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Human influenza A viruses isolated in South America: Genetic relations, adamantane resistance and vaccine strain match
Infection, Genetics and Evolution 9 (2009) 229–234
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Human influenza A viruses isolated in South America: Genetic relations, adamantane resistance and vaccine strain match§ ˜ i a,b, Jose´ Russi c, Juan Cristina a,* Natalia Gon a
Laboratorio de Virologı´a Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Igua´ 4225, 11400 Montevideo, Uruguay Centro Nacional de Referencia de Influenza, Servicio Nacional de Laboratorios de Salud Pu´blica, Ministerio de Salud Pu´blica, Av. 8 de Octubre 2720, 11200 Montevideo, Uruguay c Former Head of Servicio Nacional de Laboratorios, Ministerio de Salud Pu´blica, Repu´blica Argentina 1815, Montevideo, Uruguay b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 August 2008 Received in revised form 12 November 2008 Accepted 13 November 2008 Available online 27 November 2008
In order to gain insight into the genetic relations among H3N2 Influenza A virus (IAV) circulating in the South American region from 1999 to 2007, to investigate the presence of adamantane-resistant strains in this region, and to establish the genetic relations among that strains and vaccine strains recommended for the Southern hemisphere, 11 haemagglutinin (HA) H3 IAV sequences obtained from Uruguayan patients were aligned with corresponding sequences from 68 H3 IAV strains isolated in South America and 9 H3 IAV vaccine strains. Maximum likelihood phylogenetic tree analysis was performed using the GTR evolutionary model. The results of these studies indicate that multiple clades co-circulate during most influenza seasons in South America. Strikingly, one strain isolated in Uruguay in 2005 and all strains isolated in that country during the 2007 season bear an HA adamantane-resistant polymorphism. No other strain isolated in South America previous to the 2005 season bears that HA characteristic amino acid change. Only vaccine strains recommended for the 2007 season were assigned to the same cluster with all available IAV isolated in South America for that season. Evolution of IAV in this region appears to be shaped by re-introduction of new strains. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Influenza A virus Vaccine strains Evolution South America Adamantane resistance
1. Introduction Influenza A virus (IAV) is a member of the family Orthomyxoviridae and contains eight segments of a single-stranded RNA genome with negative polarity (Neumann et al., 2004). IAV causes 300,000–500,000 deaths worldwide each year, and in pandemic years, this number can increase to 1 million (in 1957–1958) or as high as 50 million, as was seen in 1918–1919 (Nguyen-Van-Tam and Hampson, 2003; WHO, 2007). IAV evades host immunity by accumulation of point mutations (drift) in the major surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA), or as a result of genetic reassortment of segments from different IAV strains co-infecting the same cell (shift) (Nicholson et al., 2003). New IAV pandemics may emerge through reassortation with strains from the avian reservoir or by direct introduction of an
§ Note: Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers AM991338 through AM991342. * Corresponding author. Tel.: +598 2 525 09 01; fax: +598 2 525 08 95. E-mail address: [email protected] (J. Cristina).
1567-1348/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2008.11.004
avian strain into the human population (Taubenberger and Morens, 2006). At present time only 2 of 16 possible HA subtypes (H1 and H3) and 2 of 9 possible NA (N1 and N2) described to date are circulating in the human population. H3N2 and H1N1 IAV have co-circulated in humans since the re-emergence of H1N1 in 1977. IAV H3N2 viruses have been the predominant strains during the last 20 years, with the exception of the 1988–1989 and 2000–2001 seasons where H1N1 infections dominated (Lin et al., 2004). Based on antigenic analyses of recently isolated influenza viruses, epidemiologic data, post-vaccination serologic studies in humans, and the availability of candidate vaccine strains, the World Health Organization (WHO) recommend the influenza virus strains to be included in the trivalent influenza vaccine, composed of one H1N1 and one H3N2 IAV strains plus one Influenza B virus strain, for each Northern hemisphere winter season (available at: http:// www.who.int/csr/disease/influenza/vaccinerecommendations1/ en/index.html). Since October 1998, a second meeting of WHO is held to evaluate the vaccine formula in order to recommend the influenza virus strains to be included in the vaccine for the Southern hemisphere (Pontoriero et al., 2003; available at: http://
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www.who.int/csr/disease/influenza/vaccinerecommendations1/ en/index.html). Adamantanes, like amantadine or rimantadine, have been used as anti-viral agents against IAV in developed countries (Hata et al., 2007). Adamantanes block the ion channel of IAV M2 protein and thus inhibit the pH change necessary for the uncoating process (Wang et al., 1993). A dramatic rise in the frequency of resistance to adamantane drugs by H3N2 IAV has occurred in recent years in different countries (Simonsen et al., 2007). In the influenza 2005– 2006 season, an important increase of resistant viruses in communities was observed in Japan (Saito et al., 2007), Southeast Asia and Australia (Barr et al., 2007), and North America (Bright et al., 2006). Furthermore, the resistant viruses were already detected in September 2005, just before the influenza season in Nagasaki, Japan, and even in previous 2004–2005 influenza season in Aichi prefecture in the same country (Hata et al., 2007). Phylogenetic analysis revealed that the resistant viruses belong to a single genetic lineage, sharing a mutation at amino acid position 31 of the M2 protein (S31N) (Simonsen et al., 2007; Hata et al., 2007). These analyses have also shown that these viruses also share common characteristic amino acid changes at positions 193 (S193F) and 225 (D225N) of the HA protein (Hata et al., 2007). Little is know about the genetic relations among H3N2 IAV strains circulating in the South American region, the presence and frequency of adamantane-resistant strains in this region, as well as the genetic relations among South American IAV strains and vaccine strains recommended for the Southern hemisphere. 2. Materials and methods 2.1. Human samples Nasal swabs from 11 Uruguayan patients with clinical symptoms of influenza were available at the National Influenza Reference Centre, Montevideo, Uruguay. 2.2. Virus type In order to address virus type, nasal swabs were first cultured in MDCK cells. Viral antigens were detected by an immunofluorescent assay with type-specific monoclonal antibodies (Chemicon International, Inc., CA, USA) and with the Directigen Flu A+B (Beckton Dickinson Europe, Maylan, France). Virus isolates were typed by haemagglutination inhibition assay (HAI) with the WHO Influenza reagent kit, provided by the Center for Disease Control and Prevention (CDC), Atlanta, GA, USA All 11 IAV strains isolated from the Uruguayan patients were assigned to subtype H3. 2.3. RNA extraction and RT-PCR amplification Total RNA was extracted from infected cells using Trizol (Gibco, BRL) according to manufacturer’s instructions. Extracted RNA was eluted with 15 ml of UltraPure DNAse/RNase-free distilled water (Gibco, BRL, Life Technologies) and cDNA synthesis and PCR amplification of IAV HA gene was carried out as previously described (Ellis et al., 1997). Amplicons were purified by using a QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer’s instructions prior to sequencing. 2.4. Sequencing reactions Purified PCR products were sequenced directly. The primers used for amplification were also used for sequencing the PCR fragments. The sequencing reaction was carried out using a BigDye DNA sequencing kit on a 373 DNA Sequencer Apparatus, both from PerkinElmer.
2.5. Nucleotide sequence accession numbers Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers AM991338 through AM991342. For sequence names and accession numbers see Table 1.
Table 1 Origins of the H3 IAV strains isolated in South America. Name
Accession number
Country of isolation
A/Rio de Janeiro/57/1999 A/Espirito Santo/33/1999 A/Rio Grande do Sul/25/1999 A/Rio Grande do Sul/21/1999 A/Espirito Santo/14/1999 A/Espirito Santo/3/1999 A/Buenos Aires/M6/1999 A/Chaco/140/1999 A/Buenos Aires/M14/1999 A/Mar del Plata/267/1999 A/Brazil/003/2000 A/Brazil/024/2000 A/Brazil/011/2000 A/Brazil/013/2000 A/Brazil/006/2000 A/Brazil/010/2000 A/Brazil/008/2000 A/Rio de Janeiro/28/2000 A/Espirito Santo/128/2000 A/Rio de Janeiro/172/2000 A/Rio de Janeiro/580/2001 A/Rio Grade do Sul/523/2001 A/Chile/6416/2001 A/Espirito Santo/452/2001 A/Rio de Janeiro/565/2001 A/Rio de Janeiro/471/2001 A/Rio de Janeiro/470/2001 A/Rio de Janeiro/465/2001 A/Rio de Janeiro/310/2001 A/Espirito Santo/454/2001 A/Brazil/125/2001 A/Rio de Janeiro/533/2001 A/Rio de Janeiro/478/2001 A/Rio Grande do Sul/523/2001 A/Brazil/722/2001 A/Neuquen/1016002/2001 A/Cordoba/1007333/2001 A/Neuquen/1038228/2001 A/Neuquen/2260/2001 A/Chile/6416/2001 A/Chaco/R538/2001 A/Santa Catarina/339/2002 A/Espirito Santo/88/2002 A/Santa Catarina/311/2002 A/Rio Grande do Sul/205/2002 A/Santa Catarina/327/2002 A/Rio de Janeiro/98/2003 A/Rio de Janeiro/99/2003 A/Rio de Janeiro/107/2003 A/Rio de Janeiro/346/2003 A/Uruguay/11/2003 A/Santa Catarina/379/2004 A/Minas Gerais/156/2004 A/Minas Gerais/154/2004 A/Rio de Janeiro/17/2004 A/Rio Grande do Sul/406/2004 A/Santa Catarina/380/2004 A/Parana/306/2004 A/Parana/308/2004 A/Parana/312/2004 A/Rio Grande do Sul/212/2004 A/Parana/298/2004 A/Rio Grande do Sul/417/2004 A/Rio Grande do Sul/411/2004 A/Rio de Janeiro/26/2004
AY968022 AY968021 AY968020 AY968019 AY968018 AY968017 AF534032 AF534035 AF534034 AF534040 DQ336007 DQ336007 DQ336016 DQ336017 DQ336015 DQ336013 DQ336011 AY968023 AY968024 AY968025 AY968036 AY968033 DQ865972 AY968027 AY968035 AY968031 AY968030 AY968029 AY968026 AY968028 DQ330006 AY068040 AY968032 AY968033 AF534056 AF534059 AF534058 AF534060 AF534057 DQ865972 AF534055 AY968040 AY968041 AY968038 AY968033 AY968039 AY972834 AY972832 AY972833 AY972851 AM991343 AY972848 AY972827 AY972829 AY972847 AY972846 AY972845 AY972837 AY972838 AY972849 AY972835 AY972830 AY972844 AY972841 AY972840
Brazil Brazil Brazil Brazil Brazil Brazil Argentina Argentina Argentina Argentina Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Chile Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Argentina Argentina Argentina Argentina Chile Argentina Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Uruguay Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil
N. Gon˜i et al. / Infection, Genetics and Evolution 9 (2009) 229–234 Table 1 (Continued )
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Sequences were aligned using the CLUSTAL W program (Thompson et al., 1994). Once aligned, the program Modelgenerator (Keane et al., 2006) was used to identify the optimal evolutionary model that described our sequence dataset (Akaike Information Criteria and Hierarchical Likelihood Ratio Test indicated that the GTR model best fit the sequence data). Using this model, maximum likelihood phylogenetic trees were constructed using software from the PhyML program (Guindon et al., 2005). As a measure of the robustness of each node, we used an approximate likelihood ratio test (aLRT), that demonstrate that the branch studied provides a significant likelihood against the null hypothesis that involves collapsing that branch of the phylogenetic tree but leaving the rest of the tree topology identical (Anisimova and Gascuel, 2006). We have used an aLRT non-parametric branch support based on a Shimodaira– Hasegawa-like (SH-like) procedure (Shimodaira and Hasegawa, 1999) as implemented in the PhyML program (Guindon et al., 2005). HA nucleotide sequences were translated to amino acid sequences in silico using the MEGA 3.0 program (Kumar et al., 2004).
(Bao et al., 2008), using the CLUSTAL W program (Thompson et al., 1994). Once aligned, phylogenetic trees were created using the maximum likelihood method under the GTR model (Guindon et al., 2005) and the robustness of the nodes in the trees was assessed by aLRT (Anisimova and Gascuel, 2006). The results of these studies are shown in Fig. 1. All IAV strains included in these studies can be assigned to different clusters, all of them supported by very high values of aLRT (Fig. 1). Different genetic lineages co-circulate in a particular season in a specific country, like in Brazil during the 2000 season (see Fig. 1, bottom). Co-circulation can be also observed in the region, like in the 2001 season, where two different genetic lineages can be observed, one that circulated in Brazil, while another circulated in Brazil, Argentina and Chile (Fig. 1, top and bottom). The same observations can be made for the 2004 season, where at least two distinct genetic lineages are observed, one in Brazil and Ecuador, and another in Brazil and Uruguay (Fig. 1, top). Interesting, co-circulation of IAV genetic lineages in South America are not only seen in big countries, like Brazil, but also in small ones, like Uruguay, where co-circulation can be also found, like in the 2005 season (see Fig. 1, middle). To study the possible presence of adamantine-resistant IAV strains in South America, the HA characteristic amino acid changes were observed in the H3 IAV strains isolated in this region (Hata et al., 2007). The results of these studies are shown in Fig. 2. All strains isolated in Uruguay during the 2007 season bear an adamantine-resistant polymorphism (Fig. 2). Importantly, another strain isolated in Uruguay in the 2005 season bear this changes in the HA gene, but the adamantine-resistant polymorphism is not found in three other viruses isolated in Uruguay and Peru during the same season (see Figs. 1 and 2). No other strain isolated in South America previous to the 2005 season bears the adamantineresistant polymorphism (see Figs. 1 and 2). In order to gain insight into the genetic relations among H3 IAV strains that circulated in South America from 1999 to 2007 and vaccine strains recommended for the Southern hemisphere for that seasons, the appropriate vaccine strains were included in the phylogenetic analysis (see Fig. 1 and Table 2). Only vaccine strains recommended for the 2007 season share the same cluster with available IAV strains isolated in South America in that season (Fig. 1).
3. Results
4. Discussion
In order to get insight into the genetic relations among H3 IAV isolated in South America and vaccine strains included in the influenza vaccines for the 1999–2007 influenza seasons for the Southern hemisphere, the HA sequences obtained from the Uruguayan patients were aligned with corresponding sequences from 68 H3 IAV strains isolated in South America (Table 1) and 9 H3 IAV vaccine strains (Table 2), obtained from the Influenza Database
The results of this study indicate that multiple clades cocirculates during most of the influenza seasons in South America (see Fig. 1). These results are in agreement with recent findings by Nelson et al. (2007) for Australia and New Zealand. Although some strains isolated in the region can be linked over multiple seasons (Fig. 1, bottom), suggesting that in situ evolution cannot be ruled for some seasons without further studies, viruses do not seem to have regularly evolved in geographic isolation (see for instance Brazilian strains isolated during 2000–2004 seasons in Fig. 1). Rather, evolution appears to be shaped by re-introduction of new strains. This is in agreement with recent studies that have provided evidence for regular cross-hemisphere viral migration between seasons, even among localities very distantly separated (Nelson et al., 2007; Viboud et al., 2006, 2004). Moreover, very recent studies revealed a continuous circulation of IAV in East and Southeast Asia (E-SE Asia) via a region-wide network of temporally overlapping epidemics and that epidemics in the temperate regions are seeded from this network each year. Seed strains first generally reached Oceania, North America and Europe, and later to the South American region (Russell et al., 2008). All strains isolated in Uruguay during the 2007 season bear an adamantine-resistant HA polymorphism (Fig. 2). Due to the fact of
Name
Accession number
Country of isolation
A/Parana/291/2004 A/Parana/310/2004 A/Minas Gerais/163/2004 A/Minas Gerais/163/2004 A/Ecuador/1968/2004 A/Uruguay/01/2004 A/Peru/166/2005 A/Uruguay/02/2005 A/Uruguay/03/2005 A/Uruguay/04/2005 A/Uruguay/01/2007 A/Uruguay0/4/2007 A/Uruguay/05/2007 A/Uruguay/07/2007 A/Uruguay/10/2007 A/Uruguay/716/2007
AY972842 AY972843 AY972828 AY972831 DQ265716 AM991344 DQ265708 AM991338 AM991339 AM991340 AM991335 AM991336 AM991337 AM991341 AM991342 ISDN276547
Brazil Brazil Brazil Brazil Ecuador Uruguay Peru Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay
2.6. Phylogenetic analysis
Table 2 Origins of H3 IAV vaccine strains recommended for the Southern hemisphere.a. Name
Accession number
Season
A/Sydney/5/1997 A/Moscow/10/1999 A/Panama/2007/1999 A/Fujian/411/2002 A/Kumamoto/102/2002 A/Wellington/1/2004 A/California/7/2004 A/New York/55/2004 A/Wisconsin/67/2005 A/Hiroshima/52/2005
AF180584 AY531035 DQ508865 EF541397 EF456797 CY012104 DQ865973 EF473394 EF473424 EU283414
1999 2000, 2001, 2002, 2003 2001, 2002, 2003 2004 2004 2005 2006 2006 2007 2007
a For some seasons, more than one candidate vaccine viruses may be available for vaccine formulation.
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Fig. 1. Phylogenetic analysis of the Haemagglutinin gene of Influenza A virus circulating in the South American region from 1999 to 2007. An unrooted maximum likelihood tree obtained under the GTR model is shown. Strains in the tree are shown by their names, which also show geographic location and year of isolation, according to the World Health Organization (1980) (for accession numbers see Table 1). Country of isolation and season year is indicated on the right of each name between parentheses. Vaccine strains are shown in italics. Vaccine strains recommended for a specific Southern hemisphere influenza season and the IAV strains isolated South America in that season are
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Fig. 2. Alignment of HA amino acid sequences from IAV strains isolated in South America. Strains are shown by name at the left side of the figure. R.G. means Rio Grande; Sta. Cat. means Santa Catarina. HA1 amino acid sequences from position 143 to 234 (relative to strain A/Rio Grande do Sul/406/2004) are shown. Characteristic amino acid changes at positions 193 (Ser to Phe; S193F) and 225 (Asp to Asn; D225N) of the HA protein of adamantine-resistant viruses are shown in bold. Identity to strain A/Rio Grade do Sul/406/2004 (isolated in Brazil in the 2004 season) is indicated by a dash.
the infrequent use of adamantine drugs in South American countries, that all adamantine-resistant viruses belong to a single genetic lineage (Simonsen et al., 2007), and that it is very unlikely that these changes may arise just by chance in IAV circulating in South America, this particular polymorphism may permit to shed light into the possible migration of IAV strains to our region. The results of these studies suggest that adamantine-resistant viruses, first detected in the 2004–2005 season in Japan (Hata et al., 2007; Saito et al., 2007), and then spread to South East Asia, Oceania and North America during the 2005–2006 season (Bright et al., 2006; Barr et al., 2007; Simonsen et al., 2007; Saito et al., 2008) arrived to South America in the 2005 season. This is in agreement with a cross-hemisphere viral migration (Nelson et al., 2007; Viboud et al., 2006, 2004) and supports recent evidence that seeded strains from a E-SE Asia network finally arrive to the South American region (Russell et al., 2008). Nevertheless, not all South American isolates that belong to the 2005 season may have the same origin; since isolates of Peru and Uruguay do not bear the characteristic adamantine-resistant polymorphism (see Figs. 1 and 2). This speaks of a possibility of co-circulation of viruses of at least two different origins, although the effect of genomic reassortment cannot be ruled out. Nevertheless, the results of these studies reveal how migration may be playing an important role in the IAV epidemiology history in the South American region. For most of the South American influenza seasons studied vaccine and natural strains of the same season are assigned to different clusters (see Fig. 1). This indicates that for the majority of the seasons studied vaccine and circulating IAV strains have distant genetic relations among themselves. The results of these studies speak of the need of a more profound study of the IAV strains circulating in the South American region, as well as the routes and mechanisms of virus’s geographical spread. Containing the spread of IAV will require understand how the dynamics, antigenic evolution and seasonal emergence interrelate (Nelson and Holmes, 2007). Recent studies revealed that once H3N2 IAV strains leave the E-SE Asia network, they will probably impact in the South American region (Russell et al., 2008). For this reason, to gain insight into the patterns of spread from E-SE Asia to this region is extremely important for consequent improvements to vaccine strain selection for the Southern hemisphere. The results of this work also highlight the critical importance of expanding surveillance in the South American region, in order to elucidate the geographical movements and evolution of this virus
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Human influenza A viruses isolated in South America: Genetic relations, adamantane resistance and vaccine strain match§ ˜ i a,b, Jose´ Russi c, Juan Cristina a,* Natalia Gon a
Laboratorio de Virologı´a Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Igua´ 4225, 11400 Montevideo, Uruguay Centro Nacional de Referencia de Influenza, Servicio Nacional de Laboratorios de Salud Pu´blica, Ministerio de Salud Pu´blica, Av. 8 de Octubre 2720, 11200 Montevideo, Uruguay c Former Head of Servicio Nacional de Laboratorios, Ministerio de Salud Pu´blica, Repu´blica Argentina 1815, Montevideo, Uruguay b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 August 2008 Received in revised form 12 November 2008 Accepted 13 November 2008 Available online 27 November 2008
In order to gain insight into the genetic relations among H3N2 Influenza A virus (IAV) circulating in the South American region from 1999 to 2007, to investigate the presence of adamantane-resistant strains in this region, and to establish the genetic relations among that strains and vaccine strains recommended for the Southern hemisphere, 11 haemagglutinin (HA) H3 IAV sequences obtained from Uruguayan patients were aligned with corresponding sequences from 68 H3 IAV strains isolated in South America and 9 H3 IAV vaccine strains. Maximum likelihood phylogenetic tree analysis was performed using the GTR evolutionary model. The results of these studies indicate that multiple clades co-circulate during most influenza seasons in South America. Strikingly, one strain isolated in Uruguay in 2005 and all strains isolated in that country during the 2007 season bear an HA adamantane-resistant polymorphism. No other strain isolated in South America previous to the 2005 season bears that HA characteristic amino acid change. Only vaccine strains recommended for the 2007 season were assigned to the same cluster with all available IAV isolated in South America for that season. Evolution of IAV in this region appears to be shaped by re-introduction of new strains. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Influenza A virus Vaccine strains Evolution South America Adamantane resistance
1. Introduction Influenza A virus (IAV) is a member of the family Orthomyxoviridae and contains eight segments of a single-stranded RNA genome with negative polarity (Neumann et al., 2004). IAV causes 300,000–500,000 deaths worldwide each year, and in pandemic years, this number can increase to 1 million (in 1957–1958) or as high as 50 million, as was seen in 1918–1919 (Nguyen-Van-Tam and Hampson, 2003; WHO, 2007). IAV evades host immunity by accumulation of point mutations (drift) in the major surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA), or as a result of genetic reassortment of segments from different IAV strains co-infecting the same cell (shift) (Nicholson et al., 2003). New IAV pandemics may emerge through reassortation with strains from the avian reservoir or by direct introduction of an
§ Note: Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers AM991338 through AM991342. * Corresponding author. Tel.: +598 2 525 09 01; fax: +598 2 525 08 95. E-mail address: [email protected] (J. Cristina).
1567-1348/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2008.11.004
avian strain into the human population (Taubenberger and Morens, 2006). At present time only 2 of 16 possible HA subtypes (H1 and H3) and 2 of 9 possible NA (N1 and N2) described to date are circulating in the human population. H3N2 and H1N1 IAV have co-circulated in humans since the re-emergence of H1N1 in 1977. IAV H3N2 viruses have been the predominant strains during the last 20 years, with the exception of the 1988–1989 and 2000–2001 seasons where H1N1 infections dominated (Lin et al., 2004). Based on antigenic analyses of recently isolated influenza viruses, epidemiologic data, post-vaccination serologic studies in humans, and the availability of candidate vaccine strains, the World Health Organization (WHO) recommend the influenza virus strains to be included in the trivalent influenza vaccine, composed of one H1N1 and one H3N2 IAV strains plus one Influenza B virus strain, for each Northern hemisphere winter season (available at: http:// www.who.int/csr/disease/influenza/vaccinerecommendations1/ en/index.html). Since October 1998, a second meeting of WHO is held to evaluate the vaccine formula in order to recommend the influenza virus strains to be included in the vaccine for the Southern hemisphere (Pontoriero et al., 2003; available at: http://
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www.who.int/csr/disease/influenza/vaccinerecommendations1/ en/index.html). Adamantanes, like amantadine or rimantadine, have been used as anti-viral agents against IAV in developed countries (Hata et al., 2007). Adamantanes block the ion channel of IAV M2 protein and thus inhibit the pH change necessary for the uncoating process (Wang et al., 1993). A dramatic rise in the frequency of resistance to adamantane drugs by H3N2 IAV has occurred in recent years in different countries (Simonsen et al., 2007). In the influenza 2005– 2006 season, an important increase of resistant viruses in communities was observed in Japan (Saito et al., 2007), Southeast Asia and Australia (Barr et al., 2007), and North America (Bright et al., 2006). Furthermore, the resistant viruses were already detected in September 2005, just before the influenza season in Nagasaki, Japan, and even in previous 2004–2005 influenza season in Aichi prefecture in the same country (Hata et al., 2007). Phylogenetic analysis revealed that the resistant viruses belong to a single genetic lineage, sharing a mutation at amino acid position 31 of the M2 protein (S31N) (Simonsen et al., 2007; Hata et al., 2007). These analyses have also shown that these viruses also share common characteristic amino acid changes at positions 193 (S193F) and 225 (D225N) of the HA protein (Hata et al., 2007). Little is know about the genetic relations among H3N2 IAV strains circulating in the South American region, the presence and frequency of adamantane-resistant strains in this region, as well as the genetic relations among South American IAV strains and vaccine strains recommended for the Southern hemisphere. 2. Materials and methods 2.1. Human samples Nasal swabs from 11 Uruguayan patients with clinical symptoms of influenza were available at the National Influenza Reference Centre, Montevideo, Uruguay. 2.2. Virus type In order to address virus type, nasal swabs were first cultured in MDCK cells. Viral antigens were detected by an immunofluorescent assay with type-specific monoclonal antibodies (Chemicon International, Inc., CA, USA) and with the Directigen Flu A+B (Beckton Dickinson Europe, Maylan, France). Virus isolates were typed by haemagglutination inhibition assay (HAI) with the WHO Influenza reagent kit, provided by the Center for Disease Control and Prevention (CDC), Atlanta, GA, USA All 11 IAV strains isolated from the Uruguayan patients were assigned to subtype H3. 2.3. RNA extraction and RT-PCR amplification Total RNA was extracted from infected cells using Trizol (Gibco, BRL) according to manufacturer’s instructions. Extracted RNA was eluted with 15 ml of UltraPure DNAse/RNase-free distilled water (Gibco, BRL, Life Technologies) and cDNA synthesis and PCR amplification of IAV HA gene was carried out as previously described (Ellis et al., 1997). Amplicons were purified by using a QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer’s instructions prior to sequencing. 2.4. Sequencing reactions Purified PCR products were sequenced directly. The primers used for amplification were also used for sequencing the PCR fragments. The sequencing reaction was carried out using a BigDye DNA sequencing kit on a 373 DNA Sequencer Apparatus, both from PerkinElmer.
2.5. Nucleotide sequence accession numbers Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession numbers AM991338 through AM991342. For sequence names and accession numbers see Table 1.
Table 1 Origins of the H3 IAV strains isolated in South America. Name
Accession number
Country of isolation
A/Rio de Janeiro/57/1999 A/Espirito Santo/33/1999 A/Rio Grande do Sul/25/1999 A/Rio Grande do Sul/21/1999 A/Espirito Santo/14/1999 A/Espirito Santo/3/1999 A/Buenos Aires/M6/1999 A/Chaco/140/1999 A/Buenos Aires/M14/1999 A/Mar del Plata/267/1999 A/Brazil/003/2000 A/Brazil/024/2000 A/Brazil/011/2000 A/Brazil/013/2000 A/Brazil/006/2000 A/Brazil/010/2000 A/Brazil/008/2000 A/Rio de Janeiro/28/2000 A/Espirito Santo/128/2000 A/Rio de Janeiro/172/2000 A/Rio de Janeiro/580/2001 A/Rio Grade do Sul/523/2001 A/Chile/6416/2001 A/Espirito Santo/452/2001 A/Rio de Janeiro/565/2001 A/Rio de Janeiro/471/2001 A/Rio de Janeiro/470/2001 A/Rio de Janeiro/465/2001 A/Rio de Janeiro/310/2001 A/Espirito Santo/454/2001 A/Brazil/125/2001 A/Rio de Janeiro/533/2001 A/Rio de Janeiro/478/2001 A/Rio Grande do Sul/523/2001 A/Brazil/722/2001 A/Neuquen/1016002/2001 A/Cordoba/1007333/2001 A/Neuquen/1038228/2001 A/Neuquen/2260/2001 A/Chile/6416/2001 A/Chaco/R538/2001 A/Santa Catarina/339/2002 A/Espirito Santo/88/2002 A/Santa Catarina/311/2002 A/Rio Grande do Sul/205/2002 A/Santa Catarina/327/2002 A/Rio de Janeiro/98/2003 A/Rio de Janeiro/99/2003 A/Rio de Janeiro/107/2003 A/Rio de Janeiro/346/2003 A/Uruguay/11/2003 A/Santa Catarina/379/2004 A/Minas Gerais/156/2004 A/Minas Gerais/154/2004 A/Rio de Janeiro/17/2004 A/Rio Grande do Sul/406/2004 A/Santa Catarina/380/2004 A/Parana/306/2004 A/Parana/308/2004 A/Parana/312/2004 A/Rio Grande do Sul/212/2004 A/Parana/298/2004 A/Rio Grande do Sul/417/2004 A/Rio Grande do Sul/411/2004 A/Rio de Janeiro/26/2004
AY968022 AY968021 AY968020 AY968019 AY968018 AY968017 AF534032 AF534035 AF534034 AF534040 DQ336007 DQ336007 DQ336016 DQ336017 DQ336015 DQ336013 DQ336011 AY968023 AY968024 AY968025 AY968036 AY968033 DQ865972 AY968027 AY968035 AY968031 AY968030 AY968029 AY968026 AY968028 DQ330006 AY068040 AY968032 AY968033 AF534056 AF534059 AF534058 AF534060 AF534057 DQ865972 AF534055 AY968040 AY968041 AY968038 AY968033 AY968039 AY972834 AY972832 AY972833 AY972851 AM991343 AY972848 AY972827 AY972829 AY972847 AY972846 AY972845 AY972837 AY972838 AY972849 AY972835 AY972830 AY972844 AY972841 AY972840
Brazil Brazil Brazil Brazil Brazil Brazil Argentina Argentina Argentina Argentina Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Chile Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Argentina Argentina Argentina Argentina Chile Argentina Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Uruguay Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil
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Sequences were aligned using the CLUSTAL W program (Thompson et al., 1994). Once aligned, the program Modelgenerator (Keane et al., 2006) was used to identify the optimal evolutionary model that described our sequence dataset (Akaike Information Criteria and Hierarchical Likelihood Ratio Test indicated that the GTR model best fit the sequence data). Using this model, maximum likelihood phylogenetic trees were constructed using software from the PhyML program (Guindon et al., 2005). As a measure of the robustness of each node, we used an approximate likelihood ratio test (aLRT), that demonstrate that the branch studied provides a significant likelihood against the null hypothesis that involves collapsing that branch of the phylogenetic tree but leaving the rest of the tree topology identical (Anisimova and Gascuel, 2006). We have used an aLRT non-parametric branch support based on a Shimodaira– Hasegawa-like (SH-like) procedure (Shimodaira and Hasegawa, 1999) as implemented in the PhyML program (Guindon et al., 2005). HA nucleotide sequences were translated to amino acid sequences in silico using the MEGA 3.0 program (Kumar et al., 2004).
(Bao et al., 2008), using the CLUSTAL W program (Thompson et al., 1994). Once aligned, phylogenetic trees were created using the maximum likelihood method under the GTR model (Guindon et al., 2005) and the robustness of the nodes in the trees was assessed by aLRT (Anisimova and Gascuel, 2006). The results of these studies are shown in Fig. 1. All IAV strains included in these studies can be assigned to different clusters, all of them supported by very high values of aLRT (Fig. 1). Different genetic lineages co-circulate in a particular season in a specific country, like in Brazil during the 2000 season (see Fig. 1, bottom). Co-circulation can be also observed in the region, like in the 2001 season, where two different genetic lineages can be observed, one that circulated in Brazil, while another circulated in Brazil, Argentina and Chile (Fig. 1, top and bottom). The same observations can be made for the 2004 season, where at least two distinct genetic lineages are observed, one in Brazil and Ecuador, and another in Brazil and Uruguay (Fig. 1, top). Interesting, co-circulation of IAV genetic lineages in South America are not only seen in big countries, like Brazil, but also in small ones, like Uruguay, where co-circulation can be also found, like in the 2005 season (see Fig. 1, middle). To study the possible presence of adamantine-resistant IAV strains in South America, the HA characteristic amino acid changes were observed in the H3 IAV strains isolated in this region (Hata et al., 2007). The results of these studies are shown in Fig. 2. All strains isolated in Uruguay during the 2007 season bear an adamantine-resistant polymorphism (Fig. 2). Importantly, another strain isolated in Uruguay in the 2005 season bear this changes in the HA gene, but the adamantine-resistant polymorphism is not found in three other viruses isolated in Uruguay and Peru during the same season (see Figs. 1 and 2). No other strain isolated in South America previous to the 2005 season bears the adamantineresistant polymorphism (see Figs. 1 and 2). In order to gain insight into the genetic relations among H3 IAV strains that circulated in South America from 1999 to 2007 and vaccine strains recommended for the Southern hemisphere for that seasons, the appropriate vaccine strains were included in the phylogenetic analysis (see Fig. 1 and Table 2). Only vaccine strains recommended for the 2007 season share the same cluster with available IAV strains isolated in South America in that season (Fig. 1).
3. Results
4. Discussion
In order to get insight into the genetic relations among H3 IAV isolated in South America and vaccine strains included in the influenza vaccines for the 1999–2007 influenza seasons for the Southern hemisphere, the HA sequences obtained from the Uruguayan patients were aligned with corresponding sequences from 68 H3 IAV strains isolated in South America (Table 1) and 9 H3 IAV vaccine strains (Table 2), obtained from the Influenza Database
The results of this study indicate that multiple clades cocirculates during most of the influenza seasons in South America (see Fig. 1). These results are in agreement with recent findings by Nelson et al. (2007) for Australia and New Zealand. Although some strains isolated in the region can be linked over multiple seasons (Fig. 1, bottom), suggesting that in situ evolution cannot be ruled for some seasons without further studies, viruses do not seem to have regularly evolved in geographic isolation (see for instance Brazilian strains isolated during 2000–2004 seasons in Fig. 1). Rather, evolution appears to be shaped by re-introduction of new strains. This is in agreement with recent studies that have provided evidence for regular cross-hemisphere viral migration between seasons, even among localities very distantly separated (Nelson et al., 2007; Viboud et al., 2006, 2004). Moreover, very recent studies revealed a continuous circulation of IAV in East and Southeast Asia (E-SE Asia) via a region-wide network of temporally overlapping epidemics and that epidemics in the temperate regions are seeded from this network each year. Seed strains first generally reached Oceania, North America and Europe, and later to the South American region (Russell et al., 2008). All strains isolated in Uruguay during the 2007 season bear an adamantine-resistant HA polymorphism (Fig. 2). Due to the fact of
Name
Accession number
Country of isolation
A/Parana/291/2004 A/Parana/310/2004 A/Minas Gerais/163/2004 A/Minas Gerais/163/2004 A/Ecuador/1968/2004 A/Uruguay/01/2004 A/Peru/166/2005 A/Uruguay/02/2005 A/Uruguay/03/2005 A/Uruguay/04/2005 A/Uruguay/01/2007 A/Uruguay0/4/2007 A/Uruguay/05/2007 A/Uruguay/07/2007 A/Uruguay/10/2007 A/Uruguay/716/2007
AY972842 AY972843 AY972828 AY972831 DQ265716 AM991344 DQ265708 AM991338 AM991339 AM991340 AM991335 AM991336 AM991337 AM991341 AM991342 ISDN276547
Brazil Brazil Brazil Brazil Ecuador Uruguay Peru Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay Uruguay
2.6. Phylogenetic analysis
Table 2 Origins of H3 IAV vaccine strains recommended for the Southern hemisphere.a. Name
Accession number
Season
A/Sydney/5/1997 A/Moscow/10/1999 A/Panama/2007/1999 A/Fujian/411/2002 A/Kumamoto/102/2002 A/Wellington/1/2004 A/California/7/2004 A/New York/55/2004 A/Wisconsin/67/2005 A/Hiroshima/52/2005
AF180584 AY531035 DQ508865 EF541397 EF456797 CY012104 DQ865973 EF473394 EF473424 EU283414
1999 2000, 2001, 2002, 2003 2001, 2002, 2003 2004 2004 2005 2006 2006 2007 2007
a For some seasons, more than one candidate vaccine viruses may be available for vaccine formulation.
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Fig. 1. Phylogenetic analysis of the Haemagglutinin gene of Influenza A virus circulating in the South American region from 1999 to 2007. An unrooted maximum likelihood tree obtained under the GTR model is shown. Strains in the tree are shown by their names, which also show geographic location and year of isolation, according to the World Health Organization (1980) (for accession numbers see Table 1). Country of isolation and season year is indicated on the right of each name between parentheses. Vaccine strains are shown in italics. Vaccine strains recommended for a specific Southern hemisphere influenza season and the IAV strains isolated South America in that season are
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Fig. 2. Alignment of HA amino acid sequences from IAV strains isolated in South America. Strains are shown by name at the left side of the figure. R.G. means Rio Grande; Sta. Cat. means Santa Catarina. HA1 amino acid sequences from position 143 to 234 (relative to strain A/Rio Grande do Sul/406/2004) are shown. Characteristic amino acid changes at positions 193 (Ser to Phe; S193F) and 225 (Asp to Asn; D225N) of the HA protein of adamantine-resistant viruses are shown in bold. Identity to strain A/Rio Grade do Sul/406/2004 (isolated in Brazil in the 2004 season) is indicated by a dash.
the infrequent use of adamantine drugs in South American countries, that all adamantine-resistant viruses belong to a single genetic lineage (Simonsen et al., 2007), and that it is very unlikely that these changes may arise just by chance in IAV circulating in South America, this particular polymorphism may permit to shed light into the possible migration of IAV strains to our region. The results of these studies suggest that adamantine-resistant viruses, first detected in the 2004–2005 season in Japan (Hata et al., 2007; Saito et al., 2007), and then spread to South East Asia, Oceania and North America during the 2005–2006 season (Bright et al., 2006; Barr et al., 2007; Simonsen et al., 2007; Saito et al., 2008) arrived to South America in the 2005 season. This is in agreement with a cross-hemisphere viral migration (Nelson et al., 2007; Viboud et al., 2006, 2004) and supports recent evidence that seeded strains from a E-SE Asia network finally arrive to the South American region (Russell et al., 2008). Nevertheless, not all South American isolates that belong to the 2005 season may have the same origin; since isolates of Peru and Uruguay do not bear the characteristic adamantine-resistant polymorphism (see Figs. 1 and 2). This speaks of a possibility of co-circulation of viruses of at least two different origins, although the effect of genomic reassortment cannot be ruled out. Nevertheless, the results of these studies reveal how migration may be playing an important role in the IAV epidemiology history in the South American region. For most of the South American influenza seasons studied vaccine and natural strains of the same season are assigned to different clusters (see Fig. 1). This indicates that for the majority of the seasons studied vaccine and circulating IAV strains have distant genetic relations among themselves. The results of these studies speak of the need of a more profound study of the IAV strains circulating in the South American region, as well as the routes and mechanisms of virus’s geographical spread. Containing the spread of IAV will require understand how the dynamics, antigenic evolution and seasonal emergence interrelate (Nelson and Holmes, 2007). Recent studies revealed that once H3N2 IAV strains leave the E-SE Asia network, they will probably impact in the South American region (Russell et al., 2008). For this reason, to gain insight into the patterns of spread from E-SE Asia to this region is extremely important for consequent improvements to vaccine strain selection for the Southern hemisphere. The results of this work also highlight the critical importance of expanding surveillance in the South American region, in order to elucidate the geographical movements and evolution of this virus
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