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Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast
Veterinary Microbiology 156 (2012) 429–433
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast§ Luz Alba M.G. Fornells a,*, Tatiane F. Silva a, Iliani Bianchi b, Carlos E.P.F. Travassos c, Maı´ra H.T. Liberal d, Clau´dio M. Andrade d, Melissa P. Petrucci b, Venicio F. Veiga a, Maite F.S. Vaslin a, Jose´ Nelson S.S. Couceiro a a
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Instituto Orca, Vila Velha, Espı´rito Santo, Brazil c Universidade Estadual do Norte Fluminense, Campos, RJ, Brazil d Pesagro, Rio de Janeiro, RJ, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 June 2011 Received in revised form 23 November 2011 Accepted 25 November 2011
Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espı´rito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Paramyxovirus APMV-2 APMV-10 Penguin Brazil
1. Introduction Wild birds play a major role in the dissemination of various microorganisms, especially paramyxoviruses and
§ The sequences described here were deposited in GenBank under the following accession numbers: ES-02 – HQ687899, ES-03 – HQ687900, ES05 – HQ687901 and ES-06 – HQ687902. * Corresponding author at: Laborato´rio de Virologia Molecular I, Depto. Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Centro de Cieˆncias da Sau´de, Bloco I, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro, RJ 21941-590, Brazil. Tel.: +55 2125608344 167; fax: +55 2133960701. E-mail address: [email protected] (Luz Alba M.G. Fornells).
0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.11.026
orthomyxoviruses (Olsen et al., 2006). Orthomyxoviruses are pleomorphic and enveloped structures, and their capsids contain segmented negative-sense, singlestranded RNA genomes; paramyxoviruses exhibit many similar characteristics but have non-segmented RNA genomes. Both types of these viruses have caused diseases in humans and animals for centuries, and several new viruses have been identified in recent decades. Serotypes APMV-1 to APMV-10 of the Avulavirus genus (Paramyxoviridae family, subfamily Paramyxovirinae) are able to infect a variety of wild and domestic birds worldwide (Miller et al., 2010). Their genomes encode at least six open reading frames (ORFs NP, P, M, F, HN and L), and the L (polymerase) gene is a useful target for molecular
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Fig. 1. (A) A map of the South American Atlantic coast (http://earth.google.com/intl/pt/) showing usual and exceptional (2008) migratory routes of the Magellanic penguin (Spheniscus magellanicus). Samples collection site is indicated by a star. (B) An electron microscopic image of ES-03 virus isolate, showing enveloped pleomorphic structures. The arrow indicates a viral capsid negatively stained with 4% phosphotungstic acid. Scale bar = 100 nm.
detection of these viruses. In contrast, influenza A viruses from the Orthomyxoviridae family are classified based on their hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) subtypes and identified by RT-PCR for using their matrix protein (M) as universal target for identification (Fouchier et al., 2000; Ellis and Zambon, 2001). Serological surveys have shown that ortho- and paramyxoviruses circulate among Antarctic and sub-Antarctic penguins. Although no influenza virus A has been isolated from these birds, serum antibodies to the H7 and H10 subtypes have been previously reported in Ade´lie (Pygoscelis adeliae) and other penguin species by Morgan et al. (1981), Austin and Webster (1993), Wallensten et al. (2006), and others. APMV-2 paramyxoviruses have been detected in Ade´lie and Humboldt penguins, APMV-1, also called Newcastle disease virus (NDV), in Antarctic penguins as well unclassified APMV serotypes in king (Aptenodytes patagonicus), royal (Eudyptes schlegeli), and Ade´lie penguins from Antarctica and little blue penguins (Eudyptula minor) from Australia (Morgan et al., 1981; Alexander et al., 1989; Austin and Webster, 1993; Wallensten et al., 2006; Smith et al., 2008). Recently, a new Avulavirus serotype, APMV-10, was identified in rockhopper penguins (Eudyptes chrysocome) from the Falkland Islands (Miller et al., 2010). Penguins usually live on the Antarctic continent and in the coldest regions of South America, with some species
exhibiting annual migrations. Magellanic penguins, Spheniscus magellanicus are distributed along the southernmost shores of South America, with breeding colonies extending from the coast of Chile to the Valdez Peninsula region in Argentina and on the Falkland Islands. From these breeding colonies, the birds migrate to winter habitats up to the southern coast of Brazil (Stokes et al., 1998). GarciaBorboroglu et al. (2010) detected a severe alteration in the distributional pattern of Magellanic penguins along the eastern coast of South America in 2008 (Fig. 1A), when these birds were observed near the equator in Ceara´, Brazil (ffi3S). To check the possible presence of paramyxovirus and/or influenza A virus infections among migratory penguins, we analyzed 73 cloacal samples collected from penguins captured on the coast of Espı´rito Santo state, Brazil, during the 2008 migration period. Serological and molecular assays showed the presence of APMV-2 and APMV-10 paramyxovirus in some of these birds. 2. Materials and methods 2.1. Virus isolation, purification and electron microscopy Cloacal swabs were collected from 73 Magellanic penguins (Spheniscus magellanicus) at the ORCA Institute
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(Vila Velha, Espı´rito Santo, Brazil) at 208430 48.600 S latitude and 408310 52.9600 W longitude, between September and October 2008. Swabs were suspended in DMEM (Sigma Life Science, USA) with gentamicin sulfate at 1000 IU/mL and glycerol; inoculated into specific-pathogen-free (SPF) embryonated chicken eggs and incubated at 35–37 8C for 72 h. The allantoic fluids were analyzed in duplicate by hemagglutination test (HA) with 0.5% chicken red blood cells (RBCs). HA-positive samples were propagated in eggs, and their allantoic fluid was clarified at 2800 g, concentrated at 65,000 g for 1 h, and purified in a sucrose gradient (20–60%) by centrifugation at 80,000 g for 2 h. Purified viral suspension was observed under a Morgani EM 268 electron microscope (Fei, Eindhoven, Netherlands) at 80 kV on a copper grid stained with 4% phosphotungstic acid (PTA) following Fonseca et al. (1984). 2.2. Identification of virus samples by hemagglutination inhibition assay Hemagglutination inhibition (HI) tests were performed as described in OIE (2009), using standard polyclonal antisera produced in chickens against each one of the influenza virus A H1N1 (A/New Caledonia/20/99/2000), H2N2 (A/CAP/2004), H3N8 (A/Miami/63), H7N7 (A/Prague/56), and the paramyxovirus APMV-1 (NDV-La Sota), APMV-2 (P/Ck/CA/Yucaipa/56), APMV-3 (P/turkey/Wisconsin/68), APMV-6 (DK/H.K./199/77), APMV-7 (Dove/ TN/4/75) and APMV-10, obtained from the USDA, NVSL (Ames, IA). Polyclonal antiserum produced in chicken against APMV-10 was kindly provided by Dr. P. Miller (USDA, NVSL, Athens, GA). 2.3. RNA extraction and RT-PCR Total RNA was extracted from allantoic fluid using a QIAamp mini kit (QIAGEN, Sa˜o Paulo, Brazil). Two-step RTPCRs were performed for the M (matrix protein) gene of influenza A viruses as proposed by Ellis and Zambon (2001). For paramyxovirus detection, two fragments of the L gene were amplified independently. Fragment containing the C–D motifs was amplified with the PARF2 and PAR-R primers (Tong et al., 2008) and the fragment containing the A–B motifs was amplified using the degenerate primers APMVR (50 -ACTTGRTTGTCHCCYTGAACCATTGA-30 ) and APMV823F (50 -ACAACWGAYYTKSAAAARTAYTG-30 ), design by our group, at an annealing temperature of 54.9 8C. The amplified fragments were introduced in pGEM-T Easy (Promega, Sunnyvale, CA, USA) or purified directly from the PCR and sequenced in both directions. 2.4. Sequence and phylogenetic analyses Consensus sequences were obtained for each isolate using the MultAlin program (Corpet, 1988). GeneDoc program (http://www.psc.edu/biomed/genedoc) was used for analysis of sequence identities. Phylogenetic reconstructions were performed using the neighbor-joining (NJ). GenBank accessions for paramyxovirus sequences used in
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this study were: Newcastle disease virus (NDV) B1 (APMV1) NP_071471.1, Avian paramyxovirus 2 California/Yucaipa isolate (APMV2_YC) ACA49110.1, Avian paramyxovirus 2 Bangor isolate (APMV2_BG) ADK25248.1, Avian paramyxovirus 3 Netherland isolate (APMV3_NT) ACB46872.1, Avian paramyxovirus 3 Wisconsin isolate (APMV3_WI) ACI47553.1, Avian paramyxovirus 4 (APMV4) ACJ06716.1, Avian paramyxovirus 5 (APMV5) ADD39006.1, Avian paramyxovirus 6 ACF19434.1, Avian paramyxovirus 7 (APMV7) ACN72645.1, Avian paramyxovirus 8 (APMV8) ACO48302.1, Avian paramyxovirus 9 (APMV9) ACJ82944.1, Avian paramyxovirus 10 (APMV10) HM147142.1, Human parainfluenza virus (PIV-2) NP_598406.1, Parainfluenza virus 5 (SV5) YP_138518.1, Mumps virus (MuV) NP_054714.1, Simian virus 41 (SV41) YP_138510.1, Nipah virus (NIV) NP_112028.1, and Hendra virus (HeV) NP_047113.2. 3. Results 3.1. Virus isolation and serological characterization In September and October 2008, cloacal swabs were collected from Magellanic penguins in Espı´rito Santo (Brazil), where they arrived after a long migration, probably approximately 4000 km from the Falkland Islands, were malnourished and underweight. Cloacal swabs were used to inoculate embryonated eggs to attempt virus isolation and propagation. Nine (12.3%) virus isolates from 73 cloacal swabs, which were identified as ES samples (Espirito Santo samples), showed positive results using the HA test with chicken RBCs. Hemagglutinating samples, were submitted to HI test against influenza A and paramyxovirus viruses using polyclonal antisera. Influenza virus samples could not be identified by HI test, however five paramyxovirus samples were recognized. Two samples (ES-03 and ES-05) were identified as APMV-2, both of them with HI titers equal to 40. Three samples were identified as AMPV-10 (ES-01, ES-02 and ES-06) with HI titers equal to 40, 20 and 20, respectively. HI-positive samples were purified by sucrose gradient centrifugation and showed typical paramyxovirus morphology when observed under electron microscopy. The viruses were 150–300 nm in diameter and displayed enveloped pleomorphic structures with short glycoprotein projections (5–8 nm) and free viral nucleocapsids with a herringbone pattern (400–600 nm; Fig. 1B). 3.2. Molecular characterization and phylogenetic analysis All the nine virus isolates were negative for PCR amplification of a portion of the influenza M gene. However RT-PCR assays using primers able to amplify the conserved motifs A–D in domain III of protein L of the Paramyxoviridae family showed the presence of a 909-bp fragment of the L protein in those five isolates previously positive for paramyxovirus AMPV-2 and -10 by HI test. These amplified fragments were sequenced, although ES01 isolate has not able to produce a readable sequence. However, sequencing analysis of the other four isolates reveals that ES-02 and ES-06 shared 99% identity to each
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Fig. 2. Phylogenetic analysis of the partial L protein of Avulavirus genus members. The Rubulavirus and Henipavirus genera were used as outgroups. The numbers represent the bootstrap scores from 1000 replicates. The phylogenetic tree was constructed with MEGA 4 (Molecular Evolutionary Genetics Analysis software, version 4.0, Tempe, AZ, USA), and the scale bar represents genetic distance.
other, strongly suggesting that they belonged to the same serotype. These two isolates showed highest identity (93% and 92%, respectively) to APMV-10, a serotype recently identified in another species of penguin on the Falkland Islands (Miller et al., 2010), reinforcing serological results. Similarly, the ES-03 and ES-05 isolates shared 98% amino acid identity over the amplified portion of the L protein and showed the highest identity (93% and 92%, respectively) to the APMV-2 Bangor subgroup. The phylogenetic tree obtained from the sequence alignment with the 10 APMV serotypes, using two other genera of the Paramyxoviridae family (Rubulavirus and Henipavirus) as outgroups, showed that the isolates belonged to the Avulavirus genus (Fig. 2) and confirmed that the ES-02 and ES-06 isolates were closely related to APMV-10 (bootstrap value of 100%). The ES-03 and ES-05 isolates were closely related to the AMPV-2 serotype, showing more similarity to the Bangor subgroup than to the California/Yucaipa. Although serological assays were not performed for all known Avulavirus serotypes, our results indicate that the penguin paramyxovirus isolates described here should probably be classified as new members of the APMV-2 and AMPV-10 serotypes. These results reveal, for the first time, the presence of paramyxovirus in migratory penguins at Brazilian coast.
4. Discussion Since 2005, our group has monitored the circulation of orthomyxovirus and paramyxovirus in resident and migratory birds as part of the effort to survey avian pathogens transmitted by wild birds in Brazil (MAPA, 2006). As previous studies of sub-Antarctic and Antarctic penguins have shown that these animals may become infected with a variety of avian influenza viruses and paramyxoviruses, including NDV (Morgan et al., 1981; Alexander et al., 1989; Austin and Webster, 1993; Wallensten et al., 2006), the presence of some of these viruses in penguins that arrived on the southeast coast of Brazil was analyzed. We isolate five hemagglutinating avian viruses compatible with paramyxoviruses by electron microscopy and positive for avian paramyxovirus by HI test. Two samples were identified serologically as APMV-2 and three as AMPV-10. For four of these isolates the sequencing of conserved domain of L gene confirmed serological results, putting isolates ES-03 and ES-05 as new members of APMV-2, and, ES-02 and ES-06 as new members of APMV-10 of avian paramyxoviruses. The strong sequence identity between the ES-03 and ES-05 samples reinforces the classification of these samples as members of the APMV-2 serotype, closest to
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the Bangor subgroup. This subgroup was recently identified after the complete sequencing of a strain isolated from a finch (Subbiaha et al., 2010). Two distinct subgroups of APMV-2 (Bangor and California/Yucaipa) were proposed based on their relatively weak (66.5%) identity to the L protein (Subbiaha et al., 2010). These two subgroups differ substantially in their nucleotide and amino acid sequences but show only discrete antigenic differences (Kumar et al., 2010). Additionally, ES-02 and ES-06 Domain III of L-gene sequences showed 93% homology to sequences with a proposed new serotype (APMV-10) of avian paramyxoviruses (Miller et al., 2010). Phylogenetic analysis put these new isolates together with this new serotype also, reinforcing the hypothesis that they are new members of APMV-10 serotype. Therefore, although a complete genomic analysis of the four isolates described here is still necessary, our data permit us to propose that they belong to the Bangor subgroup of APMV-2 and to APMV-10 serotype. Garcia-Borboroglu et al. (2010) reported that Magellanic penguins migrated farther north than usual in 2008, probably because of scarce food resources, and many starved. Magellanic penguins are considered sentinels of ocean conditions (Stokes et al., 1998; Boersma, 2008) and their unusual migration behavior in 2008 may be associated to the abnormal ocean conditions observed from Brazil to the northern coasts of Argentina that year potentially linked to climate variability. The isolation of paramyxoviruses from penguins demonstrates that this species may be a vector for spreading these viruses into regions distant from its original habitat. Wild avian species of the eastern South American coast may become infected by these viruses through close contact between migratory and resident birds. Although we cannot say whether the paramyxoviruses isolated from the penguins were acquired, in their breeding areas or during the migration period along Argentinean, Uruguayan and Brazilian coasts, surveillance work in Antarctica has indicated the presence of the paramyxovirus identified in this work in that region (Alexander et al., 1989; Miller et al., 2010). Thus far, only NDV paramyxovirus have been focused for Brazilian Department of Agriculture as surveillance target for wild birds (MAPA, 2006). These results reinforce the importance of continued surveys on migratory penguins to scale potential risks of virus spread from origin to unnatural habitats. Acknowledgements We thank the nongovernmental organization ORCA for field assistance and FAPERJ for financial support (E-26/ 170.642/2006). We are grateful to Dr. Patty Miller and
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Dr. David Swaine from USDA, NVSL, Athens, GA for providing APMV-10 antiserum. References Alexander, D.J., Manuell, R.J., Collins, M.S., Brockman, S.J., Westbury, H.A., Morgan, I., Austin, F.J., 1989. Characterization of paramyxoviruses isolated from penguins in Antarctica and sub-Antarctica during 1976–9. Arch. Virol. 109 (1–2), 135–143. Austin, F.J., Webster, R.G., 1993. Evidence of ortho- and paramyxoviruses in fauna from Antarctica. J. Wildl. Dis. 29 (4), 568–571. Boersma, P.D., 2008. Penguins as marine sentinels. Bioscience 58 (7), 557–560. Corpet, F., 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16 (22), 10881–10890. Ellis, J.S., Zambon, M.C., 2001. Combined PCR-heteroduplex mobility assay for detection and differentiation of influenza A viruses from different animal species. J. Clin. Microbiol. 39 (11), 4097–4102. Fonseca, M.E.F., Frimer, N., Mendonc¸a, R.E.A., Couceiro, J.N.S.S., Machado, R.D., 1984. A combined staining technique developed for virus particle observation in the electron microscope. Rev. Bras. Biol. 44 (1), 37–40. Fouchier, R.A., Bestebroer, T.M., Herfst, S., Van Der Kemp, L., Rimmelzwaan, G.F., Osterhaus, A.D., 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J. Clin. Microbiol. 38, 4096–4101. Garcia-Borboroglu, P., Boersma, D., Ruoppolo, V., Pinho-da-Silva-Filho, R., Corrado-Adornes, A., Conte-Sena, D., Velozo, R., Myaji-Kolesnikovas, C., Dutra, G., Maracini, P., Carvalho-do-Nascimento, C., Ramos-Ju´nior, V., Barbosa, L., Serra, S., 2010. Magellanic penguin mortality in 2008 along the SW Atlantic coast. Mar. Pollut. Bull. 60, 1652–1657. Kumar, S., Nayak, B., Samuel, A.S., Xiao, S., Collins, P.L., Samal, S.K., 2010. Complete genome sequence of avian paramyxovirus-3 strain Wisconsin: evidence for the existence of subgroups within the serotype. Virus Res. 149, 78–85. MAPA, 2006. Plano de Prevenc¸a˜o a` Influenza Avia´ria em Aves Silvestres e de Subsisteˆncia. Available from: http://www.agricultura.gov.br, accessed 06.06.2011. Miller, P.J., Afonso, C.L., Spackman, E., Scott, M.A., Pedersen, J.C., Senne, D.A., Brown, J.D., Fuller, C.M., Uhart, M.M., Karesh, W.B., Brown, I.H., Alexander, D.J., Swayne, D.E., 2010. Evidence for a new avian paramyxovirus serotype 10 detected in Rockhopper penguins from the Falkland Islands. J. Virol. 84 (21), 11496–11504. Morgan, I.R., Westbury, H.A., Caple, I.W., Campbell, J., 1981. A survey of virus infection in sub-Antarctic penguins on Macquarie Island, Southern Ocean. Aust. Vet. J. 57 (7), 333–335. OIE (World Organization for Animal Health), 2009. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2009. Newcastle disease, pp. 576–589In: http://www.oie.int/eng/normes/mmanual/2008/pdf/ 2.03.14_NEWCASTLE_DIS.pdf. Olsen, B., Munster, V.J., Wallensten, A., Waldenstro, J., Osterhaus, A.D., Fouchier, R.A., 2006. Global patterns of influenza A virus in wild birds. Science 312, 384–388. Smith, K.M., Karesh, W.B., Majluf, P., Paredes, R., Zavalaga, C., Reul, A.H., Stetter, M., Braselton, W.E., Puche, H., Cook, R.A., 2008. Health evaluation of free-ranging Humboldt penguins (Spheniscus humboldti) in Peru. Avian Dis. 52, 130–135. Stokes, D.L., Boersma, P.D., Davis, L.S., 1998. Satellite tracking of Magellanic penguin migration. Condor 100 (2), 376–381. Subbiaha, M., Nayaka, S., Collins, P.L., Siba, K., Samal, S.K., 2010. Complete genome sequences of avian paramyxovirus serotype 2 (APMV-2) strains Bangor, England and Kenya: evidence for the existence of subgroups within serotype 2. Virus Res. 152, 85–95. Tong, S., Chern, S.W.W., Li, Y., Pallansch, M.A., Anderson, L.J., 2008. Sensitive and broadly reactive reverse transcription-PCR assays to detect novel paramyxoviruses. J. Clin. Microbiol. 46 (8), 2652–2658. Wallensten, A., Munster, V.J., Osterhaus, A.D.M.E., Waldenstrom, J., Bonnedahl, J., Broman, T., Fouchier, R.A.M., Olsen, B., 2006. Mounting evidence for the presence of influenza A virus in the avifauna of the Antarctic region. Antarct. Sci. 18 (3), 353–356.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast§ Luz Alba M.G. Fornells a,*, Tatiane F. Silva a, Iliani Bianchi b, Carlos E.P.F. Travassos c, Maı´ra H.T. Liberal d, Clau´dio M. Andrade d, Melissa P. Petrucci b, Venicio F. Veiga a, Maite F.S. Vaslin a, Jose´ Nelson S.S. Couceiro a a
Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil Instituto Orca, Vila Velha, Espı´rito Santo, Brazil c Universidade Estadual do Norte Fluminense, Campos, RJ, Brazil d Pesagro, Rio de Janeiro, RJ, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 June 2011 Received in revised form 23 November 2011 Accepted 25 November 2011
Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espı´rito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Paramyxovirus APMV-2 APMV-10 Penguin Brazil
1. Introduction Wild birds play a major role in the dissemination of various microorganisms, especially paramyxoviruses and
§ The sequences described here were deposited in GenBank under the following accession numbers: ES-02 – HQ687899, ES-03 – HQ687900, ES05 – HQ687901 and ES-06 – HQ687902. * Corresponding author at: Laborato´rio de Virologia Molecular I, Depto. Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Centro de Cieˆncias da Sau´de, Bloco I, Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro, RJ 21941-590, Brazil. Tel.: +55 2125608344 167; fax: +55 2133960701. E-mail address: [email protected] (Luz Alba M.G. Fornells).
0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.11.026
orthomyxoviruses (Olsen et al., 2006). Orthomyxoviruses are pleomorphic and enveloped structures, and their capsids contain segmented negative-sense, singlestranded RNA genomes; paramyxoviruses exhibit many similar characteristics but have non-segmented RNA genomes. Both types of these viruses have caused diseases in humans and animals for centuries, and several new viruses have been identified in recent decades. Serotypes APMV-1 to APMV-10 of the Avulavirus genus (Paramyxoviridae family, subfamily Paramyxovirinae) are able to infect a variety of wild and domestic birds worldwide (Miller et al., 2010). Their genomes encode at least six open reading frames (ORFs NP, P, M, F, HN and L), and the L (polymerase) gene is a useful target for molecular
[(Fig._1)TD$IG]
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Fig. 1. (A) A map of the South American Atlantic coast (http://earth.google.com/intl/pt/) showing usual and exceptional (2008) migratory routes of the Magellanic penguin (Spheniscus magellanicus). Samples collection site is indicated by a star. (B) An electron microscopic image of ES-03 virus isolate, showing enveloped pleomorphic structures. The arrow indicates a viral capsid negatively stained with 4% phosphotungstic acid. Scale bar = 100 nm.
detection of these viruses. In contrast, influenza A viruses from the Orthomyxoviridae family are classified based on their hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) subtypes and identified by RT-PCR for using their matrix protein (M) as universal target for identification (Fouchier et al., 2000; Ellis and Zambon, 2001). Serological surveys have shown that ortho- and paramyxoviruses circulate among Antarctic and sub-Antarctic penguins. Although no influenza virus A has been isolated from these birds, serum antibodies to the H7 and H10 subtypes have been previously reported in Ade´lie (Pygoscelis adeliae) and other penguin species by Morgan et al. (1981), Austin and Webster (1993), Wallensten et al. (2006), and others. APMV-2 paramyxoviruses have been detected in Ade´lie and Humboldt penguins, APMV-1, also called Newcastle disease virus (NDV), in Antarctic penguins as well unclassified APMV serotypes in king (Aptenodytes patagonicus), royal (Eudyptes schlegeli), and Ade´lie penguins from Antarctica and little blue penguins (Eudyptula minor) from Australia (Morgan et al., 1981; Alexander et al., 1989; Austin and Webster, 1993; Wallensten et al., 2006; Smith et al., 2008). Recently, a new Avulavirus serotype, APMV-10, was identified in rockhopper penguins (Eudyptes chrysocome) from the Falkland Islands (Miller et al., 2010). Penguins usually live on the Antarctic continent and in the coldest regions of South America, with some species
exhibiting annual migrations. Magellanic penguins, Spheniscus magellanicus are distributed along the southernmost shores of South America, with breeding colonies extending from the coast of Chile to the Valdez Peninsula region in Argentina and on the Falkland Islands. From these breeding colonies, the birds migrate to winter habitats up to the southern coast of Brazil (Stokes et al., 1998). GarciaBorboroglu et al. (2010) detected a severe alteration in the distributional pattern of Magellanic penguins along the eastern coast of South America in 2008 (Fig. 1A), when these birds were observed near the equator in Ceara´, Brazil (ffi3S). To check the possible presence of paramyxovirus and/or influenza A virus infections among migratory penguins, we analyzed 73 cloacal samples collected from penguins captured on the coast of Espı´rito Santo state, Brazil, during the 2008 migration period. Serological and molecular assays showed the presence of APMV-2 and APMV-10 paramyxovirus in some of these birds. 2. Materials and methods 2.1. Virus isolation, purification and electron microscopy Cloacal swabs were collected from 73 Magellanic penguins (Spheniscus magellanicus) at the ORCA Institute
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(Vila Velha, Espı´rito Santo, Brazil) at 208430 48.600 S latitude and 408310 52.9600 W longitude, between September and October 2008. Swabs were suspended in DMEM (Sigma Life Science, USA) with gentamicin sulfate at 1000 IU/mL and glycerol; inoculated into specific-pathogen-free (SPF) embryonated chicken eggs and incubated at 35–37 8C for 72 h. The allantoic fluids were analyzed in duplicate by hemagglutination test (HA) with 0.5% chicken red blood cells (RBCs). HA-positive samples were propagated in eggs, and their allantoic fluid was clarified at 2800 g, concentrated at 65,000 g for 1 h, and purified in a sucrose gradient (20–60%) by centrifugation at 80,000 g for 2 h. Purified viral suspension was observed under a Morgani EM 268 electron microscope (Fei, Eindhoven, Netherlands) at 80 kV on a copper grid stained with 4% phosphotungstic acid (PTA) following Fonseca et al. (1984). 2.2. Identification of virus samples by hemagglutination inhibition assay Hemagglutination inhibition (HI) tests were performed as described in OIE (2009), using standard polyclonal antisera produced in chickens against each one of the influenza virus A H1N1 (A/New Caledonia/20/99/2000), H2N2 (A/CAP/2004), H3N8 (A/Miami/63), H7N7 (A/Prague/56), and the paramyxovirus APMV-1 (NDV-La Sota), APMV-2 (P/Ck/CA/Yucaipa/56), APMV-3 (P/turkey/Wisconsin/68), APMV-6 (DK/H.K./199/77), APMV-7 (Dove/ TN/4/75) and APMV-10, obtained from the USDA, NVSL (Ames, IA). Polyclonal antiserum produced in chicken against APMV-10 was kindly provided by Dr. P. Miller (USDA, NVSL, Athens, GA). 2.3. RNA extraction and RT-PCR Total RNA was extracted from allantoic fluid using a QIAamp mini kit (QIAGEN, Sa˜o Paulo, Brazil). Two-step RTPCRs were performed for the M (matrix protein) gene of influenza A viruses as proposed by Ellis and Zambon (2001). For paramyxovirus detection, two fragments of the L gene were amplified independently. Fragment containing the C–D motifs was amplified with the PARF2 and PAR-R primers (Tong et al., 2008) and the fragment containing the A–B motifs was amplified using the degenerate primers APMVR (50 -ACTTGRTTGTCHCCYTGAACCATTGA-30 ) and APMV823F (50 -ACAACWGAYYTKSAAAARTAYTG-30 ), design by our group, at an annealing temperature of 54.9 8C. The amplified fragments were introduced in pGEM-T Easy (Promega, Sunnyvale, CA, USA) or purified directly from the PCR and sequenced in both directions. 2.4. Sequence and phylogenetic analyses Consensus sequences were obtained for each isolate using the MultAlin program (Corpet, 1988). GeneDoc program (http://www.psc.edu/biomed/genedoc) was used for analysis of sequence identities. Phylogenetic reconstructions were performed using the neighbor-joining (NJ). GenBank accessions for paramyxovirus sequences used in
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this study were: Newcastle disease virus (NDV) B1 (APMV1) NP_071471.1, Avian paramyxovirus 2 California/Yucaipa isolate (APMV2_YC) ACA49110.1, Avian paramyxovirus 2 Bangor isolate (APMV2_BG) ADK25248.1, Avian paramyxovirus 3 Netherland isolate (APMV3_NT) ACB46872.1, Avian paramyxovirus 3 Wisconsin isolate (APMV3_WI) ACI47553.1, Avian paramyxovirus 4 (APMV4) ACJ06716.1, Avian paramyxovirus 5 (APMV5) ADD39006.1, Avian paramyxovirus 6 ACF19434.1, Avian paramyxovirus 7 (APMV7) ACN72645.1, Avian paramyxovirus 8 (APMV8) ACO48302.1, Avian paramyxovirus 9 (APMV9) ACJ82944.1, Avian paramyxovirus 10 (APMV10) HM147142.1, Human parainfluenza virus (PIV-2) NP_598406.1, Parainfluenza virus 5 (SV5) YP_138518.1, Mumps virus (MuV) NP_054714.1, Simian virus 41 (SV41) YP_138510.1, Nipah virus (NIV) NP_112028.1, and Hendra virus (HeV) NP_047113.2. 3. Results 3.1. Virus isolation and serological characterization In September and October 2008, cloacal swabs were collected from Magellanic penguins in Espı´rito Santo (Brazil), where they arrived after a long migration, probably approximately 4000 km from the Falkland Islands, were malnourished and underweight. Cloacal swabs were used to inoculate embryonated eggs to attempt virus isolation and propagation. Nine (12.3%) virus isolates from 73 cloacal swabs, which were identified as ES samples (Espirito Santo samples), showed positive results using the HA test with chicken RBCs. Hemagglutinating samples, were submitted to HI test against influenza A and paramyxovirus viruses using polyclonal antisera. Influenza virus samples could not be identified by HI test, however five paramyxovirus samples were recognized. Two samples (ES-03 and ES-05) were identified as APMV-2, both of them with HI titers equal to 40. Three samples were identified as AMPV-10 (ES-01, ES-02 and ES-06) with HI titers equal to 40, 20 and 20, respectively. HI-positive samples were purified by sucrose gradient centrifugation and showed typical paramyxovirus morphology when observed under electron microscopy. The viruses were 150–300 nm in diameter and displayed enveloped pleomorphic structures with short glycoprotein projections (5–8 nm) and free viral nucleocapsids with a herringbone pattern (400–600 nm; Fig. 1B). 3.2. Molecular characterization and phylogenetic analysis All the nine virus isolates were negative for PCR amplification of a portion of the influenza M gene. However RT-PCR assays using primers able to amplify the conserved motifs A–D in domain III of protein L of the Paramyxoviridae family showed the presence of a 909-bp fragment of the L protein in those five isolates previously positive for paramyxovirus AMPV-2 and -10 by HI test. These amplified fragments were sequenced, although ES01 isolate has not able to produce a readable sequence. However, sequencing analysis of the other four isolates reveals that ES-02 and ES-06 shared 99% identity to each
[(Fig._2)TD$IG]
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Fig. 2. Phylogenetic analysis of the partial L protein of Avulavirus genus members. The Rubulavirus and Henipavirus genera were used as outgroups. The numbers represent the bootstrap scores from 1000 replicates. The phylogenetic tree was constructed with MEGA 4 (Molecular Evolutionary Genetics Analysis software, version 4.0, Tempe, AZ, USA), and the scale bar represents genetic distance.
other, strongly suggesting that they belonged to the same serotype. These two isolates showed highest identity (93% and 92%, respectively) to APMV-10, a serotype recently identified in another species of penguin on the Falkland Islands (Miller et al., 2010), reinforcing serological results. Similarly, the ES-03 and ES-05 isolates shared 98% amino acid identity over the amplified portion of the L protein and showed the highest identity (93% and 92%, respectively) to the APMV-2 Bangor subgroup. The phylogenetic tree obtained from the sequence alignment with the 10 APMV serotypes, using two other genera of the Paramyxoviridae family (Rubulavirus and Henipavirus) as outgroups, showed that the isolates belonged to the Avulavirus genus (Fig. 2) and confirmed that the ES-02 and ES-06 isolates were closely related to APMV-10 (bootstrap value of 100%). The ES-03 and ES-05 isolates were closely related to the AMPV-2 serotype, showing more similarity to the Bangor subgroup than to the California/Yucaipa. Although serological assays were not performed for all known Avulavirus serotypes, our results indicate that the penguin paramyxovirus isolates described here should probably be classified as new members of the APMV-2 and AMPV-10 serotypes. These results reveal, for the first time, the presence of paramyxovirus in migratory penguins at Brazilian coast.
4. Discussion Since 2005, our group has monitored the circulation of orthomyxovirus and paramyxovirus in resident and migratory birds as part of the effort to survey avian pathogens transmitted by wild birds in Brazil (MAPA, 2006). As previous studies of sub-Antarctic and Antarctic penguins have shown that these animals may become infected with a variety of avian influenza viruses and paramyxoviruses, including NDV (Morgan et al., 1981; Alexander et al., 1989; Austin and Webster, 1993; Wallensten et al., 2006), the presence of some of these viruses in penguins that arrived on the southeast coast of Brazil was analyzed. We isolate five hemagglutinating avian viruses compatible with paramyxoviruses by electron microscopy and positive for avian paramyxovirus by HI test. Two samples were identified serologically as APMV-2 and three as AMPV-10. For four of these isolates the sequencing of conserved domain of L gene confirmed serological results, putting isolates ES-03 and ES-05 as new members of APMV-2, and, ES-02 and ES-06 as new members of APMV-10 of avian paramyxoviruses. The strong sequence identity between the ES-03 and ES-05 samples reinforces the classification of these samples as members of the APMV-2 serotype, closest to
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the Bangor subgroup. This subgroup was recently identified after the complete sequencing of a strain isolated from a finch (Subbiaha et al., 2010). Two distinct subgroups of APMV-2 (Bangor and California/Yucaipa) were proposed based on their relatively weak (66.5%) identity to the L protein (Subbiaha et al., 2010). These two subgroups differ substantially in their nucleotide and amino acid sequences but show only discrete antigenic differences (Kumar et al., 2010). Additionally, ES-02 and ES-06 Domain III of L-gene sequences showed 93% homology to sequences with a proposed new serotype (APMV-10) of avian paramyxoviruses (Miller et al., 2010). Phylogenetic analysis put these new isolates together with this new serotype also, reinforcing the hypothesis that they are new members of APMV-10 serotype. Therefore, although a complete genomic analysis of the four isolates described here is still necessary, our data permit us to propose that they belong to the Bangor subgroup of APMV-2 and to APMV-10 serotype. Garcia-Borboroglu et al. (2010) reported that Magellanic penguins migrated farther north than usual in 2008, probably because of scarce food resources, and many starved. Magellanic penguins are considered sentinels of ocean conditions (Stokes et al., 1998; Boersma, 2008) and their unusual migration behavior in 2008 may be associated to the abnormal ocean conditions observed from Brazil to the northern coasts of Argentina that year potentially linked to climate variability. The isolation of paramyxoviruses from penguins demonstrates that this species may be a vector for spreading these viruses into regions distant from its original habitat. Wild avian species of the eastern South American coast may become infected by these viruses through close contact between migratory and resident birds. Although we cannot say whether the paramyxoviruses isolated from the penguins were acquired, in their breeding areas or during the migration period along Argentinean, Uruguayan and Brazilian coasts, surveillance work in Antarctica has indicated the presence of the paramyxovirus identified in this work in that region (Alexander et al., 1989; Miller et al., 2010). Thus far, only NDV paramyxovirus have been focused for Brazilian Department of Agriculture as surveillance target for wild birds (MAPA, 2006). These results reinforce the importance of continued surveys on migratory penguins to scale potential risks of virus spread from origin to unnatural habitats. Acknowledgements We thank the nongovernmental organization ORCA for field assistance and FAPERJ for financial support (E-26/ 170.642/2006). We are grateful to Dr. Patty Miller and
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