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Journal of Virological Methods 165 (2010) 133–138

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

enotyping and screening of reassortant live-attenuatednfluenza B vaccine strain

un-Young Leea,1, Kwang-Hee Leea,1, Eun-Ju Junga, Yo Han Janga, Sang-Uk Seoa,yun-Ah Kimb, Baik Lin Seonga,∗

Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, South KoreaR&D Center of Pharmaceuticals, CJ Corp., 522-1, Dokpyong-Ri, Majang-Myun, Ichon-Si, Kyonggi-Do 467-180, South Korea

rticle history:eceived 13 July 2009eceived in revised form 16 October 2009ccepted 22 October 2009vailable online 31 October 2009

a b s t r a c t

Live-attenuated influenza virus vaccines can be generated by reassortment of gene segments between anattenuated donor strain and a virulent wild-type virus. The annual production schedule for the seasonalinfluenza vaccine necessitates rapid and efficient genotyping of the reassorted progeny to identify thedesired vaccine strains. This study describes a multiplex RT-PCR system capable of identifying each gene

eywords:nfluenza B viruseassortantsultiplex RT-PCRenotyping

segment from the cold-adapted attenuated donor virus, B/Lee/40ca. The specificity of the amplificationsystem was optimized by testing various wild-type influenza B viruses. The resulting RT-PCR method issensitive and efficient enough for routine identification of reassortant clones to identify the desired geneconstellation, consisting of six segments from the attenuated donor virus and the H and N genes from thewild-type virus. By providing a more rapid and efficient means of genotyping the candidate reassortantstrains, this method could be implemented to expedite the generation of each component strain and

re and

ive vaccine allow more time to cultu

. Introduction

Influenza virus is an enveloped virus with a negative-sense seg-ented RNA genome that belongs to the Orthomyxoviridae family

Beigel, 2008). Influenza A and influenza B viruses are globallymportant respiratory human pathogens that cause annual epi-emics and occasional pandemics necessitating annual vaccinationgainst newly circulating strains. Annual influenza epidemics areaused typically by antigenic drift, point mutations within geneegments, while pandemics are typically caused by antigenic shift,he reassortment of a whole gene segment that changes completelyhe antigenicity of a circulating virus (Carrat and Flahault, 2007;ampson and Mackenzie, 2006; Wright et al., 2007). Influenza spe-ific antiviral drugs are able to reduce the extent of virus spreadhroughout the population (Beigel and Bray, 2008; De Clercq,004; Intharathep et al., 2008; Moscona, 2008), but vaccinationemains the primary means of protecting people against influenza

isease.

Every year the World Health Organization (WHO) predictshe virus strains likely to circulate in the upcoming influenzaeason in each hemisphere and recommends three strains, two

∗ Corresponding author. Tel.: +82 2 2123 2885; fax: +82 2 362 7265.E-mail address: blseong@yonsei.ac.kr (B.L. Seong).

1 These authors contributed equally to this work.

166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2009.10.022

process the final seasonal influenza vaccine.© 2009 Elsevier B.V. All rights reserved.

influenza A types and one influenza B type, to be includedinto the vaccine formulation. Currently, inactivated influenzavaccines are licensed worldwide, but live-attenuated influenza vac-cines are used only in Russia and have been licensed recentlyin the USA (Bardiya and Bae, 2005; Belshe, 2004; Ohmit etal., 2008). Immunization with the live-attenuated influenzavaccine induces a broader immune response that resemblesnatural infection, which establishes better long-term immunityagainst influenza virus (Cox et al., 2004; Wareing and Tannock,2001).

The segmented nature of the influenza genome allows attenu-ated reassortant viruses to be produced by co-infecting cells withan attenuated influenza strain, which serves as the source of atten-uated genes and a circulating wild-type human influenza virus.The co-infection results in a pool of reassortant viruses with a ran-dom combination of the eight RNA genomes from the two parentalviruses. Among the 256 possible reassortants, it is necessary toisolate and identify the reassortant that inherited the two genesegments encoding the hemagglutinin (HA) and neuraminidase(NA) surface glycoproteins from the epidemic wild-type virus andthe remaining six gene segments (PB2, PB1, PA, NP, M, and NS)

from the attenuated donor virus (Maassab and Bryant, 1999; Seoet al., 2008). The tight schedule associated with the annual pro-duction of the trivalent influenza vaccine necessitates an accurateand rapid method to genotype reassortants to identify clones withthe desired genetic constellation. Recently a multiplex RT-PCR

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34 E.-Y. Lee et al. / Journal of Virol

echnique was developed to identify rapidly gene segments orig-nating from a live-attenuated influenza A virus strain (Ha et al.,006). This report presents a similar multiplex RT-PCR system forenotyping reassortant influenza B viruses. This method could bemplemented to facilitate progeny genotyping and expedite theroduction of the trivalent live-attenuated influenza vaccines eachear.

. Materials and methods

.1. Viruses and cell

The cold-adapted influenza B virus (B/Lee/40ca) was generatedrom repeated passages at low temperature in embryonateden’s eggs, as previously described (Seo et al., 2008). Wild-type

nfluenza B viruses (B/Shangdong/7/97, B/Hong Kong/330/2001,/Jillin/20/2003, B/Shanghai/361/2002, B/Yamagata/16/88,/Beijing/76/98, B/Panama/45/90, B/Hawaii/10/2001, and/Jiangsu/10/2003) were obtained from the National Insti-ute for Biological Standards and Control (NIBSC, UK). Alliruses were propagated in 11-day-old specific-pathogen-freeSPF) embryonated hen’s eggs (Charles River, Jinan, China)r in Madin–Darby canine kidney (MDCK) cells, which wereaintained in Eagle’s minimal essential medium (MEM) sup-

lemented with 10% fetal bovine serum. Virus-infected MDCKells were maintained in MEM with 0.025 �g/ml of TPCK-reated trypsin (Invitrogen, Life Technologies, Carlsbad, CA,SA).

.2. Primer selection

Primers were designed on the basis of sequence informationrom the Influenza Sequence Database of Los Alamos Nationalaboratories, Los Alamos, New Mexico (http://www.flu.lanl.gov).ultiple sequence alignments were performed using the align-ent program of MAFFT version 6 from the Medical Institute

f Bioregulation, Kyushu University (http://align.bmr.kyushu-.ac.jp/mafft/online/serv-er). Regions of naturally occurringequence diversity were identified to design virus strain-pecific primers. The primers were designed to have annnealing temperature of ∼58–60 ◦C and to minimize base-aring at the 3′-end of the primer binding site for theajority of wild-type influenza B viruses. Positive control

rimers were chosen to amplify the two internal PB2 andB1 genes that are highly conserved among all influenza

viruses (PB2-F: 5′-gcaggaataccaagagaatc-3′; PB2-R: 5′-cttgagaaaataccatgca-3′; PB1-F: 5′-tagtagttgaaaacttccc-3′; andB1-R: 5′-cagtaacttttctttttgctc-3′). All of the primers were synthe-ized using a DNA synthesizer (PerkinElmer, Foster City, CA, USAnd Cosmogenetech, Seoul, Korea).

.3. RNA isolation and cDNA synthesis

Total RNA was isolated from virus-infected MDCK cells grownn 6-well plates (about 1 × 105 cells per well) using the easy-LUETM Total RNA Extraction kit (INtRON Biotechnology, Korea)ccording to the manufacturer’s protocol. The concentration ofNA was measured spectrophotometrically at 260 nm, and storedt −70 ◦C. The reverse transcription (RT) reaction was per-ormed using the Omniscript Reverse Transcriptase kit (Qiagen,

alencia, Germany) according to the manufacturer’s protocol.ach reaction contained 5.25 �l RNase-free water, 1 �l 10× RTuffer, 1 �l 5 mM of each dNTPs, 0.5 �l Omniscript reverseranscriptase, 0.25 �l RNase inhibitor (10,000 unit), 10 pmol syn-hesized 18mer oligo-dT primer, and 800 ng total RNA (1 �l).

Methods 165 (2010) 133–138

The thermal cycler program for the RT reaction was as fol-lows (per cycle): 37 ◦C for 60 min, 95 ◦C for 5 min, and 4 ◦C for5 min.

2.4. Polymerase chain reaction (PCR)

The PCR was performed using 50 ng cDNA and 48 �l of a mastermixture containing 28.5 �l distilled water, 5 �l 1× i-Taq enzymebuffer (INtRON Biotechnology, Korea), 4 �l 4 mM of each dNTP(Takara, Japan), 10 �l of a 10 pmol mixture of each primer set,and 1 U i-Taq polymerase (0.5 �l) (INtRON Biotechnology, Korea).The reaction was performed on a PE 9700 thermocycler (AppliedBiosystems, PerkinElmer) using the following cycling conditions:94 ◦C for 5 min, 25 cycles of 94 ◦C for 1 min, 57 ◦C for 1 min, and72 ◦C for 2 min, and a final extension step at 72 ◦C for 7 min. ThePCR products were resolved by electrophoresis on 2% agarose gels,stained by ethidium bromide (0.2 �g/ml) and visualized by a UVtransilluminator.

2.5. Sequencing

Amplified RT-PCR products from the reassortant viruses weresequenced directly to confirm their origins. Sequencing was per-formed by Cosmogenetech, Seoul, Korea, using the ABI 3730XLautomated sequencing machine (Applied Biosystems, Foster City,CA, USA).

3. Results

3.1. Design of B/Lee/40ca specific primers

Strain-specific and segment-specific primer pairs were designedto match the B/Lee/40ca genome but result in mismatched basepairs at the 3′-end of the primer binding site for non-homologouswild-type influenza B viruses. The number of individual influenzaB virus sequences used for the alignment of each gene seg-ment were 56 for PB2, 65 for PB1, 64 for PA, 59 for HA,70 for NP, 110 for NA, 58 for M, and 50 for NS gene. Eachprimer pair was validated to amplify specific products fromthe attenuated vaccine donor strain B/Lee/40ca and not toamplify products from wild-type influenza B viruses. To deter-mine the specificity of the primer pairs, five influenza B strains(B/Lee/40ca, B/Panama/45/90, B/Beijing/76/98, B/Jilin/20/2003, andB/Shangdong/7/97) were examined. From the initial screening,low-specificity primer pairs were excluded and primer pairs withhigh-specificity (Table 1) were further tested using five addi-tional wild-type influenza B virus strains (B/Yamagata/16/88,B/Shanghai/361/2002, B/Jiangsu/10/2003, B/Hawaii/10/2001, andB/Hong Kong/330/2001) (data not shown). A subset of the vali-dation experiments are shown in Fig. 1, and a summary of theamplified PCR products for each virus strain/primer set combina-tion are shown in Table 2. The positive control primers were chosenfrom conserved regions of the PB2 and PB1 genes and included ineach reaction (Fig. 1, lanes 19, 20, 36, and 37).

3.2. Selection of multiplex primer sets specific for thecold-adapted donor virus

A multiplex RT-PCR format was developed that enabled thesimultaneous amplification of multiple RNA influenza gene seg-ments. Since the RT-PCR products for the different gene segments

can be easily distinguished by size, three different multiplex reac-tions were tested, each composed of two or three segment-specificprimer pairs (Table 3). All eight segment-specific PCR productswere amplified successfully when B/Lee/40ca RNA was used as atemplate and no nonspecific bands were observed. Further, no PCR

E.-Y. Lee et al. / Journal of Virological Methods 165 (2010) 133–138 135

Table 1Oligonucleotide primers used for RT-PCR amplification of B/Lee/40ca virus.

Segment PCR products Positiona Length (bp) Sense primer Anti-sense primer

PB2

B-PB2-1 37–831 795 5′-1 AAATTGAACTACTAAAGCAGC 3′-1 TGATTATCTTCCTGCAGGCTB-PB2-2 37–1024 988 5′-1 AAATTGAACTACTAAAGCAGC 3′-2 CTCTTTAGTTCAAGTCTCCCB-PB2-3 37–1708 1672 5′-1 AAATTGAACTACTAAAGCAGC 3′-3 TTCAATGTTACTAAATTTTTAAGCB-PB2-4 151–2011 1861 5′-2 AGAACCCTTCATTGAGAATG 3′-4 GGATTGTAGGAGAACAAGGG

PB1

B-PB1-1 1303–1979 677 5′-3 GGCATCTCTTTCGCCAGG 3′-3 GAGGATGCAGTAACCGTCCB-PB1-2 194–929 736 5′-1 TGATGTTACAGGATGTGTAAT 3′-1 TGTCACAGTCATACTGACCCB-PB1-3 194–1655 1476 5′-1 TGATGTTACAGGATGTGTAAT 3′-2 ATTCATTCACTCCAGCGACB-PB1-4 372–1979 1608 5′-2 ACAGTGGACAAATTGACTCAG 3′-3 GAGGATGCAGTAACCGTCCB-PB1-5 194–1979 1786 5′-1 TGATGTTACAGGATGTGTAAT 3′-3 GAGGATGCAGTAACCGTCC

PA

B-PA-1 785–1278 494 5′-2 TACACCCAAAAGTTGAAGTG 3′-2 TTTTACTTGTCAGAGTACTTAGB-PA-2 187–722 538 5′-1 TTCTTGATGAGGAAGGAAAGA 3′-1 TTGTCTATGTAACTTCTCATCTB-PA-3 785–1513 729 5′-2 TACACCCAAAAGTTGAAGTG 3′-3 ATGTCAAAGCTTTCCCCTTCB-PA-4 187–1278 1092 5′-1 TTCTTGATGAGGAAGGAAAGA 3′-2 TTTTACTTGTCAGAGTACTTAGB-PA-5 187–1513 1327 5′-1 TTCTTGATGAGGAAGGAAAGA 3′-3 ATGTCAAAGCTTTCCCCTTC

HA

B-HA-1 172–463 292 5′-1 CTAACAACAACACCTACCAG 3′-1 TCTCTGCATTGATAACATTACTB-HA-2 521–814 294 5′-2 ATAGGAACGGCTTCTTCAAC 3′-2 CAATTCTGCCGCTTTGTTTTAAB-HA-3 664–1350 687 5′-3 GACAAAACCCAAATGGAAAGA 3′-3 GAGTATTTCGTCGTGAAGCCB-HA-4 172–1350 1179 5′-1 CTAACAACAACACCTACCAG 3′-3 GAGTATTTCGTCGTGAAGCC

NP

B-NP-1 1238–1423 186 5′-3 ATGAAGATCTAAGAGTGTTA 3′-4 CTCCACTTACTTCATTCCCTB-NP-2 1238–1594 357 5′-3 ATGAAGATCTAAGAGTGTTA 3′-5 TGGTTTTCTTTGCCATCGAAB-NP-3 689–1264 576 5′-2 GATCAAAGGCACTGAAAAGG 3′-3 GTGCAGATAACACTCTTAGAB-NP-4 292–901 610 5′-1 GTCTACAACATGGTGGTAAAG 3′-1 TGCATCACGTCCTTCAACGB-NP-5 292–1030 739 5′-1 GTCTACAACATGGTGGTAAAG 3′-2 TGACAACTATCATGCTTCTGB-NP-6 292–1264 973 5′-1 GTCTACAACATGGTGGTAAAG 3′-3 GTGCAGATAACACTCTTAGA

NAB-NA-1 266–538 273 5′-2 GACATTTCCACCCCCAGAGC 3′-1 CCCAATTTGACTGATACTAGB-NA-2 220–250 1031 5′-1 ACGCATCAAATGCCCAGACT 3′-2 TACTCCACTAAGAGTAAGAG

MB-M-1 651–913 263 5′-3 TGGAGTGTTGAGATCTCTAG 3′-1 GCCTCCTTATTTGGATTCCTB-M-2 482–913 432 5′-2 AACAAGCATCGCACTCGCATA 3′-1 GCCTCCTTATTTGGATTCCTB-M-3 413–913 501 5′-1 TCATGTACCTAAACCCTGAA 3′-1 GCCTCCTTATTTGGATTCCT

NS

B-NS-1 360–526 167 5′-2 AAATCCCTTAACTAGCAAA 3′-2 CATCCCTTATCTTTTGTCGB-NS-2 446–704 259 5′-3 AGCCGGAAAATGTCGATCAC 3′-4 GCCTCCTTATTTGGATTCCTB-NS-3 360–627 268 5′-2 AAATCCCTTAACTAGCAAA 3′-3 CCTGAGGAAGGTTCCGTTCB-NS-4 168–471 304 5′-1 TCCTGGTCAAGACCGCCTAC 3′-1 AATTGGGTGATCGACATTT

a Nucleotide positions represents the nucleotide from the 5′-end of the positive sense genome.

Table 2Specificities of RT-PCR of B/Lee/40ca virusa.

Viruses Genome segments (number of primer sets for each RNA)

PB2 (1–4) PB1 (1–5) PA (1–5) HA (1–4) NP (1–6) NA (1–2) M (1–3) NS (1–4)

B/Lee/40ca All All All All All All All AllB/Panama/45/90 – – – – – – – –B/Beijing/76/98 – – – – – – – –B/Jilin/20/2003 – – – – – – – –B/Shangdong/7/97 – – – – – – – –B/Yamagata/16/88 1, 4 – 2, 4, 5 – – – – 1, 3B/Shahai/361/2002 – 1, 2, 4 – – – – 3 –B/Jiangsu/10/2003 4 – – – 4 – 2 1B/Hawaii/10/2001 2 – – – – – – –

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B/Hongkong/330/2001 – – –

a Summary of the RT-PCR amplification of various influenza B viruses with primellowed the amplification of PCR product.

roducts were generated from the wild-type B/Yamagata/16/88nder the same conditions (Fig. 2). These results indicate that theelected primer sets, even when combined in a multiplex reaction,pecifically amplified only the desired products of the expected

able 3rimer sets used for three-tube multiplex RT-PCR.

Primer Primer set (test tube)

(I) (II) (III)

Segment-specificPB2-2 (988 bp) NP-11 (610 bp) HA-16 (687 bp)PB1-6 (677 bp) PA-9 (494 bp) NA-21 (273 bp)M-14 (263 bp) NS-15 (259 bp)

– – – – –

gned for B/Lee/40ca virus. Numbers in table represents the primer set number that

sizes, which could be easily distinguished by agarose gel elec-trophoresis.

3.3. Screening of reassortant viruses

The feasibility of the three-tube multiplex RT-PCR systemwas then extended to demonstrate its utility for genotypingand selection of live-attenuated vaccine candidates. For this

purpose, reassortant viruses were generated by co-infection of10- to 11-day-old embryonated hen’s eggs with B/Lee/40ca andB/Shangdong/7/97 viruses (Maassab and Bryant, 1999; Seo et al.,2008). Then, MDCK cells were infected with each plaque purifiedvirus, and total RNA was extracted from the virus-infected cells.

136 E.-Y. Lee et al. / Journal of Virological Methods 165 (2010) 133–138

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ig. 1. RT-PCR analysis of five wild-type influenza B test strains using segment-spirus was mixed and annealed to segment-specific primers to B/Lee/40ca for RT-PCthidium bromide. Segment-specific primers are PB2 (lanes 1–4), PB1 (lanes 5–9),lanes 27 and 28), M (lanes 29–31), and NS (lanes 32–35), respectively. The data areB2 and PB1 genes that are highly conserved among all influenza B viruses.

sing the three-tube multiplex RT-PCR system, the genome originas identified for each reassortant virus (Fig. 3). From 15 indepen-ent plaque isolates, eight distinct RNA genotypes were identified.he genome composition, as deduced from the pattern of PCR prod-cts, is summarized in Table 4. Each target RNA was amplifiedpecifically without production of nonspecific PCR products (Fig. 3).he multiplex genotyping procedure enabled the identification ofreassortant with the desired 6:2 RNA constellation (Fig. 3, R-8).

his isolate contained the six genome segments that encode

he intracellular protein, inherited from the attenuated donorirus B/Lee/40ca, and two genome segments encoding the HAnd NA surface glycoproteins, inherited from the wild-type/Shangdong/9/97 virus. Other reassortant clones displayed var-

ous combinations of the RNA segments, such as 7:1 (R-1, R-4, and

primer pairs for the B/Lee/40ca strain, as shown in Table 1. Total RNA from eachplification. The PCR products were separated on a 2% agarose gel and stained withnes 10–14), HA (lanes 15–18), NA (lanes 19, 20, 36 and 37), NP (lanes 21–26), NA

arized in Table 2. Positive control (Ctrl) primers were chosen for the two internal

R-5), 6:2 (R-2 and R-3), and 5:3 (R-6 and R-7), respectively. The RT-PCR experiment was repeated three times using the same primersets with similar results (data not shown). The whole genome ofthe R-8 reassortant virus was sequenced directly, confirming theorigins of each RNA segment and verifying the accuracy of themultiplex RT-PCR method.

4. Discussion

Live-attenuated influenza virus vaccines (LAIV) provide effec-tive and broad-spectrum protection against various influenzastrains (Belshe et al., 2004; Seo et al., 2007; Suguitan et al., 2006).This report describes the development of a multiplex RT-PCR sys-tem that can identify simultaneously the origin of each genome

E.-Y. Lee et al. / Journal of Virological

Fig. 2. Simultaneous detection of influenza RNAs by a three-tube multiplex RT-PCR.B/Lee/40ca and B/Yamagata/16/88 viruses are used for comparison. Lane M: 1 kbl((

slblc(s1tO

The design of the primers to include a mismatched base at the

FMv

TG

adder molecular weight marker; lanes 1 and 4: PB2 (988 bp), PB1 (677 bp), and M263 bp); lanes 2 and 5: NP (610 bp), PA (494 bp), and NS (255 bp); lanes 3 and 6: HA687 bp) and NA (273 bp); lanes 7 and 8: positive control.

egment of reassortant isolates to facilitate the process of trivalentive-attenuated vaccine manufacturing. A number of methods haveeen reported for genotyping influenza viruses, such as polyacry-

amide gel electrophoresis (PAGE) analysis in partial denaturingonditions (Palese and Schulman, 1976), DNA–RNA hybridizationsNerome et al., 1983), multiplex RT-PCR followed by florescent

ingle-strand conformation polymorphism analysis (Cha et al.,997), RT-PCR followed by digestion of PCR products with restric-ion enzymes (Cooper and Subbarao, 2000; Klimov and Cox, 1995;ffringa et al., 2000; Sakamoto et al., 1996), and a heteroduplex

ig. 3. Genotyping of reassortant influenza B viruses by a three-tube multiplex RT-PCR. La(263 bp); lane 2: NP (610 bp), PA (494 bp), and NS (255 bp); lane 3: HA (687 bp) and NA (

irus, respectively.

able 4enotyping and identification of the origin of RNA segments in reassortant viruses.

Clone no. RNA ratio (B/Lee/40cavs. B/Shangdong/7/97)

Re-1 7:1Re-2 6:2Re-3 6:2Re-4 7:1Re-5 7:1Re-6 5:3Re-7 5:3

Methods 165 (2010) 133–138 137

mobility assay (HMA) (Ellis and Zambon, 2001). While these tech-niques are reliable, they are laborious, time-consuming, and insome cases expensive.

The present system utilizes three sets of multiple segment-specific primer pairs. Multiplex RT-PCR has been employed widelyas a highly sensitive and specific method for the detection of infec-tious diseases, including influenza viruses (Chi et al., 2007; Poddar,2002; Stockton et al., 1998). Recently reverse genetic techniqueshave been developed that allow the generation of vaccine reassor-tant strains from cDNAs (Hoffmann et al., 2000, 2002; Neumann etal., 1999). While this method has been used for the development ofa vaccine against the highly pathogenic avian influenza (HPAI) (Shiet al., 2007; Song et al., 2009; Suguitan et al., 2006), it has yet to beused to manufacture seasonal human influenza vaccines. Therefore,the selection of vaccine strains still relies on classical reassortmentof wild-type and vaccine donor viruses, and the identification of iso-lated reassortant viruses with the desired genetic composition. Thepresent multiplex RT-PCR method for identification of influenza Bvaccine strains, in combination with a similar method developed forinfluenza A vaccine strains (Ha et al., 2006), would be instrumentalfor the generation of the trivalent vaccine formula according to theannual WHO recommendations.

3′-terminus enhanced the specificity and prevented amplificationof non-homologous strains (Kwok et al., 1990). In this study, thespecificity of the primer pairs were tested against nine differentheterologous wild-type influenza B viruses and the cold-adapted

ne M: 1-kb ladder molecular weight marker; lane 1: PB2 (988 bp), PB1 (677 bp), and273 bp); lanes 4 and 5: HA (1112 bp) and NA (1382 bp) specific for B/Shandong/7/97

Segment derivedfrom B/Lee/40ca

Segment derived fromB/Shangdong/7/97

PB2, PB1, PA, NP, NA, M, NS HAPB2, PB1, PA, NP, NA, M HA, NSPB2, PB1, HA, NP, NA, M PA, NSPB2, PB1, PA, HA, NP, NA, M NSPB2, PB1, PA, HA, NP, NA, NS NAPB2, PA, HA, NP, NA, PB1, M, NSPB2, HA, NP, M, NS PB2, PA, NA

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/Lee/40ca homologous virus, and only the primers specific for/Lee/40ca virus were selected for the multiplex reactions (Table 1).

n some instances, a ∼350 bp PCR product also was amplified fromhe NA primer pair (Fig. 1, lane 27), but this did not create diffi-ulties for analyzing the results. No other nonspecific PCR productsere observed for any of the other primer pairs. The present mul-

iplex RT-PCR was adopted for up to three sets of primer pairs perube. A prerequisite for a multiplex RT-PCR assay is the generationf PCR products that can be distinguished easily by size after gellectrophoresis. A single-tube multiplex RT-PCR that allows iden-ification of all eight influenza RNAs in one reaction would be andeal experimental set-up. However, we were not able to designuitable primer combinations that enable amplification of all eightene segments in a single-tube reaction. From the five possible mul-iplex combinations tested with B/Yamagata/16/88 and B/Lee/40cairuses (data not shown), the sets of primer pairs that optimallymplify all of the RNA segments is shown in Table 3. Using thehree-tube multiplex RT-PCR system, the feasibility of this sys-em for genotyping a pool of reassortant viruses from the mixednfection of attenuated donor virus and wild-type virus (Fig. 3 andable 4) was demonstrated. The origin of each gene segment ofhe 6:2 genome composition vaccine candidates was confirmed byirect sequencing of each RT-PCR product.

In conclusion, the strain-specific multiplex RT-PCR system washown to provide an efficient method for genotyping and screeningf candidate influenza B reassortant vaccine strains. This methodould be implemented to expedite the generation of each compo-ent strain, to allow more time to culture and process the finaleasonal influenza vaccine, and to minimize the morbidity andortality associated with influenza infections.

cknowledgements

This work was supported by a contract research grant from theorea Food and Drug Administration (KFDA) (06092-KFDA346),grant from the Ministry of Health, Welfare, and Family Affairs

A085105) from the Korean Government, and from Biotrion Co.,eoul, Korea.

eferences

ardiya, N., Bae, J.H., 2005. Influenza vaccines: recent advances in production tech-nologies. Appl. Microbiol. Biotechnol. 67, 299–305.

eigel, J., Bray, M., 2008. Current and future antiviral therapy of severe seasonal andavian influenza. Antiviral Res. 78, 91–102.

eigel, J.H., 2008. Influenza. Crit. Care Med. 36, 2660–2666.elshe, R., Lee, M.S., Walker, R.E., Stoddard, J., Mendelman, P.M., 2004. Safety,

immunogenicity and efficacy of intranasal, live attenuated influenza vaccine.Expert Rev. Vaccines 3, 643–654.

elshe, R.B., 2004. Current status of live attenuated influenza virus vaccine in theUS. Virus Res. 103, 177–185.

arrat, F., Flahault, A., 2007. Influenza vaccine: the challenge of antigenic drift. Vac-cine 25, 6852–6862.

ha, T.A., Zhao, J., Lane, E., Murray, M.A., Stec, D.S., 1997. Determination of thegenome composition of influenza virus reassortants using multiplex reversetranscription-polymerase chain reaction followed by fluorescent single-strandconformation polymorphism analysis. Anal. Biochem. 252, 24–32.

hi, X.S., Li, F., Tam, J.S., Rappaport, R., Cheng, S.M., 2007. Semiquantitative one-step RT-PCR for simultaneous identification of human influenza and respiratorysyncytial viruses. J. Virol. Methods 139, 90–92.

ooper, L.A., Subbarao, K., 2000. A simple restriction fragment length polymorphism-based strategy that can distinguish the internal genes of human H1N1, H3N2,and H5N1 influenza A viruses. J. Clin. Microbiol. 38, 2579–2583.

ox, R.J., Brokstad, K.A., Ogra, P., 2004. Influenza virus: immunity and vaccinationstrategies. Comparison of the immune response to inactivated and live, attenu-ated influenza vaccines. Scand. J. Immunol. 59, 1–15.

Methods 165 (2010) 133–138

De Clercq, E., 2004. Antiviral drugs in current clinical use. J. Clin. Virol. 30, 115–133.Ellis, J.S., Zambon, M.C., 2001. Combined PCR-heteroduplex mobility assay for detec-

tion and differentiation of influenza A viruses from different animal species. J.Clin. Microbiol. 39, 4097–4102.

Ha, S.H., Kim, H.A., Kim, Y.H., Kim, J.S., Lee, K.H., Park, S.Y., Park, W.J., Seong, B.L., 2006.A multiplex RT-PCR method for screening of reassortant live influenza vaccinevirus strains. J. Virol. Methods 134, 154–163.

Hampson, A.W., Mackenzie, J.S., 2006. The influenza viruses. Med. J. Aust. 185,S39–S43.

Hoffmann, E., Krauss, S., Perez, D., Webby, R., Webster, R.G., 2002. Eight-plasmidsystem for rapid generation of influenza virus vaccines. Vaccine 20, 3165–3170.

Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Webster, R.G., 2000. A DNAtransfection system for generation of influenza A virus from eight plasmids.Proc. Natl. Acad. Sci. U.S.A. 97, 6108–6113.

Intharathep, P., Laohpongspaisan, C., Rungrotmongkol, T., Loisruangsin, A., Malais-ree, M., Decha, P., Aruksakunwong, O., Chuenpennit, K., Kaiyawet, N.,Sompornpisut, P., Pianwanit, S., Hannongbua, S., 2008. How amantadine andrimantadine inhibit proton transport in the M2 protein channel. J. Mol. Graph.Model. 27, 342–348.

Klimov, A.I., Cox, N.J., 1995. PCR restriction analysis of genome composition andstability of cold-adapted reassortant live influenza vaccines. J. Virol. Methods52, 41–49.

Kwok, S., Kellogg, D.E., McKinney, N., Spasic, D., Goda, L., Levenson, C., Sninsky, J.J.,1990. Effects of primer-template mismatches on the polymerase chain reaction:human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 18,999–1005.

Maassab, H.F., Bryant, M.L., 1999. The development of live attenuated cold-adaptedinfluenza virus vaccine for humans. Rev. Med. Virol. 9, 237–244.

Moscona, A., 2008. Medical management of influenza infection. Annu. Rev. Med. 59,397–413.

Nerome, K., Sakamoto, S., Yano, N., Yamamoto, T., Kobayashi, S., Webster, R.G., Oya,A., 1983. Antigenic characteristics and genome composition of a naturally occur-ring recombinant influenza virus isolated from a pig in Japan. J. Gen. Virol. 64(Pt 12), 2611–2620.

Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M.,Perez, D.R., Donis, R., Hoffmann, E., Hobom, G., Kawaoka, Y., 1999. Generation ofinfluenza A viruses entirely from cloned cDNAs. Proc. Natl. Acad. Sci. U.S.A. 96,9345–9350.

Offringa, D.P., Tyson-Medlock, V., Ye, Z., Levandowski, R.A., 2000. A comprehensivesystematic approach to identification of influenza A virus genotype using RT-PCRand RFLP. J. Virol. Methods 88, 15–24.

Ohmit, S.E., Victor, J.C., Teich, E.R., Truscon, R.K., Rotthoff, J.R., Newton, D.W., Camp-bell, S.A., Boulton, M.L., Monto, A.S., 2008. Prevention of symptomatic seasonalinfluenza in 2005–2006 by inactivated and live attenuated vaccines. J. Infect.Dis. 198, 312–317.

Palese, P., Schulman, J.L., 1976. Differences in RNA patterns of influenza A viruses. J.Virol. 17, 876–884.

Poddar, S.K., 2002. Influenza virus types and subtypes detection by single step singletube multiplex reverse transcription-polymerase chain reaction (RT-PCR) andagarose gel electrophoresis. J. Virol. Methods 99, 63–70.

Sakamoto, S., Kino, Y., Oka, T., Herlocher, M.L., Maassab, F., 1996. Gene analysis ofreassortant influenza virus by RT-PCR followed by restriction enzyme digestion.J. Virol. Methods 56, 161–171.

Seo, S.U., Byun, Y.H., Lee, E.Y., Jung, E.J., Jang, Y.H., Kim, H.A., Ha, S.H., Lee, K.H., Seong,B.L., 2008. Development and characterization of a live attenuated influenza Bvirus vaccine candidate. Vaccine 26, 874–8781.

Seo, S.U., Lee, K.H., Byun, Y.H., Kweon, M.N., Seong, B.L., 2007. Immediate and broad-spectrum protection against heterologous and heterotypic lethal challenge inmice by live influenza vaccine. Vaccine 25, 8067–8076.

Shi, H., Liu, X.F., Zhang, X., Chen, S., Sun, L., Lu, J., 2007. Generation of an attenu-ated H5N1 avian influenza virus vaccine with all eight genes from avian viruses.Vaccine 25, 7379–7384.

Song, M.S., Oh, T.K., Pascua, P.N., Moon, H.J., Lee, J.H., Baek, Y.H., Woo, K.J., Yoon,Y., Sung, M.H., Poo, H., Kim, C.J., Choi, Y.K., 2009. Investigation of the biologicalindicator for vaccine efficacy against highly pathogenic avian influenza (HPAI)H5N1 virus challenge in mice and ferrets. Vaccine 27, 3145–3152.

Stockton, J., Ellis, J.S., Saville, M., Clewley, J.P., Zambon, M.C., 1998. Multiplex PCRfor typing and subtyping influenza and respiratory syncytial viruses. J. Clin.Microbiol. 36, 2990–2995.

Suguitan Jr., A.L., McAuliffe, J., Mills, K.L., Jin, H., Duke, G., Lu, B., Luke, C.J., Murphy,B., Swayne, D.E., Kemble, G., Subbarao, K., 2006. Live, attenuated influenza AH5N1 candidate vaccines provide broad cross-protection in mice and ferrets.

PLoS Med. 3, 1541–1555.

Wareing, M.D., Tannock, G.A., 2001. Live attenuated vaccines against influenza; anhistorical review. Vaccine 19, 3320–3330.

Wright, P.F., Newman, G., Kawaoka, Y., 2007. Orthomyxoviruses. In: Knipe, D.M.,Howley, P.M., Griffin, D.E. (Eds.), Fields Virology. Lippincott Williams andWilkins, a Wolters Kluwer Business, Philadelphia, pp. 1691–1740.