• View in gallery

    Phylogenetic tree showing the relationships of chikungunya virus (CHIK) strains. A 434-basepair fragment from the envelope 1 gene was analyzed by using the neighbor-joining method, the number of differences model, and bootstrap percentage corresponding to 1,000 replicates with the MEGA 4 software. O'nyong nyong virus (ONNV) was used as an outgroup. Accession number, name of the strain, and place and year of isolation are indicated. Strains sequenced in this paper are shown in bold. Bootstrap values > 80 are shown.

  • 1.

    Kuniholm MH, Wolfe ND, Huang CY, Mpoudi-Ngole E, Tamoufe U, Burke DS, Gubler DJ, 2006. Seroprevalence and distribution of Flaviviridae, Togaviridae, and Bunyaviridae arboviral infections in rural Cameroonian adults. Am J Trop Med Hyg 74: 10781083.

    • Search Google Scholar
    • Export Citation
  • 2.

    Peyrefitte CN, Bessaud M, Pastorino BA, Gravier P, Plumet S, Merle OL, Moltini I, Coppin E, Tock F, Daries W, Ollivier L, Pages F, Martin R, Boniface F, Tolou HJ, Grandadam M, 2008. Circulation of chikungunya virus in Gabon, 2006–2007. J Med Virol 80: 430433.

    • Search Google Scholar
    • Export Citation
  • 3.

    Pastorino B, Muyembe-Tamfum JJ, Bessaud M, Tock F, Tolou H, Durand JP, Peyrefitte CN, 2004. Epidemic resurgence of chikungunya virus in Democratic Republic of the Congo: identification of a new central African strain. J Med Virol 74: 277282.

    • Search Google Scholar
    • Export Citation
  • 4.

    Powers AM, Logue CH, 2007. Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus. J Gen Virol 88: 23632377.

  • 5.

    Enserink M, 2006. Massive outbreak draws fresh attention to little-known virus. Science 311: 1085.

  • 6.

    Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L, Vaney MC, Lavenir R, Pardigon N, Reynes JM, Pettinelli F, Biscornet L, Diancourt L, Michel S, Duquerroy S, Guigon G, Frenkiel MP, Bréhin AC, Cubito N, Desprès P, Kunst F, Rey FA, Zeller H, Brisse S, 2006. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. PLoS Med 3: e263.

    • Search Google Scholar
    • Export Citation
  • 7.

    Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, Cardioli P, Fortuna C, Boros S, Magurano F, Silvi G, Angelini P, Dottori M, Ciufolini MG, Majori GC, Cassone A, 2007. Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet 370: 18401846.

    • Search Google Scholar
    • Export Citation
  • 8.

    Casas I, Powell L, Klapper PE, Cleator GM, 1995. New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J Virol Methods 53: 2536.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ledesma J, Fedele CG, Carro F, Lledo L, Sánchez-Seco MP, Tenorio A, Soriguer RC, Sanz JV, Dominguez G, Rosas MF, Barandika JF, Gegundez MI, 2009. Independent lineage of lymphocytic choriomeningitis virus in wood mouse (Apodemus sylvaticus), Spain. Emerg Infect Dis 15: 16771680.

    • Search Google Scholar
    • Export Citation
  • 10.

    Sánchez-Seco MP, Vázquez A, Collao X, Hernández L, Aranda C, Ruiz S, Escosa R, Gutiérrez R, Bustillo MA, Molero F, Tenorio A, 2009. Surveillance of arboviruses in Spanish wetlands: detection of new flavi and phleboviruses. Vector Borne Zoonotic Dis. 2009 Jun 1 [Epub ahead of print].

    • Search Google Scholar
    • Export Citation
  • 11.

    Powers AM, Brault AC, Tesh RB, Weaver SC, 2000. Re-emergence of chikungunya and o'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships. J Gen Virol 81: 471479.

    • Search Google Scholar
    • Export Citation
  • 12.

    Sánchez-Seco MP, Negredo AI, Puente S, Pinazo MJ, Schuffenecker I, Tenorio A, Fedele CG, Domingo C, Rubio JM, de Ory F, 2009. Diagnóstico microbiológico del virus chikungunya en España (2006–2007): detección de casos en viajeros. Enf Inf Microbiol Clin 27: 457461.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

Different Lineages of Chikungunya Virus in Equatorial Guinea in 2002 and 2006

View More View Less
  • 1 Laboratory of Arbovirus and Imported Viral Diseases, National Center of Microbiology, Instituto de Salud Carlos III, Madrid, Spain; National Center of Tropical Medicine, Madrid, Spain; Hospital de Elche, Alicante, Spain

Chikungunya virus is a mosquito-borne virus that causes an acute febrile infection and severe arthralgia and is considered a re-emergent pathogen. During a study investigating arboviruses causing febrile infection in infants in Bata, Equatorial Guinea, the genome of this virus was amplified from blood samples during near two rainy seasons (2002–2003). In 2006, this virus was isolated from a traveler returning to Spain from Equatorial Guinea. These results show that chikungunya virus is present in this country and two lineages are circulating. Thus, this virus should be considered in the differential diagnosis of febrile syndromes in inhabitants and in travelers returning from this country.

Tropical Africa is the likely site of origin of many of the arboviruses of modern medical importance. However, there is little data about the prevalence of arboviral infections in this region.

Equatorial Guinea is located in the equatorial part of Africa between Cameroon and Gabon and is divided into a continental part (area = 26,017 km2) and some islands (area = 2,034 km2). It has a population of approximately 500,000 persons, with a high percentage (45%) less than 15 years of age. This country has an equatorial climate with an annual average temperature of 25°C. The average annual precipitation (> 2,000 mm) is divided into two rainy seasons: from April to May and from October to December.

In west and central Africa, data on the circulation of chikungunya virus (CHIKV) are rare. In Cameroon, serologic prevalence assays have shown that the alphaviruses CHIKV and O'nyong nyong virus are the most common arboviral infections.1 In Gabon, an outbreak of a dengue-like syndrome caused by CHIKV was described in 2006–2007.2 The virus was phylogenetically related to strains isolated in 2006 in Cameroon and seen years earlier in the Democratic Republic of Congo, where 50,000 persons were infected.3

Chikungunya virus belongs to the family Togaviridae and the genus Alphavirus. It causes an acute infection with an abrupt onset of fever, headache and severe joint pains and is transmitted mainly by Aedes mosquitoes.4 In recent years, it has been considered a re-emergent virus and has become a public health problem in countries such as the Democratic Republic of Congo.3 More recently, CHIKV has caused large outbreaks that affected different Indian Ocean territories and continental India.5,6 This epidemic was the source of an outbreak in Italy caused by an infected traveler from India.7

In 2002–2003, a project to study the presence of some viruses in Equatorial Guinea was conducted. Samples from febrile children attending the Reference Center for the Control of Endemic Diseases located in Bata (continental region of Equatorial Guinea) were obtained. From June 2002 to January 2003, 720 blood samples were obtained. RNA was preserved in a guanidinium isothiocyanate buffer. Samples were sent to the National Center for Microbiology in Madrid, Spain, for testing. Samples used were not given personal identifiers to comply with bioethics guides.

After RNA extraction,8 samples were assayed. We used generic primers to potentially detect arboviruses in different polymerase chain reaction (PCR) assays. We searched for arenaviruses,9 hantaviruses, and orthobunyaviruses (using in-house methods); no positive results were obtained. Arboviruses belonging to the genera Flavivirus, Phlebovirus, and/or Alphavirus were tested by using a generic multiplex real-time nested PCR in which consensus primers for each of the three genera were mixed.10 Positive amplification with the generic multiplex method was achieved in eight samples obtained in the rainy seasons when vector activity is high. Sequences of amplified fragments corresponding to 195 basepairs of the non-structural protein 4 gene of alphaviruses identified a homogeneous cluster of CHIKV belonging to the Central–East/South Africa genotype. Chikungunya virus was found in 2002 (three samples in June, one sample in July, and two samples in December) and 2003 (two samples in January). Four of these eight virus-positive samples were also positive for Plasmodium falciparum.

To obtain a sequence with more phylogenetic information, primers designed by Powers and others (sense 10246 5′-TTACCCNTTYATGTGGGG-3′10262 and antisense 10793 5′-CTTACSGGGTTTGTYGC-3′10777),11 were used in combination with primer10714 5′-TRAAGCCAGATGGTGCC-3′0698 to amplify a fragment of a region of the envelope 1 (E1) gene often used for CHIKV phylogenetic analysis. Three of the eight positive samples (probably those with the highest concentration of virus) showed a 469-basepair product whose sequences formed a unique cluster within the Central/East African genotype clade (Figure 1).

Figure 1.
Figure 1.

Phylogenetic tree showing the relationships of chikungunya virus (CHIK) strains. A 434-basepair fragment from the envelope 1 gene was analyzed by using the neighbor-joining method, the number of differences model, and bootstrap percentage corresponding to 1,000 replicates with the MEGA 4 software. O'nyong nyong virus (ONNV) was used as an outgroup. Accession number, name of the strain, and place and year of isolation are indicated. Strains sequenced in this paper are shown in bold. Bootstrap values > 80 are shown.

Citation: The American Society of Tropical Medicine and Hygiene 82, 3; 10.4269/ajtmh.2010.09-0435

In Spain, CHIKV was considered in the differential diagnosis of a febrile syndrome in travelers returning from Equatorial Guinea. In 2006, one of three such travelers returning from this area was diagnosed in Spain as being infected with CHIKV on the basis of a positive PCR result, which showed amplification of part of the E1 gene.12 This sequence is similar to others described at the same time from Cameroon and Gabon,1,2 but is different from sequences obtained in Equatorial Guinea in 2002. There is evidence of endemic circulation of a genetically stable monophyletic CHIKV population during 2000–2007 in west central Africa.2

Our results suggest that a paraphyletic population of CHIKV was circulating in Equatorial Guinea in 2002. Our results indicate that CHIKV is likely endemic in Equatorial Guinea, and suggest that either two (or more) CHIKV populations were co-circulating in Equatorial Guinea or the western Central African lineage has replaced the lineage detected during 2002–2003. Our data clearly identified three genotypes but cannot be used to unequivocally demonstrate the endemicity of CHIKV in Equatorial Guinea. To conduct a robust phylogenetic analysis, complete sequences of viruses belonging to each group are needed. Therefore, studies of CHIKV in patients infected in Equatorial Guinea are ongoing.

Acknowledgments:

We thank the staff of the Center for the Control of Endemic Diseases (Bata, Equatorial Guinea) for their collaboration in this study and Fionna Westbury for grammatical assistance.

  • 1.

    Kuniholm MH, Wolfe ND, Huang CY, Mpoudi-Ngole E, Tamoufe U, Burke DS, Gubler DJ, 2006. Seroprevalence and distribution of Flaviviridae, Togaviridae, and Bunyaviridae arboviral infections in rural Cameroonian adults. Am J Trop Med Hyg 74: 10781083.

    • Search Google Scholar
    • Export Citation
  • 2.

    Peyrefitte CN, Bessaud M, Pastorino BA, Gravier P, Plumet S, Merle OL, Moltini I, Coppin E, Tock F, Daries W, Ollivier L, Pages F, Martin R, Boniface F, Tolou HJ, Grandadam M, 2008. Circulation of chikungunya virus in Gabon, 2006–2007. J Med Virol 80: 430433.

    • Search Google Scholar
    • Export Citation
  • 3.

    Pastorino B, Muyembe-Tamfum JJ, Bessaud M, Tock F, Tolou H, Durand JP, Peyrefitte CN, 2004. Epidemic resurgence of chikungunya virus in Democratic Republic of the Congo: identification of a new central African strain. J Med Virol 74: 277282.

    • Search Google Scholar
    • Export Citation
  • 4.

    Powers AM, Logue CH, 2007. Changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus. J Gen Virol 88: 23632377.

  • 5.

    Enserink M, 2006. Massive outbreak draws fresh attention to little-known virus. Science 311: 1085.

  • 6.

    Schuffenecker I, Iteman I, Michault A, Murri S, Frangeul L, Vaney MC, Lavenir R, Pardigon N, Reynes JM, Pettinelli F, Biscornet L, Diancourt L, Michel S, Duquerroy S, Guigon G, Frenkiel MP, Bréhin AC, Cubito N, Desprès P, Kunst F, Rey FA, Zeller H, Brisse S, 2006. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak. PLoS Med 3: e263.

    • Search Google Scholar
    • Export Citation
  • 7.

    Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, Cardioli P, Fortuna C, Boros S, Magurano F, Silvi G, Angelini P, Dottori M, Ciufolini MG, Majori GC, Cassone A, 2007. Infection with chikungunya virus in Italy: an outbreak in a temperate region. Lancet 370: 18401846.

    • Search Google Scholar
    • Export Citation
  • 8.

    Casas I, Powell L, Klapper PE, Cleator GM, 1995. New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J Virol Methods 53: 2536.

    • Search Google Scholar
    • Export Citation
  • 9.

    Ledesma J, Fedele CG, Carro F, Lledo L, Sánchez-Seco MP, Tenorio A, Soriguer RC, Sanz JV, Dominguez G, Rosas MF, Barandika JF, Gegundez MI, 2009. Independent lineage of lymphocytic choriomeningitis virus in wood mouse (Apodemus sylvaticus), Spain. Emerg Infect Dis 15: 16771680.

    • Search Google Scholar
    • Export Citation
  • 10.

    Sánchez-Seco MP, Vázquez A, Collao X, Hernández L, Aranda C, Ruiz S, Escosa R, Gutiérrez R, Bustillo MA, Molero F, Tenorio A, 2009. Surveillance of arboviruses in Spanish wetlands: detection of new flavi and phleboviruses. Vector Borne Zoonotic Dis. 2009 Jun 1 [Epub ahead of print].

    • Search Google Scholar
    • Export Citation
  • 11.

    Powers AM, Brault AC, Tesh RB, Weaver SC, 2000. Re-emergence of chikungunya and o'nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships. J Gen Virol 81: 471479.

    • Search Google Scholar
    • Export Citation
  • 12.

    Sánchez-Seco MP, Negredo AI, Puente S, Pinazo MJ, Schuffenecker I, Tenorio A, Fedele CG, Domingo C, Rubio JM, de Ory F, 2009. Diagnóstico microbiológico del virus chikungunya en España (2006–2007): detección de casos en viajeros. Enf Inf Microbiol Clin 27: 457461.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to María-Paz Sánchez-Seco, Laboratory of Arbovirus and Imported Viral Diseases, National Center of Microbiology, Instituto de Salud Carlos III, Ctra Pozuelo-Majadahonda Km 2, 28220 Madrid, Spain. E-mail: paz.sanchez@isciii.es†These authors contributed equally to the article.

Financial support: This study was supported by the Instituto de Salud Carlos III (MPY 1207/04) and Fondo de Investigaciones Sanitarias (FIS PI080834) and is part of the work carried out within the Red de Investigación Cooperativa en Enfermedades Tropicales network (FIS C03/04 and RD06/0021) of the Spanish Ministry of Health. Ximena Collao is supported by the Higher Education Quality and Equity Improvement Program (MECESUP) project, Chile.

Authors' addresses: Ximena Collao, Ana I. Negredo, Antonio Tenorio, Fernando de Ory, and María-Paz Sánchez-Seco, Laboratory of Arbovirus and Imported Viral Diseases, National Center of Microbiology, Instituto de Salud Carlos III, Ctra Pozuelo-Majadahonda Km 2, 28220 Madrid, Spain, E-mails: xime.collao@isciii.es, anabelnegrdo@isciii.es, atenorio@isciii.es, fory@isciii.es, and paz.sanchez@isciii.es. Jorge Cano and Agustin Benito, National Center of Tropical Medicine, Instituto de Salud Carlos III, C/Sinesio Delgado no. 6, 28029 Madrid, Spain, E-mails: jcano@isciii.es and abenito@isciii.es. Mar Masia, Hospital de Elche, Elche, Alicante, Spain, E-mail: marmasia@ya.com.

Save