remaining eight samples were positive for both H1N1and H3N2viruses.The results of multiplex RT-PCR were 100%in agreement with those of virus isolation.These results demonstrate the usefulness of multiplex RT-PCR for detection and identification of influenza A virus subtypes.The results also indicate an increased occurrence of H1N2in US swine population.©2002Elsevier Science B.V.All rights reserved.Keywords :Swine influenza virus;Multiplex RT-PCR;Subtype H1N2 /locate /jviromet1.IntroductionSwine influenza is an acute respiratory disease of pigs and is characterized by high fever,lethargy,nasal discharge,coughing,dyspnea and*Corresponding author.Tel.:+1-612-625-0235;fax:+1-612-625-1210.E -mail address :jooxx001@ (H.S.Joo).0166-0934/02/$-see front matter ©2002Elsevier Science B.V.All rights reserved.PII:S 0166-0934(01)00442-6Y.K.Choi et al./Journal of Virological Methods102(2002)53–59 54weight loss.The severity of clinical illness appears to be dependent upon the infecting influenza virus strain,the immune status and age of the animal, and the presence of concomitant pathogens or environmental stress factors such as chilling and poor ventilation.Type A swine influenza viruses also cause respiratory disease in humans(Went-worth et al.,1994,1997),and zoonotic transmis-sion of swine influenza viruses from pigs to people has been well documented(Smith et al.,1976; Hinshaw et al.,1978;Dasco et al.,1984;Rota et al.,1989;Wentworth et al.,1994,1997).To date,15different hemagglutinin(H)and nine neuraminidase(N)subtypes of influenza A viruses have been identified in mammals and birds (Rohm et al.,1996).In swine,three major influ-enza A virus subtypes(H1N1,H1N2,and H3N2) have been identified in different countries,al-though genetic reassortment of other subtypes is possible.In North America,clinical disease in pigs has been associated mainly with classical H1N1 subtype(Chambers et al.,1991).However,during 1998,many swine farms in mid-western states of the US experienced severe respiratory and/or re-productive diseases attributed to the infection with a H3N2subtype.Subsequently,isolation of H3N2 subtype from clinically affected pigs has been reported from many states including Minnesota, North Carolina,Texas,Illinois,Colorado,Ne-braska,Oklahoma and Wisconsin(Zhou et al., 1999;Webby et al.,2000).In addition,an isolation of H1N2subtype has been reported from a sample collected in November1999during an outbreak of respiratory disease and abortion on an Indiana swine farm(Karasin et al.,2000).This H1N2virus was demonstrated to be a second-generation reas-sortment between H3N2and H1N1subtypes.In veterinary diagnostic laboratories,the detec-tion of type A swine influenza virus infection has been routinely carried out by virus isolation in embryonated chicken eggs or Madin–Darby ca-nine kidney(MDCK)cells with subsequent sub-type determination by hemagglutination inhib-ition(HI)and neuraminidase inhibition tests using monospecific antiserum to each subtype(Murphy and Webster,1996).Although the cell culture method is sensitive,it requires viable virus and several days for cytopathic effects to be observed.Diagnostic tests based on the detection of nucle-oprotein antigen,such as immunofluorescent staining and enzyme-linked immunosorbent as-says,have also been used(Takimoto et al.,1991). Single reverse transcription-polymerase chain re-action(RT-PCR)assays have been reported for the detection of type A influenza viruses in hu-mans(Bressoud et al.,1990;Yamada et al.,1991) and swine(Schorr et al.,1994).However,there is no report on the use of multiplex RT-PCR assays to detect and subtype swine influenza viruses.The purpose of this study was to develop two multiplex RT-PCR assays that can identify and subtype H1 and H3or N1and N2directly from clinical specimens.Then,the two multiplex RT-PCR as-says were used to investigate the occurrence of H1N1,H1N2and H3N2subtypes in clinical spec-imens from pigs.2.Materials and methods2.1.Samples and6irusesTwo hundred cell culture isolates of type A influenza virus,100virus-positive nasal swabs and 60lung samples were obtained randomly from archived isolates/samples at the Minnesota Veteri-nary Diagnostic Laboratory(MVDL).Thirty infl-uenza virus-negative samples(15nasal swabs and 15lung tissues)were also obtained.These samples were collected between1999and2001from pigs with respiratory diseases.Nasal swab was placed in a tube with1–2ml of Eagle’s minimum essen-tial medium(MEM)and vortexed vigorously.Ap-proximately10%suspension of lung homogenate was prepared in Eagle’s MEM.The nasal swabs or lung homogenates were centrifuged at3000rpm for20min,and the supernatants were used for virus isolation and RNA extraction.The virus was isolated from these samples using MDCK cells as described previously(Meguro et al.,1979).Two reference strains of swine influenza virus H1N1 (A/Swine/New Jersey/11/76)and H3N2(A/Swine/ Minnesota/9088-2/98)were used in the multiplex RT-PCR assays.An isolate designated as A/ Swine/Minnesota/1713/00was used as a reference strain of H1N2subtype.Y.K.Choi et al./Journal of Virological Methods102(2002)53–59552.2.Primer constructionTwo oligonucleotide primer sets suitable for H gene multiplex RT-PCR were designed from pub-lished sequences of swine influenza virus H1N1 (H1F,H1R)(GenBank accession no.K00992) and H3N2(H3F,H3R)(GenBank accession no. AF153234).Two primer sets for N1(N1F,N1R) and N2(N2F,N2R)genes were constructed based on GenBank data(AF250363,AF153238).An-other H1-1F primer was designed for sequencing of H1fragment from H1N1(GenBank accession no.K00992).The primers used for two multiplex RT-PCR assays are shown in Table1.2.3.Viral RNA extraction and multiplexRT-PCR assaysViral RNAs were extracted using QIAamp Vi-ral RNA Mini Kit(Qiagen,Valencia,CA)from 200m l volume of each viral isolate,nasal swab or lung homogenate.The RT-PCR was performed using One-Step RT-PCR Enzyme Mix(Qiagen, Valencia,CA)containing a specially formulated enzyme blend for both reverse transcription and PCR.Amplification of DNA was carried out at 95°C for15min for reverse transcription fol-lowed by30cycles of denaturing at95°C for1 min,annealing at60°C for30s and extension at 72°C for1min.The PCR was ended with afinal extension step at72°C for10min.The PCR amplified products were analyzed by0.7%agarose gel electrophoresis.Two multiplex RT-PCR assays using the four primer sets were also tested for specificity using five bacteria and seven viruses commonly infect-ing swine.Bacterial pathogens tested were My-coplasma hyopneumoniae(ATCC27719)and M. hyorhinis(ATCC23839)andfield isolates of Streptococcus suis,Hemophilus parasuis and Bor-detella bronchiseptica.Viruses tested were porcine reproductive and respiratory syndrome virus (ATCC VR-2332),transmissible gastroenteritis virus(ATCC VR-743),encephalomyocarditis virus(ATCC VR129),porcine parvovirus (NADC-8),psuedorabies virus(Shope strain), porcine circovirus(PCV)type-1(ATCC CCL33) and afield isolate of PCV type-2(ISUVDL98-15237).2.4.Cloning and sequencing of PCR productsA pGEM®-T Easy Vector system I(Promega, Madison,WI)was used for cloning PCR prod-ucts.The PCR products were purified from low melting point agarose gels with a QIAEXII kit (Qiagen,Valencia,CA)according to the manufac-turer’s instructions.Purified PCR product was ligated with50ng of pGEM®-T Easy Vector at 15°C overnight and was then transferred into pBlueScript Escherichia coli cells.The Luria Bertani agar plates containing ampicillin(50m g/ ml)and X-Gal(5-bromo-4-chloro-3-indolyl-D-galactopyranoside)(40m g/ml)were used to select clones.Plasmid DNA was isolated from E.coli by QIAprep®Spin Miniprep kit(Qiagen,Valencia,Table1Primers used for two multiplex RT-PCR assaysPrimers Sequence(5%–3%)Positions SIV subtypes345–365H1F GGG ACA TGT TAC CCA GGA GAT H1GCA TTG TAT GTC CAA ATA TCC AH1R1351–1330CCA AGG TGA GAG GTC AAG CAGH1-1F a736–756TAT GCC TGG TTT TCG CTC AAH3F61–80H3724–705TTC GGG ATT ACA GTT TGT TGH3RN1324–346GGT TCC AAA GGA GAC ATT TTT GN1FCTA TCC AAA CAC CAT TGC CAT AN1R1078–1057TGC GAT CCT GAC AAG TGT TAT C364–385N2F N2CAG ACA CAT CTG ACA CCA GGA TN2R866–845a Primer used for sequencing only.Y .K .Choi et al ./Journal of Virological Methods 102(2002)53–5956Fig.1.Detection and subtyping of type A in fluenza virus by multiplex RT-PCR ne M-Hi-Low size DNA marker;lanes 1,3,5,7and 9—MDCK control cells,H1N1(A /Swine /New Jersey /11/76),H1N2(A /Swine /Minnesota /1877/00),H3N2(A /Swine /Minnesota /9088-2/98),and both H1N1and H3N2with H1and H3primers;lanes 2,4,6,8and 10—MDCK control cells,H1N1,H1N2,H3N2and both H1N1and H3N2with N1and N2primers.CA).To evaluate the speci ficity of the assays,size-speci fic PCR products obtained from the multiplex RT-PCR were sequenced using a stan-dard dye terminator cycle sequencing method and the Applied Biosystems 373A sequencer (Perkin –Elmer,Forest city,CA).The sequences were ana-lyzed with the CLUSTAL W (1.74)multiple sequence alignment program.3.Results3.1.Multiplex RT -PCRFour primer sets were designed for two multi-plex RT-PCR assays (Table 1).Two primer sets (H1F,H1R and H3F,H3R)for H genes permit-ted ampli fication of fragments of the expected sizes:1006bp of H1and 663bp of H3.Another two primer sets (N1F,N1R and N2F,N2R)for N genes permitted ampli fication of fragments of 754bp of N1and 502bp of N2.An isolate designated as A /Swine /Minnesota /1713/00was identi fied as H1N2by single RT-PCR and partial sequencing (GenBank accession no.AY052778;AY052779).Two multiplex RT-PCR methods were tested for reference strains of swine in fluenza virus:H1N1,H1N2and H3N2,and the expected fragments were visualized in 0.7%agarose gel (Fig.1).The speci ficity of RT-PCR products was con fi-rmed by sequencing (data not shown).The speci fi-city was demonstrated further by the absence ofampli fication when the RT-PCR products were tested with DNA or RNA templates from seven viruses and five bacteria or with RNA prepared from mock-infected MDCK cells.In addition,no positive ampli fication was observed when the two multiplex RT-PCR assays were performed on 30in fluenza A virus-negative samples (15nasal swabs and 15lung tissues).The primer sets were evaluated for the detection of three reference in fl-uenza A viruses in serial 10-fold dilutions of RNA extracted from 200m l of each virus stock that was adjusted to contain 105TCID 50/ml of H1N1,H1N2or H3N2.A positive ampli fication could be visualized with 10−5dilution of each of the three reference viruses.3.2.Detection and subtyping of influenza A 6irus in field casesUsing the two multiplex RT-PCR for 360swine in fluenza virus-positive samples (culture isolates,nasal swabs or lung specimens),200(55.6%),13(3.6%)and 139(38.6%)were subtyped as H1N1,H1N2and H3N2,respectively (Table 2).The remaining eight (2.2%)samples were a mixture of both H1N1and H3N2.The results of virus detec-tion using the multiplex RT-PCR were 100%in agreement with those of virus ing the HI test,216and 142of the 360virus-positive samples were subtyped as H1N1and H3N2,re-spectively.The remaining two samples were diag-nosed as a mixture of both H1N1and H3N2.In order to con firm the 13H1N2isolates,singleY.K.Choi et al./Journal of Virological Methods102(2002)53–5957RT-PCR assay was carried out,but no detectable amplified band of H3or N1gene was observed (data not shown).Two amplified bands of H1and H3genes were detected when eight dually positive samples were tested by single RT-PCR assay.The results of single RT-PCR assay for a nasal swab and two lung samples that were positive for both H1N1and H3N2are shown in Fig. 2.Each sample was tested with four different primers of H1,H3,N1and N2,and positive amplifications with the four gene segments were observed for each sample.4.DiscussionDifferent methods have been used to detect influenza A virus infection in pigs.Traditionally, swine influenza virus isolation fromfield samples has been carried out using embryonated chicken eggs.As a rapid method,a commercially available antigen-capture ELISA(Directigen®,Becton–Dickinson,Spark,MD)that was developed for the detection of influenza type A virus in throat or nasal swabs in humans has been used in veteri-nary diagnostic laboratories.In order to detect influenza virus antigen in infected tissues,fluores-cent antibody(FA)test using specific antiserum has been applied to frozen sections.Immunohisto-chemical method is similar in principle to the FA test,and influenza virus antigen can be visualized in infected cells of formalized tissue sections. However,none of these techniques can distinguish among influenza A virus subtypes of H1N1, H1N2and H3N2.Two multiplex RT-PCR assays in the present study could differentiate the three subtypes by the use of two pairs of specific primer sets for H1and H3,and N1and N2.A multiplex RT-PCR assay that can differentiate between N1and N2sub-types is of particular interest because the subtype H1N2was identified recently from a pig in this country.Although the isolation of H1N2subtype has been reported from pigs in different countries including Japan(Sugimura et al.,1980;Nerome et al.,1985),France(Gourreau et al.,1994)and U.K.(Brown et al.,1995),thefirst isolate of H1N2subtype in the US was made from a pig in November1999(Karasin et al.,2000).Since then, no other report on the isolation or detection of H1N2is available in the US.The results of this study show additional occurrences of subtype H1N2in this country.Two of the13H1N2 subtype positive samples were collected from farms in Minnesota in1999.The H1N2cases in Indiana and Minnesota indicate that subtype H1N2has been prevalent in mid-western states since1999.A rapid detection and subtyping method is necessary to obtain detailed information on the prevalence of different subtypes of influenza A virus and to establish effective control measures for the swine industry.Identification and subtyp-ing are also important for tracking prevalent strains in a region of the country.Because swine are often viewed as‘mixing vessels’for both avian and human subtypes of influenza virus (Scholtissek et al.,1985;Webster et al.,1992),it is of particular interest to know which subtypes areTable2Subtyping of type A influenza virus-positive cell culture isolates,nasal swabs or lung samples from pigs by two multiplex RT-PCR assaysSamples collected No.of samples H1N1and H3N2Subtype by multiplex HIN1and H1N2and H3N2H1N2RT-PCRH3N2H1N2H1N1101032000Cell culture isolates8205100592Nasal swabs380016038119Lung samples002Total8360200131390Y .K .Choi et al ./Journal of Virological Methods 102(2002)53–5958Fig.2.Single RT-PCR assay from a nasal swab (lanes 2–5)and lung samples 1(lanes 6–9)and 2(lanes 10–13)positive for both H1N1and ne M-Hi-Low size DNA marker;lane 1MDCK control cells;lanes 2,6and 10showing H1gene ne 3,7and 11showing H3gene fragments;lane 4,8and 12showing N1gene fragment;lane 5,9and 13showing N2gene fragments.prevalent in local swine populations and compare the subtypes with those prevalent in the human population.Two multiplex PCR assays developed in this study could provide in fluenza virus diagno-sis with easier identi fication and more rapid sub-typing than other methods.In addition,these assays can detect infection with dual in fluenza A virus subtypes in swine.It is important to note that dual infection has been detected in clinical field samples indicating the possibility of concurrent or sequential infec-tion in a pig.Dual infection was seen for subtypes H1N1and H3N2only,which may be due to the lack of cross protective immunity between in flu-enza virus H1and H3proteins.In other words,pigs immunized with H1N1can be infected with H3N2and vice versa.Concurrent or sequential infection with subtypes H1N2and H3N2is possi-ble but less likely to occur under field conditions because the prevalence of H1N2between 1999and 2001was very low,and /or the sequential infections may be reduced due to cross protective immunity to N2.Only 3.6%of 360in fluenza type A virus-positive samples were positive for subtype H1N2.If the prevalence of H1N2increases in future,sequential infection of H1N2with other subtypes can be expected.In that situation,pre-ventive measures using polyvalent vaccine would have to be considered for type A in fluenza virus infection in pigs.AcknowledgementsThe authors thank Wendy Wiese and Sigrun Haugerud for technical assistance.ReferencesBressoud,A.,Whitcomb,J.,Pourzand,C.,Haller,O.,Cerutti,P.,1990.Rapid detection of in fluenza virus H1by the polymerase chain 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