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Short Communication |
1 Institut für Virologie und Antivirale Therapie, Universitätsklinikum, Friedrich Schiller Universität, Hans-Knöll-Str. 2, D-07745 Jena, Germany
2 Impfstoffwerk Dessau-Tornau (IDT), Bereich Forschung und Entwicklung, Streetzer Weg 15a, D-06861 Dessau-Roßlau, Germany
Correspondence
Roland Zell
Roland.Zell{at}med.uni-jena.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this study are EU053130–EU053151 and EU163946–EU163949.
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). Sixteen haemagglutinin and nine neuraminidase subtypes have been described so far, resulting in more than 100 circulating FLUAV subtypes, most of which are restricted to avian hosts, preferentially to aquatic birds.
Swine influenza was first recognized as a clinical entity during the 1918 influenza pandemic; in addition to the numerous human victims, many pigs suffered from flu-like symptoms (Shope, 1931). Virus strains isolated from pigs since 1930 were either human-influenza-like or belonged to a distinct genetic lineage designated ‘classical swine’ H1N1 influenza A virus. In the mid-1970s, swine influenza in Europe was caused by human A/Port Chalmers/1/73-like H3N2 viruses and by classical swine H1N1 influenza viruses (Nardelli et al., 1978; Donatelli et al., 1991; Castrucci et al., 1993, 1994). In 1979, novel porcine influenza A viruses (FLUAVsw) of the H1N1 subtype emerged (Scholtissek et al., 1983). These viruses (avian-like FLUAVsw, H1N1) are characterized by eight avian influenza genome segments (Fig. 1). Between 1983 and 1985, porcine H3N2 viruses appeared in Italy, which were described as being the result of a reassortment of human H3N2 and avian-like H1N1 viruses (Castrucci et al., 1993). The genes of the surface proteins HA and NA were human-like (human-like FLUAVsw, H3N2; Fig. 1, left), whereas the gene segments encoding the internal proteins were of avian origin (Campitelli et al., 1997). Another reassortment event occurred in 1994 (human-like FLUAVsw, H1N2; Fig. 1, centre), when H1N2 viruses arose in the UK with human-like HAH1 and NAN2 genes (Brown et al., 1995, 1997). After the initial bird-to-swine transmission and subsequent reassortments with human influenza viruses, the European porcine influenza A viruses underwent a region-wide spread in swine, which raises the possibility of further reassortment events. If novel reassortant strains are capable of establishing within the swine population, they may have a great impact on the epidemiological situation. Recently, we isolated H1N2 FLUAVsw strains with unusual antigenic properties during continuously running swine influenza surveillance in Germany. These viruses were associated with disease and displayed a mixture of the characteristics of porcine H1N2 and H3N2 strains. The aim of this study is the molecular and serological characterization of this novel H1N2 reassortant. In addition, sequences of the novel reassortant were compared to sequence data retrieved from the GenBank database.
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80 kg body weight) with symptoms of disease. Isolate A/sw/Cloppenburg/IDT4777/05 originated from a pig farm in Cloppenburg, Lower Saxony. The first signs of disease were also observed in December 2005. The pigs developed laboured breathing, loss of appetite and a cough. Four days after disease had become apparent in the pig herd, nasal swabs were taken from 6- to 7-week-old pigs. The two locations are approximately 30 km apart, indicating a spread of this strain within this region during December 2005. Isolation of A/sw/Bakum/1832/00 (the first H1N2 strain isolated in Germany) has been described previously (Schrader & Suess, 2003). Another H1N2 isolate used for establishment of pig hyperimmune sera (A/sw/Visbek/IDT3311/04) was isolated during the course of the IDT surveillance. Viruses were passaged on Madin–Darby bovine kidney (MDBK) or Madin–Darby canine kidney (MDCK) cells as described previously (Schmidtke et al., 2006).
Both H1N2 isolates became conspicuous by their unusual serological properties. Results of haemagglutination inhibition (HI) assays and neutralization tests are presented in Table 1. Monospecific hyperimmune sera used in this study were established in pigs by fourfold immunization with viral antigens and Freund's adjuvant. The pigs were derived from a pig herd that had been free from influenza virus infections for several years. The following antigens were used: (i) two recent vaccine strains [A/IDT/Re220/92 (H3N2) and A/IDT/Re230/92 (H1N1)], (ii) three vaccine strains from a swine flu vaccine in development [A/sw/Bakum/1832/00 (H1N2), A/sw/Haselünne/IDT2617/03 (H1N1) and A/sw/Bakum/IDT1769/03 (H3N2)] and (iii) a recent A/sw/Bakum/1832/00-like H1N2 isolate from Germany [A/sw/Visbek/IDT3311/04 (H1N2)]. All H1N2 strains examined in this study belong to the genetic lineage with avian-like internal genes and human-like HAH1 and NAN2 genes prevalent in Europe (Brown et al., 1997). Furthermore, negative sera were prepared from pigs that had never had any FLUAV infection. For HI assay, a threefold pretreatment of sera was carried out: pretreatment with neuraminidase (Sigma N-3001) for 14–18 h at 37 °C, inactivation for 30 min at 56 °C after addition of sodium citrate and finally adsorption of sera with chicken red blood cells for 1 h at 4–8 °C. Serial twofold dilution of sera was performed on microtitre plates. Standardized antigen (with a haemagglutination titre of eight haemagglutinating units of each antigen investigated) was then added to all wells and the mixture was incubated at 20–25 °C for 30 min and a standardized chicken red blood cell solution (0.5 %) was then added. The reaction was incubated at 20–25 °C for 30 min. Negative and positive controls as well as sera controls and a red blood cell control were included. Finally, HI titres were recorded.
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) is less efficient. In the HI test, the cross-reaction in sera of recovered animals was noticeably lower with A/sw/Bakum/1832/00, which served as a typical representative of German H1N2 FLUAVsw, whereas the reaction was more pronounced with the homologous isolate. This low level of reaction indicated diagnostic difficulties in detecting infected pigs with heterologous A/sw/Bakum/1832/00-like antigens in the HI test. In contrast, serum-neutralization experiments revealed a substantial inactivation of both viruses by A/sw/Bakum/1832/00 hyperimmune sera. Moreover, typical H1N2 hyperimmune sera of the A/sw/Bakum/1832/00 group exhibited a clear reaction with the novel H1N2 reassortants in HI. The serological results indicate that these strains are deviant from though antigenically related to the group of human-like H1N2 FLUAVsw viruses. In particular, this property is obvious in sera of recovered pigs, whereas the hyperimmune sera used in HI disguise the antigenic drift. Furthermore, it is noteworthy that antigens of the novel H1N2 type induced a strong background of non-specific reactions in HI. For sequencing, total RNA was prepared from virus-infected MDCK cells by employing the Qiagen RNeasy Mini kit. Reverse transcription was conducted with oligo(dT)20 primer, 20 U reverse transcriptase (Fermentas) and 5 µg RNA in a final reaction volume of 20 µl. A set of specific oligonucleotide primers and cDNA was used for the amplification of DNA using standard PCR cycling conditions. Amplification products were subjected to agarose gel electrophoresis and extracted using the QIAquick gel extraction kit (Qiagen). After elution in 50 µl buffer, purified products were stored at –20 °C. Cycle sequencing was performed with the DYEnamic ET Terminator kit (Amersham Biosciences) and the CEQ DTCS Quick Start kit (Beckman Coulter). Nucleotide sequences were analysed on a Prism 310 Genetic Analyzer (Applied Biosystems) and a CEQ8000 DNA Genetic Analysis System (Beckman Coulter). The 5'-terminal sequences were determined using 5'-specific oligonucleotides as primers. The sequence of the 3' end was determined with 3'-RACE (random amplification of cDNA ends). Sequencing of the complete genomes of both viruses confirmed the presence of human-like HAH1 and NAN2 segments and avian-like PB1, PB2, PA, NP, M and NS segments. However, the sequence of the NAN2 gene differed significantly from that of other European H1N2 FLUAVsw isolates.
For further characterization, the sequences of the complete NA gene (1410 nt) were aligned to those of other influenza virus isolates retrieved from GenBank and used for phylogenetic tree inference. Sequences were aligned manually or with MEGA version 3.1 (Kumar et al., 2004). Unrooted tree topology from multiple alignments and branch lengths were obtained by using (i) the fast neighbour-joining method (MEGA 3.1) and (ii) the maximum-likelihood method as implemented in TreePuzzle 3.0 (Strimmer & von Haeseler, 1997). Bootstrap values/posterior probabilities were calculated in order to test the consistency of branching. At least 10 000 resamplings of the data using MEGA 3.1 and 25 000 puzzling steps for the quartet puzzling algorithm of TreePuzzle were performed. For tree visualization, TreeView version 1.6.6 was used (Page, 1996).
In the first approach, 137 NA sequences representing European, Asian and American isolates of human, swine and birds were analysed by the neighbour-joining method (data not shown). Genetically distinct American and Asian FLUAVsw sequences were then removed for clarity. In addition, all avian sequences were also removed except A/duck/Hong Kong/7/1975 (H3N2) and A/mallard duck/New York/170/1982 (H1N2), which served as outgroups. The resulting dataset (60 sequences) consisted of all available European FLUAVsw NAN2 sequences published in GenBank and a number of human NAN2 sequences representing H2N2, H3N2 and H1N2 isolates. This NAN2 sequence dataset was subjected to tree inference. The resultant maximum-likelihood tree (Fig. 2a) demonstrates clustering of A/sw/Dötlingen/IDT4735/05 and A/sw/Cloppenburg/IDT4777/05 with the prevalent European H3N2 FLUAVsw but not with the European H1N2 viruses. Interestingly, the NA sequence of A/sw/UK/119404/91 (H3N2) clustered with the H1N2 viruses, suggesting that this virus might be the NA donor of the human-like H1N2 FLUAVsw, which emerged in the UK in 1994. For the HAH1 phylogenetic tree, 112 sequences including all available HAH1 sequences from European FLUAVsw, several classical swine sequences, avian H1N1 sequences and a representative number of human H1N1 and H1N2 sequences were aligned and analysed using the fast neighbour-joining method (data not shown). Asian and American isolates clustering with the human HA sequences as well as some of the avian-like FLUAVsw isolates were then removed for clarity to yield a set of 61 sequences. The maximum-likelihood analysis was conducted with this dataset and is presented in Fig. 2(b). As is evident from the tree, the German H1N2 isolates cluster together with other European human-like H1N2 FLUAVsw. The avian-like H1N1 FLUAVsw are genetically distinct. Notably, the isolate A/sw/Italy/2064/99 (H1N2) has an avian-like HAH1, whereas its NAN2 clusters with the H1N2 viruses.
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. Here, isolation and characterization of the novel reassortant are described for the first time. The findings indicate that the process of reassortment of FLUAVsw in pigs is continuing. The reassortant viruses are associated with flu-like clinical symptoms in pigs. They were isolated in areas of Germany that are densely populated with pigs, which are more prone to double and multiple FLUAV infections than regions less densely populated with pigs. The isolations occurred after a period of strong H3N2 activity. From spring to autumn 2005, H3N2 was the most frequently isolated subtype in that region, whereas the H1N2 subtype had not been isolated for more than a year, although it was obvious from serological investigations that it has been continuing to co-circulate since its first detection in Germany in 2000. The results of the present investigations lead to the following conclusions: (i) a novel H1N2 reassortant has become established in the German pig population; (ii) within this novel reassortant, the human-like NAN2 of the H1N2 clade has been replaced by another human-like NAN2 from the H3N2 FLUAVsw clade; (iii) despite the close relationship of the HAH1 gene to those of other H1N2 viruses circulating in Germany since 2000, serological cross-reaction may be limited, indicating a need to incorporate these reassortant H1N2 strains into serological investigations for swine flu surveillance; and (iv) veterinary surgeons performing serological diagnosis by HI have to take into account that there are background reactions that require an increase in cut-off values. To date, porcine influenza viruses have been thought to be relatively stable. However, the isolation of the novel reassortant H1N2 viruses reveals two important issues. Firstly, in dense European pig populations, the frequency of reassortment events may have increased in recent years, indicating a requirement for improved surveillance activities in future. Secondly, the observed antigenic variation may be indicative of an ongoing antigenic drift of the haemagglutinin of the H1N2 FLUAVsw, which may have consequences for vaccination policy.
To date, no commercial vaccines are available that protect against the H1N2 subtype (see Van Reeth et al., 2003). Nevertheless, such a trivalent vaccine is in development. It is based on avian-like swine H1N1, human-like swine H3N2 and swine H1N2 [A/sw/Bakum/1832/00 (H1N2)-like] vaccine strains. Although there are signs of cross-protection, the lowered neutralizability of the novel H1N2 reassortants by A/sw/Bakum/1832/00 (H1N2)-like strains and the impaired detection of antibodies in recovered pigs indicate the need for further investigations to test the protection from the novel H1N2 reassortant by the H1N2 vaccine strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Brown, I. H., Ludwig, S., Olsen, C. W., Hannoun, C., Scholtissek, C., Hinshaw, V. S., Harris, P. A., McCauley, J. W., Strong, I. & Alexander, D. J. (1997). Antigenic and genetic analyses of H1N1 influenza A viruses from European pigs. J Gen Virol 78, 553–562.[Abstract]
Campitelli, L., Donatelli, I., Foni, E., Castrucci, M. R., Fabiani, C., Kawaoka, Y., Krauss, S. & Webster, R. G. (1997). Continued evolution of H1N1 and H3N2 influenza viruses in pigs in Italy. Virology 232, 310–318.[CrossRef][Medline]
Castrucci, M. R., Donatelli, I., Sidoli, L., Barigazzi, G., Kawaoka, Y. & Webster, R. G. (1993). Genetic reassortment between avian and human influenza A viruses in Italian pigs. Virology 193, 503–506.[CrossRef][Medline]
Castrucci, M. R., Campitelli, L., Ruggieri, A., Barigazzi, G., Sidoli, L., Daniels, R., Oxford, J. S. & Donatelli, I. (1994). Antigenic and sequence analysis of H3 influenza virus haemagglutinins from pigs in Italy. J Gen Virol 75, 371–379.
Donatelli, I., Campitelli, L., Castrucci, M. R., Ruggieri, A., Sidoli, L. & Oxford, J. S. (1991). Detection of two antigenic subpopulations of A (H1N1) influenza viruses from pigs: antigenic drift or interspecies transmission? J Med Virol 34, 248–257.[Medline]
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Nardelli, L., Pascucci, S., Gualandi, G. L. & Loda, P. (1978). Outbreaks of classical swine influenza in Italy in 1976. Zentralbl Veterinarmed B 25, 853–857.[Medline]
Page, R. D. M. (1996). TreeView: An application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357–358.
Palese, P. & Shaw, M. L. (2007). Orthomyxoviridae: the viruses and their replication. In Fields Virology, 5th edn, vol. 2, pp. 1647–1689. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Schmidtke, M., Zell, R., Bauer, K., Krumbholz, A., Schrader, C., Suess, J. & Wutzler, P. (2006). Amantadine resistance among porcine H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany between 1981 and 2001. Intervirology 49, 286–293.[CrossRef][Medline]
Scholtissek, C., Burger, H., Bachmann, P. A. & Hannoun, C. (1983). Genetic relatedness of hemagglutinins of the H1 subtype of influenza A viruses isolated from swine and birds. Virology 129, 521–523.[CrossRef][Medline]
Schrader, C. & Suess, J. (2003). Genetic characterization of a porcine H1N2 influenza virus strain isolated in Germany. Intervirology 46, 66–70.[CrossRef][Medline]
Shope, R. E. (1931). Swine influenza. I. Experimental transmission and pathology. J Exp Med 54, 349–359.[Abstract]
Strimmer, K. & von Haeseler, A. (1997). Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc Natl Acad Sci U S A 94, 6815–6819.
Van Reeth, K., Van Gucht, S. & Pensaert, M. (2003). Investigations of the efficacy of European H1N1- and H3N2-based swine influenza vaccines against the novel H1N2 subtype. Vet Rec 153, 9–13.
Received 27 July 2007;
accepted 27 September 2007.
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