Reassortment of influenza A virus genes linked to PB1 polymerase gene

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1 International Congress Series 1263 (2004) Reassortment of influenza A virus genes linked to PB1 polymerase gene Jean C. Downie* Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW 2145, Australia Abstract. Influenza virus generates most new antigenic variants in two ways, either by antigenic drift of the Haemagglutinin (HA) and Neuraminidase (NA) or by antigenic shift involving genomic reassortment. Influenza pandemics like those that occurred in 1957 and 1968 are thought to have involved reassortment of HA and NA genes between human and avian or swine viruses. The factors controlling reassortment are not known. The aim of this study was to investigate an observed reassortment restriction between two influenza viruses. In numerous experiments no reassortants were found in mixed infections of A/USSR/90/77 (H1N1) and A/Shearwater/37/72 (H6N5). However, A/USSR/77 is known to have formed reassortants with human H3N2 strains in nature. In mixed infection experiments involving A/USSR/90/77, A/Shearwater/37/72, and a human H3N2 virus strain, an H6N1 reassortant was isolated. RNA RNA hybridization and gene sequence analysis showed that the only gene segment transferred from the H3N2 parent to the H6N1 reassortant was the H3 PB1 polymerase segment. These results indicate that the PB1 (gene or polymerase) may be required for gene reassortment in influenza viruses. Crown Copyright D 2003 Published by Elsevier B.V. All rights reserved. Keywords: Reassortment; PB1 polymerase; Influenza A 1. Introduction Influenza virus generates most new antigenic variants in two ways. The first, and most common, is by single nucleotide changes in the genes encoding the antigenic sites of the Haemagglutinin (HA) and Neuraminidase (NA). Changes of this sort are called antigenic drift. Larger genetic changes occur when two Influenza virus strains exchange genome segments by reassortment. When this involves the HA or NA genes it causes the antigenic shift often associated with pandemics like those that occurred in 1957 and All pandemics are thought to have involved reassortment of genes between human and avian or swine viruses [1]. The factors controlling reassortment are not known. In numerous experiments to produce HA/NA hybrids, no reassortants were found in mixed infections of A/USSR/90/77 (H1N1) and A/Shearwater/Australia/37/72 (H6N5). * Tel.: ; fax: address: jeand@icpmr.wsahs.nsw.gov.au (J.C. Downie) / Crown Copyright D 2003 Published by Elsevier B.V. All rights reserved. doi: /j.ics

2 The aim of this study was to investigate this observed reassortment restriction. By understanding the mechanism of how reassortment occurs one may hopefully be able to predict which viruses can reassort and which may not and why. 2. Materials and methods J.C. Downie / International Congress Series 1263 (2004) Viruses, preparation of reassortants and RNA hybridization analysis The viruses used in this study were: A/Shearwater/Australia/37/72 (H6N5) (A/Shearwater/72), A/Queensland/5/72 (H3N2) (A/Qld/72), and A/USSR/90/77 (H1N1) (A/USSR/ 77). Preparation of reassortants was done using standard methods as described by Webster [2] and used previously by Downie [3] to prepare high-yielding reassortants for vaccine production. RNA hybridization analysis described by Hay et al. [4] was used to determine the HA/NA and internal gene constellations of the influenza viruses isolated. Fig. 1. Hybridization analysis of the reassortant (X) H6N1 and parent viruses (S) A/Shearwater/72, (Q) A/Qld/72 and (U) A/USSR/77. 3 H c-rna was prepared for the reassortant H6N1 and hybridized with unlabeled vrna from each parent. After SI nuclease digestion the double stranded RNAs were separated by electrophoresis on 7.5% polyacrylamide gels at 40 V for 48 h and detected by fluorography. The double stranded molecules formed between the reassortant and the parental viruses RNA segments are indicated by numbers at the gel margin.

3 716 J.C. Downie / International Congress Series 1263 (2004) RNA preparation, RT-PCR and sequencing Viral RNA was extracted from 400 Al infected allantoic fluid using Invitrogen Micro-to- Medi Total RNA Purification System. Primers were designed for the polymerase genes based on published sequences [5] for A/NT/60/68 (J02138) and A/FM/1/47 (X99037). Full length sequences of the PB1 genes were done in four over-lapping stages. The RT-PCR reaction was carried out using Invitrogen SuperScript One-Step RT-PCR Kit with Platinum Taq. The products of RT-PCR were checked by gel electrophoresis and purified using Millipore Multiscreen PCR plates. The products were then sequenced using sequencing primers on an ABI Prism Genetic Analyser model 3100 using ABI Prism BigDye Terminator Chemistry (version 3.1). Preliminary analysis was done using Sequencher analysis software. 3. Results 3.1. Reassortants After repeated experiments, no HA/NA or internal gene reassortants between A/Shearwater/72 (H6N5) and A/USSR/77 (H1N1) were obtained by the standard methods Fig. 2. Combined PB1 tree analysis for H6N1 reassortant and parental viruses. The sequences of each of the PB1 segments were edited, aligned by CLUSTALX using default parameters and the published sequences of A/Fort Monmouth/1/47 and A/PR/8/34 for comparison. They gave alignments of 589, 432, 764 and 120 nucleotides (nts) making 1905 nts in total starting at nts 130, 673, 1178 and 2138 of the 2351 nts aligned published sequences (81%). Trees were inferred by neighbor-joining. All four PB1 segments gave trees with the same topology, so the sequences were concatenated, and gave the combined tree (see above). The PB1 gene of the reassortant gave an identity of 99.5% with A/Qld/72, 97% with A/PR/8/34, 84.7% with A/Shearwater/72 and 70.8% with A/USSR/77.

4 J.C. Downie / International Congress Series 1263 (2004) Table 1 H6N1 reassortant gene constellation Gene segment Gene Gene parental origin 1 PB2 A/Shearwater/72 2 PB1 A/Qld/72 3 PA A/Shearwater/72 4 HA A/Shearwater/72 5 RNP A/Shearwater/72 6 NA A/USSR/77 7 M To be determined 8 NS A/Shearwater/72 previously described. Only parental viruses were isolated. However, A/USSR/77 is known to have formed reassortants with human H3N2 strains in nature [6] and when A/Qld/72 (H3N2) was added to the reassortant mix, an H6N1 reassortant was isolated H6N1 RNA/RNA hybridization analysis The RNA/RNA hybridization results for H6N1 against the parental viruses are shown in Fig. 1. The only segment which came from the H3 virus was a polymerase gene. Because the three polymerase genes are difficult to assess on gel electrophoresis, the identity of the polymerase gene segments were confirmed by sequencing. Tree analysis of the sequence data of the PB1 segments of the three possible parents (A/Shearwater/72, A/ Qld/72, A/USSR/77) and the H6N1 reassortant is shown in Fig. 2 and indicates that the reassortant PB1 segment was identical (99.5%) to the parent A/Qld/72. Partial sequence analysis for PB2 and PA showed identity between the reassortant and A/Shearwater/72 but not with A/USSR/77 or A/Qld/72. The sequence of the matrix gene has yet to be determined between A/Shearwater/72 and A/USSR/77. Table 1 shows the H6N1 reassortant gene constellation. 4. Discussion These results indicate that the PB1 (gene or polymerase) may be required for gene reassortment involving human influenza viruses. The human H3 PB1 (avian origin) would appear to be highly successful in human reassortment combinations. The origin of the present successful human H3 PB1 gene is not known. There is evidence that reassortment is not random [7] and certain segments are seen to co-segregate [7,8]. Even host cell specificities may be linked to this co-segregation [9]. In both of the two most recent pandemics of 1957 and 1968, the PB1 and HA/NA came from avian sources [8] and were the result of an avian/human reassortment. A/USSR/77 was seen to exchange all of its internal segments with human H3N2 virus a year after its isolation [6]. The reassortment restriction seen between A/Shearwater/72 and A/USSR/77 may have been due to an incompatibility with the host cell (hen s egg). This incompatibility may have been overcome by the human H3 (avian origin) PB1 gene. Reassortants have been isolated without using serological selection [10]. Therefore, theoretically in a non-immune host, reassortment can occur if there are simultaneous

5 718 J.C. Downie / International Congress Series 1263 (2004) multiple infections. Scholtissek has proposed that the pig is a candidate for the mixing vessel [9]. Mixing vessels could also be young children or immune-compromised adults living in rural situations in close proximity to pigs, and/or avian species especially ducks. The number of influenza viruses capable of reassortment may be restricted. Reassortment involving human influenza viruses may depend on the compatibility of the PB1 and other internal genes in combination with host cell specificities. Acknowledgements The author wishes to thank Dr. A.J. Gibbs, Division of Botany and Zoology, Faculty of Science, ANU, Canberra ACT 2601, Australia for the sequence tree analysis of the PB1 genes of the parents and reassortant. Sequences were done at the Westmead Millennium Institute DNA Sequencing Facility (Westmead DNA) by Mark Wheeler and Ilya Henner. The author also thanks Nina Santiago, Eric Kapsalis for their technical support and Amelia Lacsamana for typing of manuscript. The author was a recipient of a Westmead Millennium Institute grant and previously by an NH and MRC grant. References [1] R.G. Webster, W.G. Laver, The origin of pandemic influenza, Bull. W.H.O. 47 (1972) [2] R.G. Webster, Antigenic hybrids of influenza A viruses with surface antigens to order, Virology 42 (1970) [3] J.C. Downie, A genetic and monoclonal analysis of high-yielding reassortants of influenza A virus used for human vaccines, J. Biol. Stand. 12 (1984) [4] A.J. Hay, et al., Procedures for characterisation of the genetic material of candidate vaccine strains, International Symposium on influenza immunization II, Geneva, Dev. Biol. Stand. 39 (1977) [5] Influenza sequence database, [6] P. Palase, J.F. Young, Variation of influenza A B and C viruses, Science 215 (1982) [7] M.D. Lubeck, P. Palase, J.L. Schulman, Non-random association of parental genes in influenza A virus recombinants, Virology 95 (1979) [8] Y. Kawaoka, S. Krauss, R.G. Webster, Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics, J. Virol. 63 (11) (1989) [9] C. Scholtissek, Pandemic influenza: antigenic shift, in: C.W. Potter (Ed.), Perspectives in Medical Virology 7 Influenza, Elsevier, Amsterdam, 2002, pp [10] W.G. Laver, J.C. Downie, Influenza virus recombination: I. Matrix protein markers and segregation during mixed infection, Virology 70 (1) (1976)

Existence of reassortant A (H1N2) swine influenza viruses in Saitama Prefecture, Japan

International Congress Series 1263 (2004) 749 753 Existence of reassortant A (H1N2) swine influenza viruses in Saitama Prefecture, Japan Shin ichi Shimada a, *, Takayasu Ohtsuka b, Masayuki Tanaka b, Munehito