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Short Communication |
Mechnikov Research Institute for Vaccines and Sera, 115088, 1st Dubrovskaja Str. 15, Moscow, Russia
Correspondence
T. M. Tsfasman
tanya.tsfasman{at}gmail.com
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ABSTRACT |
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Sequences of the primers used in this study are available with the online version of this paper.
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MAIN TEXT |
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). The A/Leningrad/134/47/57 ca strain that had undergone 47 passages at suboptimal temperature acquired mutations coding for amino acid substitutions in the PB2, PB1, PA, NP, M1, M2 and NS2 proteins (Klimov et al., 1992). Complementation-recombination tests with fowl plague virus (FPV) ts (temperature-sensitive) mutants revealed ts mutations in genes of the A/Leningrad/134/47/57 strain coding for the PB2, PB1, NP, M1/M2 and NS1/NS2 proteins (Ghendon et al., 1981).
Several techniques are currently used in order to control stability of the above-mentioned mutations in the genome of ca reassortant vaccine strains during vaccine production and their reproduction in vaccinees, such as PCR restriction analysis (Klimov & Cox, 1995), single strand conformation polymorphism (SSCP) (Cha et al., 1997), complementation-recombination test (Ghenkina & Ghendon, 1979), etc. However, these methods give no information about the possible appearance of suppressive mutations (Scholtissek & Spring, 1982; Ghendon et al., 1982; Treanor et al., 1991, 1994) in the viral genome that might lead to reversion of the attenuated phenotype of the virus as well as true reversions of ts mutations.
In our previous study (Markushin et al., 2006), investigating phenotypic and genetic stability of the ca influenza virus strains, we obtained by serial passages in chick embryos at gradually elevated temperatures a ts+ revertant of A/Leningrad/134/47/57 ca ts master-strain that was used for live influenza vaccine production in the Soviet Union. After 18 passages at elevated temperatures (up to 40 °C), the virus virtually lost the ability to reproduce effectively at 26 °C, gained the ability to reproduce to high titres at 40 °C in 9–10 days old embryonated chicken eggs and could reproduce in mouse lungs like the wild-type virus (see Table 1). Evaluation of viral pathogenicity for mice was performed in the lungs of Balb/c mice as described by Nevedomskaia et al. (1992).
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It was shown previously by complementation-recombination analysis that the A/Leningrad/134/47/57 ca master-strain has ts mutations in genes coding for the PB2, PB1, NP, M1/M2 and NS1/NS2 proteins (Ghenkina & Ghendon, 1979; Ghendon et al., 1981). Complementation-recombination analysis of the revertant (see Table 2), performed as described by Ghenkina & Ghendon (1979), showed that during high-temperature passages the revertant lost phenotypic manifestation of the ts mutations in three internal genes encoding the PB2, NP and NS1/NS2 proteins. However, PCR restriction analysis revealed a single true reversion in the PB2 gene. All other mutations acquired by the ca A/Leningrad/134/47/57 strain in the process of adaptation to suboptimal temperature were preserved. We conjectured that the discrepancy in the results obtained by the two different techniques may be due to some extra- or intragenic suppressive mutations acquired by the ts+ revertant during passages at elevated temperatures (Markushin et al., 2006).
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For genomic sequence analysis of the revertant, viral RNA segments were reverse transcribed into cDNA and amplified by PCR using virus gene-specific primers designed with the VectorNTI (Invitrogen) and Omiga (Accelrys) programs using the A/Leningrad/134/47/57 ca master-strain sequence (GenBank accession nos M81583–M81587). Primer sequences are shown in Supplementary Table S1, available with the online version of this paper. PCR products were purified from 1–1.5 % low melting temperature agarose using a Promega Wizard PCR Preps DNA Purification System Kit. Sequence reactions were carried out according to the ‘Protocol for Fluorescent Sequencing with Big Dye Terminators’ (see http://www.bioinformatics.vg/Methods/sequencing.shtml) and were analysed on a MegaBACE 500 analyser (GE Healthcare). Only mutations present in the sequences obtained from two or more different primers corresponding to both DNA strands were considered. Gene sequences were viewed using the ChromasLite program (Technelysium).
We sequenced all the internal genes of the revertant of the A/Leningrad/134/47/57 ca strain [A/Leningrad/134/47/ts+18/1957(H2N2), GenBank accession nos EF633437–EF633442] and compared the sequences obtained with that of the A/Leningrad/134/47/57 ca strain (accession nos M81583–M81587) using VectorNTI (CLUSTAL W mechanism). The results are presented in Table 3. Sequencing revealed 29 mutations (15 of them resulting in an amino acid substitution) in all the internal genes acquired by the ts+ revertant during high-temperature passages (see Table 3). Sequencing confirmed the true reversion of a ts mutation in the PB2 gene [1459G-T-G (478Val-Leu-Val)] detected previously by PCR restriction assay (Markushin et al., 2006), revealed another true reversion in the PB1 gene [819G-T-A (265Lys-Asn-Lys)] that was not detected previously by PCR restriction assay, and confirmed the maintenance of all the other coding and silent mutations acquired by A/Leningrad/134/47/57 while adapting to suboptimal temperature in its internal genes. Hence, the data obtained were consistent with the hypothesis concerning the appearance of new mutations in the genome of the revertant.
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), PCR restriction assay allowed us to detect only one true reversion in the PB2 gene of the revertant, and did not detect the reversion in PB1. The explanation is in the peculiarities of the method applied. The Tru91 and HindIII restriction enzymes were chosen by Klimov & Cox (1995) so that they could cleave the sites present in the PCR products obtained from the PB2 and PB1 genes, respectively, of the wild-type A/Leningrad/134/57 strain and could not cleave the corresponding sites with a mutation present in the A/Leningrad/134/47/57 ca strain. The true reversion in position 1459 of the PB2 gene (G-T-G, amino acid substitution 478Val-Leu-Val) resulted in reappearance of the Tru9I restriction site. However, the true reversion in position 819 of the PB1 gene (G-T-A, amino acid substitution 265Lys-Asn-Lys) could not be detected by PCR restriction assay, because the mutation (819G-T-A) did not lead to reappearance of a HindIII restriction site.
We assume that the true reversion in the PB2 gene is the reason for the loss of phenotypic manifestation of the ts phenotype of this gene revealed by complementation-recombination test. However, no reversion of the ts phenotype could be detected for the PB1 gene (Markushin et al., 2006) (see Table 2
). According to the results of Klimov et al. (1992), during its adaptation to suboptional temperature the A/Leningrad/134/47/57 ca strain acquired four mutations during its adaptation to suboptimal temperature (360G-A, 819G-T, 975G-T, 1795G-A) in the PB1 gene, the last three of them coding for amino acid changes (see Table 3). We assume that position 819T (265Asn) is not critical for the ts phenotype of the PB1 gene and of the corresponding protein.
According to the data about the loss of phenotypic manifestation of the ts mutation in the NP and NS genes (having no true reversions in them), some of the new mutations might apparently suppress the phenotypic manifestation of ts mutations in the above-mentioned genes. The 101Asp-Asn substitution in the NP gene lies in its region that interacts with the PB2 protein (Biswas et al., 1998). Albo et al. (1995) determined the role of the region from 79 to 180 aa of the NP N terminus in RNA-binding. Both new coding mutations found in the NP gene of the revertant (101Asp-Asn and 180Ala-Gly) lie within this region. However, more recent experiments revealed the role of several arginine and aromatic acid residues along the length of NP in RNA binding (Elton et al., 1999), so that the suppressive role of these mutations is unclear.
The NP protein plays a multifunctional role in the viral cycle. It interacts with a variety of viral and cellular proteins, including M1 and polymerase proteins (Portela & Digard, 2002). It is quite possible that some new mutations in other genes (e.g. in the M gene or in the genes coding for the polymerase proteins) that did not lead to the reversion of the ts phenotype of the corresponding gene suppressed the ts mutation in the NP gene.
Thus, it remains unclear whether the ts mutation (1066C-A) in the NP gene of the revertant lost its phenotypic manifestation due to extra- or intragenic suppression.
Two proteins (NS1 and NS2) are encoded by the NS gene of influenza viruses. The only mutation in the NS gene of the ca A/Leningrad/134/47/57 strain lies in the NS2 coding region (see Table 3). Two coding mutations acquired by the revertant during high temperature passages in the NS gene lie within the region coding for the NS1 protein. The NS2 protein is known to be more conserved than the NS1 protein (Lamb & Krug, 2001). This might explain the lack of new mutations in the NS2 protein of the revertant. The fact that no mutations were observed in the region of the NS gene encoding the NS2 protein allows us to assume an extragenic suppression of the ts mutation in the NS2 protein of the revertant. It is known that a mutation in the NS gene could be suppressed by a mutation in one of the polymerase genes (Scholtissek & Spring, 1982), so that we may assume that some of the mutations in the PA or PB1 genes might have suppressed the ts phenotype of the NS gene in our case as well.
NS1 is a multifunctional protein (Lamb & Krug, 2001). One of the mutations in the NS1 gene of the revertant (23Pro-Ala) lies within the regions required for mRNA-binding (Qian et al., 1994; Marion et al., 1997) and for interaction with some cellular proteins (Falcon et al., 1999). Considering the dramatic character of this change, its influence on the functions of NS1 is quite probable.
The second coding mutation in NS1 (164Pro-Leu) lies within the C-terminal region (from 125–126 aa to the end) of the NS1 protein that has recently been shown to impair interferon production in vitro (Quinlivan et al., 2005; Stasakova et al., 2005). Because of its non-conservative nature, it could drastically influence the structure of NS1 and its ability to inhibit interferon production. Considering the character of the above-mentioned changes in NS1, we conjectured that they contributed somehow to the loss of ts, ca and att (attenuated) phenotype in mice by the revertant. For instance, increased ability to inhibit the interferon response could contribute to efficient virus accumulation in mouse lungs.
During passages at suboptimal temperature, the A/Leningrad/134/47/57 ca master-strain acquired two coding changes in the M gene, one of which lies within the M1, and another within the M2 coding region. One of the coding mutations in the M1 protein (144Phe-Leu) of the revertant lies within the ninth 
-helix of its dimerization domain [see 3D structure of M1 (1–158 aa) from PDB, 1AA7]. Substitution of an aromatic amino-acid with a smaller hydrophobic one could influence M1 dimerization and therefore could influence the alteration of the phenotype of the revertant. However, two coding mutations detected in the region of the M gene encoding the M1 protein did not lead to the reversion of phenotypic manifestation of the ts mutations in the same gene. The explanation might be in the ts mutation in the M2 protein, in which no new mutations occurred during high-temperature passages of the revertant.
The majority of coding mutations acquired by the revertant (including both true reversions) are in the polymerase genes. It is known that the polymerase genes are most crucial in the ts phenotype maintenance of the A/Leningrad/134/47/57 ca strain (Klimov et al., 2001). The true reversion in the PB2 gene of the ts+ revertant led to the reversion of ts phenotype of this gene. It should be noted that no other coding mutations appeared in the PB2 gene, besides the true reversion, which allows us to state the significant impact of 478Leu on the ts phenotype of the A/Leningrad/134/47/57 ca strain. Mutations in the PA and PB1 genes (including the true reversion in PB1) are candidates for mutations suppressing the ts phenotype of the NS and NP genes.
Thus, according to all the data obtained, our studies ascertain the sufficient phenotypic stability of ca strains. Only numerous passages at gradually elevated temperatures led to a significant alteration of the ts– (40 °C) and ca+ (26 °C) properties of the A/Leningrad/134/47/57 ca strain.
Direct sequencing combined with complementation-recombination tests allowed us to conclude that intra- and extragenic suppressive mutations are apparently more important for the reversion of the ts and ca phenotype compared with the true reversions of the ts and other mutations. Only two true reversions of a mutation were observed, one of which did not lead to the reversion of the phenotypic manifestation of ts phenotype of the corresponding gene (PB1).
Analysis of genetic stability of ca live influenza vaccines was performed by investigating the maintenance of the mutations present in the genome of the ca master-strain in virus isolated from vaccinees (Klimov et al., 1995; Buonagurio et al., 2006 and references therein). Our results show that it is sometimes impossible to detect a true reversion of a mutation using PCR restriction analysis (Klimov & Cox, 1995), so that this method is not thoroughly reliable. Moreover, our study shows that the reason for alteration of ca and ts phenotype of the virus as well as ts phenotypes of individual genes could possibly lie not in true reversions but in suppressive mutations in corresponding and other genes. Thus, in order to control the genetic stability of ca vaccine strains, it is necessary to analyse the maintenance of mutations present in the ca master-strain as well as the influence of other mutations which can result in phenotypic reversion via extra- or intragenic suppression of a mutation required for attenuation of the ca strain.
Our investigations show that mutations in the NP and NS genes were suppressed. However, to get unequivocal evidence of the nature of suppression of these mutations, further investigation is needed.
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Received 20 March 2007;
accepted 8 June 2007.
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