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1 Virology 412 (2011) Contents lists available at ScienceDirect Virology journal homepage: Genetic bases of the temperature-sensitive phenotype of a master donor virus used in live attenuated influenza vaccines: A/Leningrad/134/17/57 (H2N2) Irina Isakova-Sivak a,1, Li-Mei Chen a, Yumiko Matsuoka a,2, J. Theo M. Voeten c, Irina Kiseleva b,c, Jacco G.M. Heldens c, Han van den Bosch c, Alexander Klimov a, Larisa Rudenko b, Nancy J. Cox a, Ruben O. Donis a, a Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA b Institute of Experimental Medicine Russian Academy of Medical Sciences, 12 Acad. Pavlov Street, St. Petersburg, , Russia c Nobilon International BV, Wim de Körverstraat 35, 5831 AN, Boxmeer, the Netherlands article info abstract Article history: Received 28 September 2010 Returned to author for revision 28 October 2010 Accepted 5 January 2011 Available online 18 February 2011 Keywords: Influenza A virus Live attenuated influenza vaccine Temperature-sensitive mutant, cold-adapted virus, viral polymerase Trivalent live attenuated influenza vaccines whose type A components are based on cold-adapted A/Leningrad/ 134/17/57 (H2N2) (calen17) master donor virus (MDV) have been successfully used in Russia for decades to control influenza. The vaccine virus comprises hemagglutinin and neuraminidase genes from the circulating viruses and the remaining six genes from the MDV. The latter confer temperature-sensitive (ts) and attenuated (att) phenotypes. The ts phenotype of the vaccine virus is a critical biological determinant of attenuation of virulence. We developed a plasmid-based reverse genetics system for MDV calen17 to study the genetic basis of its ts phenotype. Mutations in the polymerase proteins PB1 and PB2 played a crucial role in the ts phenotype of MDV calen17. In addition, we show that calen17-specific ts mutations could impart the ts phenotype to the divergent PR8 virus, suggesting the feasibility of transferring the ts phenotype to new viruses of interest for vaccine development. Published by Elsevier Inc. Introduction Influenza A and B viruses are responsible for annual epidemics and result in increased morbidity and mortality in the human population worldwide (Simonsen et al., 1997 #2230){Thompson et al., 2003 #18434}. Vaccination is the principal intervention for preventing influenza and reducing the public health impact of epidemics and pandemics. Various types of influenza vaccines have been available and used for more than 60 years (Francis et al. 1947).Theyaresafeand effective in preventing both mild and severe outcomes of influenza (Fiore et al., 2009 #21592){Nichol, 2008 #18436}. Seasonal influenza vaccines contain two type A viruses (H1N1 and H3N2) and one type B virus to elicit immunity to currently circulating influenza viruses. Two major types of influenza vaccines are licensed for human use inactivated influenza vaccine (IIV) and live attenuated influenza vaccine (LAIV). IIV is administered parenterally and induces humoral IgG antibodies to the hemagglutinin (HA). High serum antibody titers are correlated with Corresponding author at: Mailstop G-16, Center for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA. Fax: address: rdonis@cdc.gov (R.O. Donis). 1 Current Address: Institute of Experimental Medicine Russian Academy of Medical Sciences, 12 Acad. Pavlov Street, St. Petersburg, , Russia. 2 Current Address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, North Drive, MSC 3203, Bethesda, MD 20892, USA. host protection against infection with antigenically similar viruses but are less effective against antigenic variants of the virus. In contrast to inactivated vaccines, LAIV administered intranasally (i.n.) mimics natural infection and is capable of inducing a much broader immune response. Besides eliciting humoral antibodies, LAIV induces mucosal immunity that prevents virus infection at the portal of entry and subsequent replication and spread in the respiratory tract. In addition, LAIV can generate specific cytotoxic CD8 + T lymphocytes that may confer heterosubtypic immunity, thus offering a unique advantage over inactivated vaccines (Belshe et al., 2000a; Belshe et al., 2000b; Cox et al., 2004; Kreijtz et al., 2009; Lu et al., 2006; Nichol and Treanor, 2006; Powell et al., 2007; Rudenko et al., 1996; Seo et al., 2007; Tamura et al., 2005). Development of LAIV became possible with the generation of coldadapted viruses such as A/Ann Arbor/6/60 and A/Leningrad/134/17/ 57 (Alexandrova and Smorodintsev, 1965; Maassab, 1967; Maassab et al., 1969; Smorodintseff et al., 1937). These viruses met two critical requirements for development of LAIV and their licensure for human use (Glezen, 2004). First, the attenuating mutations present in the attenuated master donor virus (MDV) must be within one or more of the six RNA segments that do not encode the HA and NA glycoproteins (Belshe et al., 1992; Jin et al., 2003; Snyder et al., 1988). Second, the transfer of the six RNA segments encoding the internal viral proteins (i.e., those that do not encode for HA or NA) from the attenuated MDV to the new epidemic virus must retain the attenuating capacity, /$ see front matter. Published by Elsevier Inc. doi: /j.virol

2 298 I. Isakova-Sivak et al. / Virology 412 (2011) without loss of immunogenicity to protect against illness caused by wild-type influenza virus (Belshe et al., 1992; Murphy and Coelingh, 2002). Finally, the attenuated phenotype of the vaccine viruses must be stable, virulent revertant viruses should not appear in vaccinees. The LAIV currently licensed in the Russian Federation consists of reassortant viruses with the HA and NA gene segments from currently circulating wild-type (wt) viruses recommended by the WHO and the six internal protein-encoding gene segments from the cold-adapted MDVs A/Leningrad/134/17/57 (H2N2) (calen17) or B/USSR/60/69 (B60) (Aleksandrova, 1977; Aleksandrova et al., 1971; Wareing et al., 2001). The six genes encoding the internal proteins of the MDV impart the cold-adapted (ca), temperature-sensitive (ts) and attenuation (att) phenotypes to the vaccine viruses by preventing replication in the lower respiratory tract of vaccinees (Aleksandrova et al., 1971; Maassab et al., 1982; Wareing et al., 2001). The MDV calen17 was derived from wild-type A/Leningrad/134/57 (H2N2) (Len134) virus after 17 sequential passages in embryonated chicken eggs at 25 C and was first used in the 1960s as a LAIV for immunization of children (Alexandrova and Smorodintsev, 1965). Genome sequence comparison between parent Len134 and its cold-adapted derivative revealed that a number of non-synonymous mutations in internal protein-encoding genes occurred during low temperature propagation in eggs (Klimov et al., 1992a). Some of these mutations imparted ts, ca and att phenotypes of the MDV (Klimov et al., 1992a). The ts phenotype is considered to be the most significant contributor to attenuation of virulence for humans since it restricts viral replication to the upper respiratory tract. Mapping the genetic loci responsible for the ts phenotype relied on single-gene reassortants (SGR) derived by replacing each parental Len134 gene with the corresponding gene from a ca donor virus using classical reassortment techniques (Kilbourne, 1969). These studies revealed that the PB2 and PB1 genes imparted the ts phenotype (Klimov et al., 2001). Analysis of single and multiple gene reassortants between calen17 and heterologous subtype H1N1 and H3N2 viruses confirmed the critical role of PB2 in conferring ts phenotype and the significant contribution of PB1 and PA, by so-called gene constellation effects (Kiseleva et al., 2003). However, the ts phenotype of calen17 was not mapped to specific amino acids within the polymerase proteins where more than one mutation was present. In this study, we developed an eight plasmid-based reverse genetics system for the MDV calen17 and used it to identify the role of each amino acid substitution in the ts phenotype (Hoffmann et al., 2000a). We found that mutations within PB2 and PB1 play a crucial role in the ts phenotype with possibly a minor additional role for NS2 and conversely, restoration of mutations in these genes was required to fully revert a ts 6:2 genotype vaccine virus to that of wild-type virus. Results Generation of calen17-rg virus from plasmid DNA A set of eight ambisense plasmids carrying the genes from the original MDV calen17 was constructed by inserting full-length viral cdnas between the Pol I and Pol II promoters (Hoffmann et al., 2000b). Infectious virus was recovered from the transfected cells and termed calen17-rg. Full genome sequencing of the rescued virus confirmed complete identity with MDV calen17 in all viral genes. Analysis of virus replication in MDCK cells at restrictive temperatures showed that the calen17-rg virus derived by reverse genetics (RG) exhibited a ts phenotype like its parental calen17 virus; both viruses replicated efficiently at 33 C but their replication at 38 C, and even at 37 C was severely impaired (Fig. 1A). In addition, these viruses formed distinct plaques in MDCK cells at 33 C while either few or no plaques were observed at 38 C (Fig. 1B). These data indicate that the established reverse genetics system for MDV calen17 allows recovery of phenotypically indistinguishable viruses entirely from plasmid Fig. 1. Infectious viral titers and plaque morphology of parental and RG-derived viruses at permissive and restrictive temperatures. (A) Virus stocks propagated in MDCK cells at the permissive temperature (33 C) were titrated by plaque assay at the permissive or restrictive temperatures (37 and 38 C). The bars represent virus titers at indicated temperature and the error lines represent standard deviation. (B) Plaque morphology in MDCK cell monolayers incubated at the indicated temperatures, fixed at 72 h after infection and stained with crystal violet. DNA and provides the means to manipulate the viral genome using standard molecular genetics techniques. Generation of Len134-rg virus To generate a reverse genetics system for the wild-type (wt) parent of the MDV calen17, we modified the plasmid set used to derive calen17-rg by site-directed mutagenesis to obtain the sequences of the ancestral wt Len134 genes. Alignment of the two genomes revealed 15 non-synonymous nucleotide changes distributed throughout the genome (Table 1). Mutation in codon 492 of the NP gene and codon 314 of the HA1 gene were noted in the MDCK-grown MDV calen17 but were absent in the original egg-grown calen17 stock. However, the latter could not affect the ts phenotype of Table 1 Amino acid differences between calen17 and Len134 virus sequences. Protein Position wt A/Leningrad/134/57 a ca A/Leningrad/134/17/57 b Amino acid Codon Amino acid Codon PB2 478 Val GTA Leu TTA PB1 265 Lys AAG Asn AAT 591 Val GTT Ile ATT PA 28 Leu CTG Pro CCG 341 Val GTA Leu TTA HA1 c 181 Asn AAT Thr ACT 197 Val GTT Ile ATT 225 Met ATG Ile ATA 314 d Thr ACA Ala GCA HA2 c 18 Ile ATT e Val GTT NP 492 d Asn AAT Ser AGT NA 366 Ile ATC Thr ACC M1 15 Ile ATC Val GTC 144 Phe TTT Leu TTG NS2 100 Met ATG Ile ATA a b c d e Wild-type parental virus from CDC repository. MDV derived from adaptation of egg-grown calen17 to MDCK cells. Numbering from the N-terminal position of the H2 HA after cleavage by signalase. Mutations introduced during adaptation of egg-grown calen17 to MDCK cells. Amino acid change specific for the wt strain used in this study and not reported before.

3 I. Isakova-Sivak et al. / Virology 412 (2011) reassortants with heterologous HA; therefore only 14 of the 15 nonsynonymous changes were changed in the calen17-rg plasmid set to obtain parental wt Len134 genes (Table 1). A virus designated Len134-rg was rescued at 37 C with a genomic sequence identical to that of the transfected plasmid DNAs. Comparative analyses of Len134-rg and wt Len134 virus replication in MDCK cells revealed that they both had similar titers in plaque assays at 33, 37 and 38 C (Fig. 1A); i.e., statistically, their titers were not significantly different. The two viruses also yielded morphologically identical plaques at all tested temperatures (Fig. 1B). The newly established reverse genetics systems for calen17 (calen17-rg) and the parental wt virus Len134 (Len134-rg) are suitable for evaluating the genetic basis of the ts phenotype. Identification of the viral genes that determine the ts phenotype Early genetic analyses suggested the possible involvement of five viral genes in the ts phenotype of calen17: PB2, PB1, PA, M1/M2 and NS1/NS2 proteins (Ghendon et al., 1981). However, subsequent molecular studies revealed that the PA, M and NS proteins did not contribute to this trait (Klimov et al., 2001). As a first step towards dissecting the genetic basis of the ts phenotype of calen17, we used a genetic approach to study the contribution of each of the six internal gene segments (PB2, PB1, PA, NP, M, NS). To this end, we created single-gene reassortants (SGRs) carrying one gene from calen17-rg and the rest of the genome from Len134-rg. Comparable infectious titers were obtained for the PA, NP or M SGR viruses at permissive and restrictive temperatures; titer reduction values at 38 C did not exceed 0.7 log 10 and the difference with Len134- rg virus was not statistically significant (Fig. 2A; P values of 0.147, and 0.38, respectively). In contrast, the introduction of ca PB2 or PB1 segments resulted in viruses that were ts at 38 C (~ fold reduction of infectious titer; P = and , respectively, when compared to Len134-rg). Nevertheless, these SGR viruses showed relatively minor titer reductions at 37 C; their subtle replication impairment was reflected in plaque morphology alterations (compare Len134-rg with PB2-SGR and PB1-SGR in Fig. 2B). Noteworthy, the SGR with the ca NS gene had 10-fold lower infectivity at the restrictive temperature of 38 C. This mutant virus was significantly more temperature sensitive than Len134-rg (Fig. 2A; P = 0.023), but also significantly (P = 0.005) less temperature sensitive than PB2-SGR and PB1-SGR, suggesting that the M100I amino acid substitution in the NS2 protein may play a minor role in the calen17 ts phenotype. To ascertain the maintenance of the ts phenotype in viruses with wild-type HA and NA, i.e., with the genotype of vaccine reassortants, a prototype virus which carries six genes encoding the internal proteins from cold-adapted calen17 and HA and NA from wt Len134 virus (socalled 6:2 reassortant genotype) was analyzed. The 6:2 RG virus showed a 20,000-fold reduced titer at 38 C compared to Len134-rg, and was indistinguishable from the calen17-rg, confirming that the wt HA and NA genes do not interfere with expression of the ts phenotype of calen17 (Fig. 2A, vaccine 6:2 reassortant compared to Len134-rg and calen17-rg viruses). Identification of specific amino acid substitutions responsible for the viral ts phenotype To determine the specific residues responsible for expression of the ts phenotype by the calen17 virus, we generated RG viruses including one or more amino acid substitutions from the calen17 virus into a wt Len134 background. Three mutations in PB2 and PB1 genes were of particular interest since their SGRs showed the most significant alteration of virus replication at restrictive temperatures. The separate introduction of the PB1 265N or PB1 591I amino acid substitutions into wt Len134 virus decreased virus titers at 38 C by 1.8 and 0.7 log 10, respectively (P = for PB1 265N and P =0.028forPB1 591I mutants compared to Len134-rg), but had no effect on virus replication at 37 C (Fig. 3A). Interestingly, the K265N substitution alone significantly impaired plaque formation at 38 C, decreasing plaque size, whereas the V591I change in PB1 only slightly affected plaque morphology at the restrictive temperature (Fig. 3B, mutants PB1 265N and PB1 591I compared to Len134-rg). Nevertheless, these two substitutions combined, resulted in a significant replicative impairment at the Fig. 2. Replication of parental (Len134-rg and calen17-rg) and reassortant (6:2 and SGRs) viruses in MDCK cells at permissive and restrictive temperatures. (A) Virus stocks propagated in MDCK cells at the permissive temperature (33 C) were titrated by plaque assay at the permissive or restrictive temperatures (37 and 38 C). The bars represent the fold reduction of virus titers (log 10 ) at 37 and 38 C relative to 33 C and the error lines represent standard deviation. * denotes statistically significant difference (P b 0.05) between Len134-rg and indicated virus. (B) Plaque morphology in MDCK cell monolayers, as described in Fig. 1.

4 300 I. Isakova-Sivak et al. / Virology 412 (2011) Fig. 3. Replication of wt and mutant viruses at permissive and restrictive temperatures. RG viruses possessing one or more mutations from MDV calen17 in Len134-rg background compared to 6:2 genotype vaccine prototype virus. (A) Virus stocks propagated in MDCK cells at the permissive temperature (33 C) were titrated by plaque assay at the permissive or restrictive temperatures (37 and 38 C). The bars represent the fold reduction of virus titers (log 10 ) at 37 and 38 C relative to 33 C and the error lines represent standard deviation. * denotes statistically significant difference (P b 0.05) between Len134-rg and indicated virus. (B) Plaque morphology at indicated temperatures as in Fig. 1. restrictive temperature 38 C (P = if PB1 265N mutant in Fig. 3A (1.7 log 10 decrease) is compared with PB1-SGR (3.1 log 10 )infig. 2A). Surprisingly a RG virus combining the single PB2 substitution and K265N change in PB1 failed to yield a genetically stable population of PB2 478L /PB1 265N virus; full-genome sequence analysis of viruses rescued from several independent transfections revealed multiple heterogeneous sequence variants (with N30% mixed sequence populations) within the polymerase genes. In contrast, the PB1 591I substitution was compatible with PB2 478L, the dual-substitution virus was severely impaired at the restrictive temperature of 38 C (4.5 log 10 titer reduction) indicating that the V591I PB1 change enhanced the effect caused by PB2 478L (3 log 10 titer reduction) (P = if PB2-SGR in Fig. 2A iscomparedwithmutantpb2 478L /PB1 591I in Fig. 3A). Furthermore, the three mutations in PB2 and PB1 combined resulted in a virus with replication characteristics virtually undistinguishable from those of the 6:2 genotype vaccine virus prototype (P values of 0.88 and 0.25 when vaccine 6:2 reassortant is compared to PB2 478L /PB1 265N,591I mutant at 37 and 38 C, respectively; Fig. 3A). The triple-substitution failed to form plaques at 38 C and yielded extremely small plaques at 37 C, whereas all mutants with only one or two substitutions (viruses PB2-SGR, PB1-SGR, PB1 265N,PB1 591I and PB2 478L /PB1 591I )wereableto form readily visible plaques at 37 C, and in some cases even at 38 C (Figs. 2B and 3B). analyzed a panel of viruses with single or clustered substitutions that revert the amino acid(s) to wild-type in a calen17 background. In an attempt to increase the relevance of the study for vaccine reassortant viruses, these changes were introduced by RG into the genome of a 6:2 reassortant with wt HA and NA and the remaining genes from calen17. Replacement of the PB2 or the PB1 gene with the corresponding wild-type gene segment changed the vaccine 6:2 virus ts phenotype at 37 C; i.e., titers at 37 C were similar to those at the permissive temperature 33 C. In contrast, these viruses remained highly impaired at 38 C, showing ~4 log 10 reduction in replication at this temperature (Fig. 4A, mutants PB2 478V and PB1 265K,591V ) whereas the PB2 478V /PB1 265K double mutant was essentially indistinguishable from the Len134-rg strain in terms of infectious titers at the restrictive temperature (Fig. 4A, P = 0.21). However, this mutant formed pinpoint plaques at 38 C, unlike the wild-type virus (compare mutant PB2 478V /PB1 265K with Len134-rg in Fig. 4B). Interestingly, the reversion of the third ts mutation PB1-591V changed plaque formation at restrictive temperatures although plaque morphology of the PB2 478V /PB1 265K,591V mutant still seemed to differ somewhat from Len134-rg virus (Fig. 4B). Since experiments with single-gene reassortants revealed that amino acid substitution M100I in NS2 protein could affect plaque morphology at high temperature, we reverted this mutation as well, in addition to the three mutations in the PB2 and PB1 genes. Noteworthy, the mutant PB2 478V /PB1 265K,591V /NS2 100M showed clear and distinct plaque formation at 38 C comparable to Len134-rg (Fig. 4B). These data, therefore, show that mutations in the two polymerase genes, PB2 and PB1, are the major determinants of the ts phenotype. In PB1, the mutation at position 591 seems to contribute to a lesser extend as compared to the mutation at position 265. These results also suggest that there may be a minor additional role for the mutation in NS2. We conclude that reversion of all these mutations is required for complete restoration of a wt phenotype in a 6:2 reassortant vaccine virus. Identification of mutations critical for maintenance of the ts phenotype in a 6:2 reassortant The safety of LAIV based on MDV calen17 could be seriously compromised by the emergence of revertant viruses which lost the attenuation phenotype imparted by ts mutations. To determine which mutations are critical for the expression of the ts phenotype, we Fig. 4. Replication of wt, 6:2 genotype vaccine prototype virus and mutants with one or more reverted mutations from vaccine 6:2 reassortant. (A) Virus stocks propagated in MDCK cells at the permissive temperature (33 C) were titrated by plaque assay at the permissive or restrictive temperatures (37 and 38 C). The bars represent the fold reduction of virus titers (log 10 ) at 37 and 38 C relative to 33 C and the error lines represent standard deviation. (B) Plaque morphology at indicated temperatures as in Fig. 1.

5 I. Isakova-Sivak et al. / Virology 412 (2011) Transference of the ts phenotype from MDV calen17 into a divergent virus To determine whether transferring the genetic signature of the calen17 ts phenotype into a divergent strain would impart this phenotype, four ts mutations were introduced by RG into the wildtype PR8 virus: PB2 (V591I), PB1 (K265N; V591I) and NS2 (M100I). Altogether, eight mutant viruses were generated with amino acid substitutions as shown in Fig. 5. The temperature-sensitive phenotype of the RG viruses was examined by plaque assay in MDCK cells at various temperatures. As expected, the introduction of calen17 ts mutations into a PR8 backbone had dramatic effect on plaque titers at restrictive temperatures. Thus, the PB2 478L mutation itself was enough to impair the replication of PR8 virus at 38 C; a 2.0 log 10 plaque titer reduction and significantly smaller plaque size were observed, compared to those at permissive temperature (Fig. 5A, B). In contrast, two other mutations within the PB1 gene (variants PB1 265N and PB1 591I ) failed to significantly reduce the virus titer at 38 C. Nevertheless, these two mutations combined (mutant PB1 265N,591I ), reduced virus titer at 38 C by as much as 1.25 log 10 (Fig. 5A).EventhoughmutationPB1 265N did not change significantly the virus titer at restrictive temperatures, when combined with the ts mutation in the PB2 protein (mutant PB2 478L /PB1 265N )a dramatic effect on virus titer at 37 and 38 C was observed, with 1.8 and 4.2 log 10 reductions, respectively. The other ts mutation PB1 591I had a somewhat lesser effect on virus titer at restrictive temperatures when combined with PB2 478L (variant PB2 478L /PB1 591I ) and/or PB1 265N (mutant PB1 265N,591I ) ts mutations. In summary, these results show that replication of a RG-derived mutant virus possessing these three ts mutations (PB2 478L /PB1 265N,591I ) was severely impaired at both 38 and 37 C, like the original MDV calen17 (Fig. 5A). Additionally, each of the three mutations in different combinations affected plaque formation at restrictive temperatures. The PB1 265N mutation inserted singly into PR8 virus imparted small size plaques at 38 and 37 C (Fig. 5B), and a similar outcome was noted when this mutation was introduced in combination with one or two other ts mutations (Fig. 5B, mutants PB1 265N,591I, PB2 478L /PB1 265N and PB2 478L / PB1 265N,591I ). As expected, the PB1 591I mutation had the least impact on plaque formation, which could be observed only in combination with the PB2 478L and/or PB1 265N mutation. Interestingly, introducing the M100I mutation into the NS2 protein of PR8 virus had no effect on virus plaque formation at 38 C (Fig. 5A, B; mutant NS2 100I ). In summary, in agreement with the findings from MDV calen17, ts mutations in both PB2 and PB1 genes could impart a ts phenotype into a divergent virus such as PR8, and confirmed the dominant role of PB2 478L and PB1 265N ts mutations. Discussion Live attenuated influenza vaccine (LAIV) based on ca viruses derived from the wt A/Leningrad/134/57 (H2N2) (Len134) virus has been used in Russia for decades and proven to be safe, immunogenic and efficacious in children, adults and elderly (Rudenko and Alexandrova, 2001; Rudenko et al., 1996; Rudenko et al., 1993). Two different viruses were used as master donors for type A LAIV until 2004: A/Leningrad/134/17/57 (calen17) to produce vaccine for adults (Alexandrova and Smorodintsev, 1965) and A/Leningrad/134/47/57 (calen47) for children (Garmashova et al., 1984). MDV calen17 was derived from Len134 virus by 30 passages in eggs at 32 C followed by 17 passages at 25 C, resulting in multiple mutations (Table 1). The calen47 MDV was derived from calen17 by 30 passages in eggs at 25 C, resulting in four additional amino acid changes: PB2-S492R, PB1-M317I, NP-L341I and M2-A86T (Klimov et al., 1992a). Currently, the type A components of Russian LAIVs contain the six gene segments encoding the internal proteins (PB2, PB1, PA, NP, M and NS) of the cold-adapted master donor virus calen17, which confer temperature sensitivity (ts), cold adaptation (ca) and attenuation (att) phenotypes, while the antigenic characteristics are transferred with the HA and NA genes from a circulating wild-type virus (6:2 vaccine genotype). Reduced replication at core body temperature will restrict replication of LAIV viruses to the upper respiratory tract of vaccinees and minimize disease symptoms (Richman and Murphy, 1979). Ferrets inoculated with selected Fig. 5. Infectious viral titers and plaque morphology of wtpr8 virus and PR8 mutants carrying one or more calen17-specific mutations at various temperatures. (A) Virus stocks propagated in eggs at the permissive temperature (35 C) were titrated by plaque assay at the permissive (33 C) or restrictive temperatures (37 and 38 C). The bars represent virus titers at indicated temperature and the error lines represent standard deviation. (B) Plaque morphology at indicated temperatures as in Fig. 1.

6 302 I. Isakova-Sivak et al. / Virology 412 (2011) calen17-based reassortants possessing either ts and/or ca phenotype revealed that the ca phenotype was not correlated with virulence in this model (Klimov et al., 2001). In contrast, all viruses with ts phenotype were attenuated for animals, i.e., no viral replication was detected in lungs, whereas all reassortants with non-ts phenotype could replicate efficiently in ferret lungs. Therefore, the ts phenotype is directly linked to attenuation and identification of the underlying mutations is of considerable interest. The early attempts to identify the genes of calen17 that impart the ts phenotype to reassortant vaccine viruses utilized classical genetic reassortment between calen17 and genetically divergent epidemic viruses. The use of wild-type (wt) viruses which differed from each other with respect to replication properties at varying temperatures has led to diverse conclusions depending on the virus chosen for reassortment (Kiseleva et al., 2003). Nevertheless, elaborate studies of large numbers of reassortants between calen17 and diverse wt viruses, led to the conclusion that the PB2 protein was a major determinant of the ts phenotype, with the other polymerase genes, PB1 and PA, as significant contributors (Kiseleva et al., 2003). More precise experiments with single-gene reassortants (SGRs) between genetically related viruses, pointed towards a leading role for PB2 and PB1 genes in conferring the ts phenotype (Klimov et al., 2001). The genetic composition of reassortant viruses in these studies was determined by RT-PCR followed by RFLP (Klimov et al., 1992b), without full-genome sequencing. Selection for desired genotypes often required multiple passages at restrictive temperature, increasing the risk of unwanted mutations within any gene segment, complicating the interpretation of results. In addition, the origin of the HA and NA genes was often unknown. In one study a ts mutation has been mapped to the HA gene (Medvedeva et al., 1991), but this is considered irrelevant as vaccine viruses do not contain HA of calen17. Here we report the use of reverse genetics system for MDV calen17 to systematically map the mutations that determine the ts phenotype. After constructing a RG system for calen17, we engineered the original wt parental Len134 virus from calen17 by reverting 14 nucleotide changes in the latter virus. The creation of two highly isogenic recombinant viruses with a defined number of amino acid differences allowed us to map the virus ts phenotype to specific mutations, avoiding any problems of gene constellation effects. The ts viral phenotypes of mutant viruses were assessed at 37 and 38 C, since the replication of calen17 in MDCK cells is significantly impaired at these temperatures (as compared to 32 C). In contrast, wild-type Len134 virus replicates efficiently at 37 and 38 C (Kiseleva et al., 2004). However, incubation at 39 C restricted Len134 virus replication (Kiseleva et al., 2004). Interestingly, the cut-off temperatures for A/Ann Arbor/6/60 (H2N2) (A/AA) MDV were 38 and 39 C, optimal for replication of wt strain and restrictive for caa/aa (Jin et al., 2003; Jin et al., 2004). Furthermore, a study by Kiseleva et al. indicated that the same viruses could have different temperature sensitivity profiles in eggs or MDCK cells (Kiseleva et al., 2004). We confirmed that mutations in the PB2 and PB1 proteins were critical contributors to the ts phenotype, as reported previously (Klimov et al., 2001). Both PB2 and PB1 genes inserted individually into the wt genetic background could impart the ts phenotype. Furthermore, Len134 SGRs with the calen17 PB2 or PB1 (PB2-SGR and PB1-SGR) were completely attenuated for ferrets, emphasizing the association between attenuation and the ts phenotype (Klimov et al., 2001). We further showed that mutations within PA, NP and M genes had no effect on the expression of the ts phenotype, which also correlated well with an earlier study (Klimov et al., 2001). Using a reverse genetics approach we identified the role of single mutations within the PB1 gene. Mutation PB1 265N had greater impact on virus ts phenotype than PB1 591I. Interestingly, a recent study of ts+ revertants from a related MDV A/Leningrad/134/47/57 (H2N2) reached different conclusions (Tsfasman et al., 2007) possibly resulting from the use of fowl plague virus as a reassortment partner to analyze the ts mutations. In concordance with previous studies, we found that introduction of all three ts mutations into PB2 and PB1 genes of wt Len134 virus was sufficient to completely transfer the ts phenotype from MDV calen17 (Kiseleva et al., 2003). Unlike previous studies, we noticed some effect of mutation M100I in the NS2 protein, which caused virus titer reduction at 38 C as much as 1.0 log 10 PFU/ml, and reduced plaque size at this temperature compared to 33 C. Probably, this discrepancy with previous studies could be explained by the criteria chosen for ts phenotype assessment. Originally, the phenotype of mutant viruses was assessed by comparing their titers in eggs at 33 C versus 40 C (Klimov et al., 2001), and lately, in MDCK cells at 33 C versus 37 C (Kiseleva et al., 2003). Here the effect of NS2 mutation was observed in MDCK cells only at 38 C whereas no significant virus titer decrease at 37 C was noticed. Importantly, according to the regulations of Russian Ministry of Health, in addition to 6:2 reassortants, vaccine reassortants for LAIV may also contain five genes from MDV and three genes from a wild-type strain (i.e., 5:3 genotype) (Anonymous, 2004). The identification of a mutation in NS with a role (albeit a minor one) in imparting the ts phenotype indicates that LAIV should not include this gene segment from wt virus. The stability of this vaccine virus ts phenotype has been demonstrated in numerous studies including in vitro experiments (Kiseleva and Klimov, 2002), animal models (Marsh et al., 2003) and clinical trials (Klimov et al., 2001; Klimov et al., 1995). In our study we attempted to understand the genetic basis of this stability, or in other words, the elimination of which ts mutations within a vaccine strain would be sufficient to revert the virus to a wt phenotype. Our results show that the removal of the two main ts mutations (PB2 478L and PB1 265N ) in a 6:2 genotype vaccine virus had critical influence on the virus ts phenotype. Nevertheless, the exclusion of two additional mutations (PB1 591I and NS2 100I ) was required to completely revert the virus phenotype to that of wt strain. These data support the idea that a combination of mutations was selected during the cold adaptation process, This co-evolution of changes may explain the demonstrated genetic stability of vaccine strains after replication in susceptible hosts. To determine whether the ts mutations of MDV calen17 can impart the ts phenotype to a divergent virus, we created A/PR/8/34 (H1N1) viruses by RG with different combinations of ts mutations from MDV calen17. The fact that introducing certain amino acid changes within polymerase genes of the PR8 strain led to loss of the wt phenotype, reinforce the evidence that those changes act together in an interdependent fashion. Unlike polymerase genes, the ts mutation in the NS gene of calen17 had no effect on the ts phenotype of PR8 virus, suggesting that this mutation may only play an accessory role. It would be of great interest to find out whether the calen17- specific mutations in PR8 background are preserved during multiple passages in host cells. Analogous studies have been done for A/Ann Arbor/6/60, the master donor virus for the LAIV (FluMist) licensed in the US for use in healthy individuals aged 2 49 years. The ts phenotype of this strain was mapped to five ts loci distributed in three proteins: PB1 (E391, G581, T661), PB2 (S265) and NP (G34) (Jin et al., 2003; Jin et al., 2004). The fact that both cold-adapted strains have the strongest ts determinants in the polymerase proteins suggests a critical role of those mutations in alteration of viral polymerase activity at restrictive temperatures. Experiments with an influenza minigenome assay indeed demonstrated reduced polymerase activity of A/ AA MDV at 39 C (Jin et al., 2004), but more comprehensive analysis of molecular mechanisms of A/AA temperature sensitivity did not reveal significant effect of impaired polymerase function on viral mrna transcription and protein synthesis at restrictive temperature (Chan et al., 2008). More probably, the interaction of polymerase

7 I. Isakova-Sivak et al. / Virology 412 (2011) proteins with some host factors plays a key role in the inability of mutant viruses to grow efficiently at high temperature. As was demonstrated for caa/aa strain, ts mutations located in the PB1 protein could significantly affect nuclear-cytoplasmic export of vrnp and reduce M1 protein incorporation into virions at restrictive temperature, which requires interaction with host cell proteins, such as Hsp70 (Chan et al., 2008). The precise molecular mechanisms underlying the ts phenotype of MDV calen17 are not known and worthy of further research. Materials and methods Cells, viruses and plasmids Madin Darby canine kidney (MDCK) and 293T human embryonic kidney (HEK293T) cells were obtained from American Type Culture Collection (ATCC) and were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. Coldadapted A/Leningrad/134/17/57 (H2N2) MDV (calen17) was obtained from Nobilon Schering-Plough (the Netherlands). This virus was derived from an original egg-isolated calen17 stock (Aleksandrova et al., 1965) and propagated in MDCK cells at permissive temperature (32 34 C) followed by plaque purification. A working stock of calen17 virus was prepared in MDCK cells by infection at an MOI of at permissive temperature. Wild-type A/Leningrad/134/57 (H2N2) (Len134) virus was from the CDC repository. This virus was amplified in MDCK cells at 37 C to prepare a working stock. A set of eight dual-promoter plasmids carrying all gene segments of A/PR/8/34 (H1N1) (PR8) was used in this study to generate mutant viruses with ts signatures from MDV calen17 (Hoffmann et al., 2000a; Subbarao et al., 2003). Site-directed mutagenesis of PR8-PB2, PR8-PB1 and PR8-NS plasmids was performed using QuikChange XL Multi sitedirected mutagenesis kit (Stratagene, La Jolla, CA) to introduce calen17-specific mutations into the PR8 background. Cloning of all genes from MDV calen17 into dual-promoter plasmids Viral RNA of calen17 was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA). Viral cdna segments representing the complete genome of calen17 virus were amplified by RT-PCR with a set of universal segment-specific primers (Hoffmann et al., 2001) using One-Step RT-PCR Kit (Qiagen, Valencia, CA). The amplified DNA segments subsequently were cloned into a dual-promoter plasmid for influenza A reverse genetics (Fodor et al., 1999; Hoffmann et al., 2001; Subbarao et al., 2003). The sequence of all the inserts was verified and site-directed mutagenesis performed to render them identical to the original virus. Site-directed mutagenesis was performed when needed using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Derivation of influenza viruses by reverse genetics DNA from a set of eight plasmids was transfected into co-cultured HEK293T/MDCK cells using TransIT LT-1 transfection reagent (Mirus, Madison, WI) according to the manufacturer's protocol. Four days after transfection, culture supernatants and cell lysates were collected and the recovered viruses were amplified twice in MDCK cells infected at an MOI of in the presence of 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich, St. Louis, MO). Culture supernatants were harvested when ~95% of the cells showed morphologic changes, clarified by lowspeed centrifugation, and stored at 80 C until use. Viruses were propagated at the permissive temperature (33 C) after transfections (HEK293T) or infections (MDCK). Reassortant H2N2 viruses were generated in compliance with the Institutional Biosafety Committee and NIH Guidelines for Research Involving Recombinant DNA Molecules. Wild-type H2N2 viruses were handled in biosafety level 3 containment facilities (CDC/NIH, 2007). To generate mutant viruses with the A/PR/8/34 (PR8) genomic backbone, 293T cells were transfected with eight plasmid DNAs carrying wild-type or mutated genes from PR8. Three to four days later, transfected cells were resuspended in the culture media with a scraper and inoculated into eggs. Allantoic fluid was harvested 48 h after inoculation and the presence of virus was revealed by agglutination of turkey red blood cells (WHO, 2002). Working stocks of reverse genetics-derived viruses were amplified in eggs and stored at 80 C. The complete genomes of all viruses derived by reverse genetics were sequenced and any stocks containing spontaneous mutations (including quasispecies) in any of the eight viral genes were discarded and re-derived with a confirmed correct sequence. Virus replication studies To determine virus replication at different temperatures, the infectious viral titers were measured by plaque assay in MDCK cells. To this end, six-well plates were seeded with cells per well the day before virus inoculation. Cell monolayers were rinsed twice with PBS prior to inoculation with 200 μl of 10-fold dilutions of virus in DMEM (Invitrogen, Carlsbad, CA) supplemented with TPCK-treated trypsin (1 μg/ml). After adsorption at 33 C for 1 h, the virus inocula were removed, cells rinsed with PBS and immediately overlayed with 3 ml of 0.8% agarose mixture (prepared by mixing equal volumes of 1.6% agarose (Lonza, Rockland, ME) and 2 MEM (Invitrogen, Carlsbad, CA) supplemented with TPCK trypsin at final concentration of 1 μg/ml) per well. Plates were incubated at 33, 37 or 38 C. After 3 days of incubation, the agarose overlay was carefully removed and the cell monolayers were stained with Crystal-Violet (0.05% solution in formalin) to visualize and enumerate plaques. Virus endpoint dilution titers were expressed as log 10 PFU/ml. The lower limit of detection was 0.7 log 10, and therefore, temperaturesensitive viruses which did not form plaques at 10-3 dilution were assigned a hypothetical maximum titer of 3.7 log 10, in order to allow calculation of reduction in virus titer. For statistical analysis, titration of each virus at different temperatures was performed at least three times. Phenotypic and genetic analyses The ts phenotype of the parent and RG viruses was assessed by evaluating viral replication in MDCK cells at permissive (33 C) and restrictive temperatures (37 and 38 C). Viruses that displayed 100-fold or greater reduction in viral titers at the restrictive temperatures compared with that observed at the permissive temperature were considered ts. Genetic analysis of all viruses was performed by nucleotide sequencing using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and a 3130xl Genetic Analyzer (Applied Biosystems) according to the instructions of the manufacturer. Sequences were analyzed using BioEdit software. Statistical analyses Data were analyzed with the Statistica software (version 6.0; Statsoft Inc.). Statistical significance between log 10 reduction values in viral titers of different viruses was determined by Student's t-test. P values of b0.05 were considered significant. Acknowledgments We gratefully acknowledge Ervin Fodor and Erich Hoffman for providing RG plasmids. We thank Daniil Korenkov for statistics consultation. The findings and conclusions in this report are those of

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