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Cacipacore virus as an emergent mosquito-borne Flavivirus

Abstract

INTRODUCTION:

Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil.

METHODS:

Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons.

RESULTS:

Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region.

CONCLUSIONS:

This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

Keywords:
Cacipacore vírus; Mosquitoes; Emerging Flavivirus

Cacipacore virus (CPCV) is a member of the Japanese encephalitis virus (JEV) complex belonging to the genus Flavivirus of the family Flaviviridae11. Kuno G, Chang G, Tsuchiya K, Karabatsos N, Cropp C. Phylogeny of the genus Flavivirus. J Virol. 1998;72(1):73-83.. It was originally isolated in 1977, from the blood of a bird (Percnostola rufifrons) in the Amazon region of Brazil22. Rosa JFST, Rosa APAT, Vasconcelos PFC, Pinheiro FP, Rodrigues SG, Rosa EST, et al. Arboviruses isolated in the Evandro Chagas Institute, including some described for the first time in the Brazilian Amazon region, their known hosts, and their pathology for man. In: Travassos da Rosa APA, Vasconcelos PFC, Rosa JFST, editors. An overview of arbovirology on Brazil and neighboring countries. Belem: Instituto Evandro Chagas; 1998. p. 19-31.. Recently, antibodies against CPCV have been reported in equines, non-human primates, and water buffalo in the central region of Brazil, suggesting virus circulation33. Pauvolid-Corrêa A, Campos Z, Juliano R, Velèz J, Nogueira RMR, Komar N. Serological evidence of widespread circulation of West Nile virus and other flaviviruses in equines of the Pantanal, Brazil. PLoS Negl Trop Dis. 2014;8(2):e2706.

4. Batista PM, Andreotti R, Almeida PS, Marques AC, Rodrigues SG, Chiang JO, et al. Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil. Rev Soc Bras Med Trop. 2013,46(6):684-90.
-55. Casseb AR, Cruz AV, Jesus IS, Chiang JO, Martins LC, Silva SP, et al. Seroprevalence of flaviviruses antibodies in water buffaloes (Bubalus bubalis) in Brazilian Amazon. J Venom Anim Toxins Incl Trop Dis. 2014;20:9.. In addition, one patient from the Amazon region exhibiting an acute febrile illness was found to be infected by CPCV in 201166. Batista WC, Tavares GS, Vieira DS, Honda ER, Pereira SS, Tada MS. Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia, Brazil. Rev Soc Bras Med Trop . 2011;44(4):528-30.. Here, we present the first report of CPCV-infected mosquitoes collected from the environment.

As part of the arbovirus surveillance programs (2002 to 2006), approximately 3,253 mosquitoes (Culicidae) were collected from urban and rural areas in Manaus (Amazonas State) and Monte Negro County (Rondonia State), using attraction traps including the CO2, the cluster of differentiation (CD4) night model, and the Shannon. Captured specimens were identified based on morphological characteristics and grouped into 264 pools (including 10-13 specimens per pool) and stored at −80 °C77. Forattini OP. Culicidologia Médica: Identificação, Biologia, Epidemiologia. Vol. 2. São Paulo: Editora da Universidade de São Paulo; 2002. 864p..

Reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing for arbovirus diagnosis and identification were performed using ribonucleic acid (RNA) extracts from the mosquito pools while taking precautions to avoid contamination. The mosquito samples were triturated at −4°C temperatures using plastic pestles and divided into two aliquots: one for RNA extraction and the other for future virus isolation. Aliquots were stored at −80°C until ready for use. RNA was extracted from the mosquito pools, using the PureLink Viral ribonucleic acid/deoxyribonucleic acid (RNA/DNA) Kit (Invitrogen, USA) according to the manufacturer’s instructions. Subsequently, RT and Hemi-Nested-PCR Flavivirus assays that differentiate dengue, yellow fever, and other viruses based on amplicon size, were performed as previously reported88. Morais Bronzoni RV, Baleotti FG, Ribeiro Nogueira RM, Nunes M, Moraes Figueiredo LT. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. 2005;43(2):696-702..

The PCR products were purified using the QIAquick Gel Extraction Kit (QIAGEN, Germany), following the manufacturer's protocol and subsequently sequenced with the ABI PRISM 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences obtained in this study were submitted to GenBank under access numbers GU811223 to GU811230, as follows: CPCVBR/RO/Culex sp 54C/2002 - GU811223; CPCVBR/RO/Anopheles sp 43A5/2002 - GU811224; CPCVBR/AM/Aedes aegypti 17/2005 - GU811225; CPCVBR/AM/Aedes aegypti 46/2005 - GU811226; and CPCVBR/AM/Aedes aegypti 47/2005 - GU811227. CPCV and others flavivirus sequences retrieved from GenBank were aligned using the BioEdit v 7.09 program99. Hall T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999;41:95-8.. Phylogenetic trees based on Neighbor-joining (NJ) methods were constructed using the Mega 5 software1010. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731-9..

Captured specimens were identified based on morphological characteristics as Aedes aegypti, Aedes albopictus, Anopheles sp., Culex sp., Coquillettidia sp., Haemagogus janthinomys, Haemagogus leucocelaenus, Haemagogus spegazzinii, Psorophor albigenus, Psorophora albipes, and Psorophora ferox77. Forattini OP. Culicidologia Médica: Identificação, Biologia, Epidemiologia. Vol. 2. São Paulo: Editora da Universidade de São Paulo; 2002. 864p., as shown in Table 1.

TABLE 1:
Number of Culicidea (Diptera) collected for the study from 2002 to 2006.

For virus detection, we used a Hemi-Nested-PCR that did not include a specific primer for Cacipacore virus. Interestingly, CPCV amplicons were obtained using the primer for dengue virus (DENV) type 2 and the CACV specific origin of the amplicons was only recognized following nucleotide sequencing88. Morais Bronzoni RV, Baleotti FG, Ribeiro Nogueira RM, Nunes M, Moraes Figueiredo LT. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. 2005;43(2):696-702.. Amplicons ~200-300bp in size, corresponding to a section of the NS5 gene region of Flavivirus were obtained from five mosquito pools (1.89% positivity). Following further amplification and purification, it was possible to sequence the amplicons. The pools infected by Flavivirus included Aedes aegypti, Anopheles sp., and Culex sp., as shown in Table 2.

TABLE 2:
Flavivirus amplicons obtained by reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assays for mosquitoes collected in the Brazilian Amazon region, 2002-2006.

The sequences, ranging from 216 to 275 nucleotides, were aligned with other sequences retrieved from GenBank. The sequences exhibited 98-100% similarity with those of the CPCV originally isolated in 1977 from a bird11. Kuno G, Chang G, Tsuchiya K, Karabatsos N, Cropp C. Phylogeny of the genus Flavivirus. J Virol. 1998;72(1):73-83.. The Flavivirus phylogenetic tree, shown in Figure 1, includes sequences of the viruses infecting our pools of mosquitoes, all of which cluster within the same CPCV branch, confirming this virus is the causative agent of the infections. The tree also shows that CPCV is related to Japanese encephalitis virus, corroborating a previous report by Kuno et al.11. Kuno G, Chang G, Tsuchiya K, Karabatsos N, Cropp C. Phylogeny of the genus Flavivirus. J Virol. 1998;72(1):73-83.also based on the NS5 gene sequence. Unfortunately, despite numerous attempts that exhausted our samples, we were unable to obtain larger nucleotide sequences from our mosquito pools.

FIGURE 1
- Phylogenetic tree based on NS5 partial gene sequences. The tree was constructed using the Neighbor-joining method with 1,000 bootstrap replications. Branch lengths are proportional to the percentage of divergence. The Tamura-Nei nucleotide substitution model was used with a gamma distribution (shape parameter = 1). GenBank accession numbers, species, country of origin, and year of isolation are detailed in the tree. CPCVs isolated from mosquito samples are indicated with a square (▄). SLEV: Japanese encephalitis; ROCV: Rocio virus; ILHV: lhéus virus; KUNV: Kunjin virus; USUV: Usutu virus; MVEV: Murray Valley encephalitis virus; JEV: Japanese encephalitis; BR/RO: Brazil/Rondonia; CPCV: Cacipacoré virus; BR/AM/A: Brazil/Amazonas/aedes; YFV: yellow fever virus;. LGTV: Langat virus; LIV: Louping ill virus .

Japanese encephalitis virus (JEV), Ilhéus virus (ILHV), Rocio virus (ROCV), Saint Louis encephalitis virus (SLEV), and West Nile virus (WNV) are all zoonotic avian viruses transmitted by Culicidae mosquitoes. Thus, it is possible that CPCV, a closely related virus, shares the same zoonotic characteristics because it was originally isolated from a bird.

Aedes aegypti, an urban anthropophilic mosquito that lives around human houses, is the main vector for DENV in Brazil and has been involved in huge outbreaks over the last 30 years, with more than 10 million reported cases. Over the last three years, Aedes aegypti has been involved in the transmission of Chikungunya virus (CHIKV) and Zika virus (ZIKV), which have also resulted in large outbreaks and thousands of infected cases. In the present study, CPCV was detected in pools of Aedes aegypti collected in Manaus, a city of two million inhabitants located at the center of the Amazon rain forest. Therefore, it is possible that sylvatic CPCV could have been introduced into this city and infected Aedes aegypti; CPCV could have been transmitted to humans through this mosquito vector, but remained unnoticed amidst the widespread outbreaks of dengue and other tropical febrile diseases. It is important to note that serologic diagnostic tests for dengue could be positive for flavivirus cross-reactivity in patients with CPCV.

Culex pipiens are found worldwide, breeding in water contaminated with organic matter. The conditions in poorly sanitized urban areas of Brazil support the propagation of this mosquito. Culex pipiens feed at night and are zoophilic; they are particularly more ornithophilic. Alphaviruses such as Western Equine Encephalitis (WEEV), Venezuelan Equine Encephalitis (VEEV), and Eastern Equine Encephalitis (EEEV); Orthobunyaviruses such as Caraparu virus and Oropouche virus; and Flaviviruses, such as SLEV, have been isolated from Culex species in different regions of Brazil and Argentina. SLEV has also been isolated from birds, including migratory species, as well as rodents22. Rosa JFST, Rosa APAT, Vasconcelos PFC, Pinheiro FP, Rodrigues SG, Rosa EST, et al. Arboviruses isolated in the Evandro Chagas Institute, including some described for the first time in the Brazilian Amazon region, their known hosts, and their pathology for man. In: Travassos da Rosa APA, Vasconcelos PFC, Rosa JFST, editors. An overview of arbovirology on Brazil and neighboring countries. Belem: Instituto Evandro Chagas; 1998. p. 19-31.,1111. Coimbra TLM, Rocco IM, Suzuki A, Pereira L, Souza L, Nassar E, et al. Arthropod- and Rodent-borne viruses detected in São Paulo State, Brazil. In: Travassos da Rosa APA, Vasconcelos PFC, Travassos da Rosa JFST, editors. An overview of arbovirology on Brazil and neighboring countries. Belem: Instituto Evandro Chagas ; 1998. p. 168-176.

12. Sabattini MS, Aviles G, Monath TO. Historical, epidemiological and ecological aspects of arboviruses in Argentina: Flaviviridae, Bunyaviridae and Rhabdoviridae. In: Travassos da Rosa A, Vasconcelos PFC, Travassos da Rosa JFS, editors. An Overview of Arbovirology on Brazil and Neighboring Countries. Belem, Pará, Brazil: Instituto Evandro Chagas; 1998. p. 113-34.
-1313. Vasconcelos P, Travassos da Rosa A, Pinheiro F, Shope RE, Travassos da Rosa J, Rodriges S, et al. Arboviruses pathogenic for man in Brazil. In: Travassos da Rosa A, Vasconcelos PFC, Travassos da Rosa JFS, editors. An Overview of Arbovirology on Brazil and Neighboring Countries. Belem, Pará, Brazil: Instituto Evandro Chagas; 1998. p. 72-99.. Thus, because CPCV has been found miles apart, it is conceivable that the virus has a natural cycle involving Culex species as vectors and birds, including those with migratory habits, as reservoirs.

Anopheles mosquitoes, especially Anopheles darlingi, are important vectors of parasites of the genus Plasmodium in Brazil, which cause malaria. These mosquitoes have also been shown to be infected with alphaviruses, (VEEV), orthobunyaviruses (Guaroa virus and Tacaiuma virus), and the flavivirus SLEV22. Rosa JFST, Rosa APAT, Vasconcelos PFC, Pinheiro FP, Rodrigues SG, Rosa EST, et al. Arboviruses isolated in the Evandro Chagas Institute, including some described for the first time in the Brazilian Amazon region, their known hosts, and their pathology for man. In: Travassos da Rosa APA, Vasconcelos PFC, Rosa JFST, editors. An overview of arbovirology on Brazil and neighboring countries. Belem: Instituto Evandro Chagas; 1998. p. 19-31.,1111. Coimbra TLM, Rocco IM, Suzuki A, Pereira L, Souza L, Nassar E, et al. Arthropod- and Rodent-borne viruses detected in São Paulo State, Brazil. In: Travassos da Rosa APA, Vasconcelos PFC, Travassos da Rosa JFST, editors. An overview of arbovirology on Brazil and neighboring countries. Belem: Instituto Evandro Chagas ; 1998. p. 168-176.

12. Sabattini MS, Aviles G, Monath TO. Historical, epidemiological and ecological aspects of arboviruses in Argentina: Flaviviridae, Bunyaviridae and Rhabdoviridae. In: Travassos da Rosa A, Vasconcelos PFC, Travassos da Rosa JFS, editors. An Overview of Arbovirology on Brazil and Neighboring Countries. Belem, Pará, Brazil: Instituto Evandro Chagas; 1998. p. 113-34.
-1313. Vasconcelos P, Travassos da Rosa A, Pinheiro F, Shope RE, Travassos da Rosa J, Rodriges S, et al. Arboviruses pathogenic for man in Brazil. In: Travassos da Rosa A, Vasconcelos PFC, Travassos da Rosa JFS, editors. An Overview of Arbovirology on Brazil and Neighboring Countries. Belem, Pará, Brazil: Instituto Evandro Chagas; 1998. p. 72-99.. Thus, it is possible that Anopheles mosquitoes could also transmit CPCV. As observed in the phylogenetic analysis, CPCV is closely related to JEV, which has also been reported in Anopheles and Aedes spp. However, further studies are necessary to check vector competence of these mosquitoes and the risk of CPCV emergence producing outbreaks1414. Huang YJ, Higgs S, Horne KM, Vanlandingham DL. Flavivirus-mosquito interactions. Viruses. 2014;6(11):4703-30..

The report of one patient from the Amazon region with an acute febrile illness caused by CPCV could resemble reports warning of ZIKV infections (only 13 reported human cases in six decades)1515. Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika Virus. N Engl J Med. 2016;374(16):1552-63.. However, over the past few years, ZIKV has emerged as an important pathogen causing worldwide epidemics associated with Aedes mosquitoes, especially in Brazil. Thus, CPCV may represent an important public health threat similar to ZIKV.

Cacipacore virus is a zoonotic flavivirus that infects different species of mosquitoes, including the urban anthropophilic Aedes aegypti, which possesses great potential to emerge in urban Brazilian areas and cause human disease. Thus, further studies are required to confirm the importance of CPCV and its vectors. This study, using molecular biology technique, shows Aedes aegypti, Anopheles sp and Culex sp mosquitoes from the Amazon region can be infected by Cacipacoré virus, a Flavivirus until recently obscure but recently reported as causative of human acute febrile illness. These findings highlight a potential vectorial condition for emergence of this Flavivirus as a public health problem in Brazil.

Acknowledgements

We wish to thank Dr. Luis Marcelo Aranha, coordinator of the Center for Advanced Instituto de Ciências Biomédicas/Universidade de São Paulo (ICB/USP) in Rondônia (ICB5/USP) for assistance in capturing mosquitoes.

REFERENCES

  • 1
    Kuno G, Chang G, Tsuchiya K, Karabatsos N, Cropp C. Phylogeny of the genus Flavivirus. J Virol. 1998;72(1):73-83.
  • 2
    Rosa JFST, Rosa APAT, Vasconcelos PFC, Pinheiro FP, Rodrigues SG, Rosa EST, et al. Arboviruses isolated in the Evandro Chagas Institute, including some described for the first time in the Brazilian Amazon region, their known hosts, and their pathology for man. In: Travassos da Rosa APA, Vasconcelos PFC, Rosa JFST, editors. An overview of arbovirology on Brazil and neighboring countries. Belem: Instituto Evandro Chagas; 1998. p. 19-31.
  • 3
    Pauvolid-Corrêa A, Campos Z, Juliano R, Velèz J, Nogueira RMR, Komar N. Serological evidence of widespread circulation of West Nile virus and other flaviviruses in equines of the Pantanal, Brazil. PLoS Negl Trop Dis. 2014;8(2):e2706.
  • 4
    Batista PM, Andreotti R, Almeida PS, Marques AC, Rodrigues SG, Chiang JO, et al. Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil. Rev Soc Bras Med Trop. 2013,46(6):684-90.
  • 5
    Casseb AR, Cruz AV, Jesus IS, Chiang JO, Martins LC, Silva SP, et al. Seroprevalence of flaviviruses antibodies in water buffaloes (Bubalus bubalis) in Brazilian Amazon. J Venom Anim Toxins Incl Trop Dis. 2014;20:9.
  • 6
    Batista WC, Tavares GS, Vieira DS, Honda ER, Pereira SS, Tada MS. Notification of the first isolation of Cacipacore virus in a human in the State of Rondônia, Brazil. Rev Soc Bras Med Trop . 2011;44(4):528-30.
  • 7
    Forattini OP. Culicidologia Médica: Identificação, Biologia, Epidemiologia. Vol. 2. São Paulo: Editora da Universidade de São Paulo; 2002. 864p.
  • 8
    Morais Bronzoni RV, Baleotti FG, Ribeiro Nogueira RM, Nunes M, Moraes Figueiredo LT. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. 2005;43(2):696-702.
  • 9
    Hall T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999;41:95-8.
  • 10
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28(10):2731-9.
  • 11
    Coimbra TLM, Rocco IM, Suzuki A, Pereira L, Souza L, Nassar E, et al. Arthropod- and Rodent-borne viruses detected in São Paulo State, Brazil. In: Travassos da Rosa APA, Vasconcelos PFC, Travassos da Rosa JFST, editors. An overview of arbovirology on Brazil and neighboring countries. Belem: Instituto Evandro Chagas ; 1998. p. 168-176.
  • 12
    Sabattini MS, Aviles G, Monath TO. Historical, epidemiological and ecological aspects of arboviruses in Argentina: Flaviviridae, Bunyaviridae and Rhabdoviridae. In: Travassos da Rosa A, Vasconcelos PFC, Travassos da Rosa JFS, editors. An Overview of Arbovirology on Brazil and Neighboring Countries. Belem, Pará, Brazil: Instituto Evandro Chagas; 1998. p. 113-34.
  • 13
    Vasconcelos P, Travassos da Rosa A, Pinheiro F, Shope RE, Travassos da Rosa J, Rodriges S, et al. Arboviruses pathogenic for man in Brazil. In: Travassos da Rosa A, Vasconcelos PFC, Travassos da Rosa JFS, editors. An Overview of Arbovirology on Brazil and Neighboring Countries. Belem, Pará, Brazil: Instituto Evandro Chagas; 1998. p. 72-99.
  • 14
    Huang YJ, Higgs S, Horne KM, Vanlandingham DL. Flavivirus-mosquito interactions. Viruses. 2014;6(11):4703-30.
  • 15
    Petersen LR, Jamieson DJ, Powers AM, Honein MA. Zika Virus. N Engl J Med. 2016;374(16):1552-63.
  • Financial support: This work was supported by the São Paulo State Research Council (FAPESP) Grant No. 2015/04882-3 and the Brazilian Government Research Council [Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)] Grant No. 158828/2010-0

Publication Dates

  • Publication in this collection
    Jul-Aug 2017

History

  • Received
    23 Nov 2016
  • Accepted
    28 Mar 2017
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