International Journal of Infectious Diseases 33 (2015) 177–178
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Letter to the Editor Virological confirmation of concurrent dengue virus serotypes 1 and 4 by virus isolation using Fc-gamma receptor-expressing BHK cells
Keywords: Dengue Concurrent infection Virus isolation Neutralization Virological confirmation
Dengue virus (DENV) is estimated to infect approximately 400 million people annually. In 2014, 160 cases of autochthonous and 173 imported DENV infection cases were confirmed in Japan.1 Concurrent infection by two DENV serotypes is not an uncommon event in dengue hyperendemic areas where several DENV serotypes co-circulate.2 However, neither of the studies by previous investigators has clearly confirmed concurrent infection by virus isolation and separation.2,3 In the present study, we identified and confirmed a case of concurrent DENV-1 and DENV-4 infection in a patient by a method of virus isolation and separation for concurrent infection, using an Fc-gamma receptor (FcgR)expressing BHK cell line.4 The results present evidence of the presence of infectious virus particles of two virus serotypes in a patient.
A serum sample from a dengue patient who returned to Japan from a visit to Cambodia in 2013, was positive for DENV-1 (genotype 1; Genbank Accession number AB873104) and DENV-4 (genotype 1; Genbank Accession number AB873105) virus genome, and negative for anti-DENV IgM and IgG.5 Initial attempts to isolate and separate DENV using non FcgR-expressing Vero and BHK cells resulted in the isolation of DENV-1 only. To isolate DENV-4, the patient’s serum sample was treated with pooled human serum samples with neutralizing activity to DENV-1 but not to DENV-4 (DENV-1 PRNT50 1:10, DENV-4 PRNT50 < 10; as determined by FcgR-expressing BHK cells).6 DENV-1 and DENV-4 strains were isolated and separated, and confirmed by RT-PCR and sequencing of the E-protein gene7 (Table 1). Neutralizing antibodies to one DENV serotype may possess antibody-dependent enhancement (ADE) activity to heterologous serotypes in FcgR-positive cells, resulting in higher levels of progeny virus production.8 ADE activity also hampers serotype cross-reactive neutralizing antibody titers in FcgR-expressing cells but not in FcgR-negative cells.6 In the present study, both neutralization and the ADE mechanism were applied to virus isolation in a concurrent infection using FcgR-expressing cells. The serotype cross-reactive pooled serum samples used in this
Table 1 Virus isolation using FcgR-expressing cells and neutralizing antibodya Passage number
0 1 2
3
Sample number
Patient 1-1* 1-2 1-2-1 1-2-2 1-2-3 1-2-4 1-2-5 1-2-6 1-2-7 1-2-3-1 1-2-3-2 1-2-3-3* 1-2-3-4 1-2-3-5 1-2-3-6 1-2-3-7 1-2-3-8 1-2-3-9 1-2-3-10 1-2-3-11
Isolation method
None (patient serum sample) 10 ml serum sample + 10 ml 1:10 Nt Ab 10 ml serum sample + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml undiluted Nt Ab 1 ml supernatant + 10 ml undiluted Nt Ab 50 ml supernatant only 10 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 0.5 ml supernatant + 10 ml 1:10 Nt Ab 0.5 ml supernatant + 10 ml 1:10 Nt Ab 0.1 ml supernatant + 10 ml 1:10 Nt Ab 0.1 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml undiluted Nt Ab 10 ml supernatant + 10 ml undiluted Nt Ab 1 ml supernatant + 10 ml undiluted Nt Ab
Log10 genome copies/ml DENV-1
DENV-4
9.8 7.5 6.0 8.8 4.0 4.4 5.0 7.4 4.9 10.0 -
6.1 7.3 8.6 8.2 8.5 7.9 8.1 8.4 8.8 8.0 8.5 8.2 8.3 7.8 8.1 8.6 8.8 8.0
http://dx.doi.org/10.1016/j.ijid.2015.02.004 1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Letter to the Editor / International Journal of Infectious Diseases 33 (2015) 177–178
178 Table 1 (Continued ) Passage number
Sample number
Isolation method
Log10 genome copies/ml
1-2-3-12 1-2-3-13 1-2-3-14 1-2-3-15 1-2-3-16 1-2-3-17
1 ml supernatant + 10 ml undiluted Nt Ab 0.5 ml supernatant + 10 ml undiluted Nt Ab 0.5 ml supernatant + 10 ml undiluted Nt Ab 0.1 ml supernatant + 10 ml undiluted Nt Ab 0.1 ml supernatant + 10 ml undiluted Nt Ab 10 ml supernatant only
DENV-1
DENV-4
-
8.5 7.9 8.5 7.8 7.9 8.7
FcgR, Fc-gamma receptor; DENV, dengue virus. a Use of non-FcgR-expressing Vero cells for virus isolation in the first passage resulted in the isolation of DENV-1 isolate only, as determined by conventional RT-PCR. In the presence of neutralizing antibodies, only DENV-1 was isolated (10 ml supernatant + 10 ml 1:10 Nt Ab, DENV-1 = 6.6 log10 genome copies/ml; 1 ml supernatant + 10 ml 1:10 Nt Ab, DENV-1 = 4.7 log10 genome copies/ml) using sample 1-2, by BHK cells. ‘Nt Ab’ denotes pooled sera with neutralizing activity to DENV-1 but not to DENV-4 using FcgRexpressing cells as assay cells; ‘-’ denotes ‘not detected’; underlined numbers indicate supernatant used for further passage (supernatant with a low DENV-1 titer and a high DENV-4 titer was chosen for further passage); ‘*’ indicates sample used for sequencing and comparison to the DENV-1 and DENV-4 sequences of serum sample. The E-protein sequences of the DENV-1 (sample number 1-1) and DENV-4 (sample number 1-2-3-3) isolates were 100% identical to those DENV-1 and DENV-4 sequences using serum samples.
study demonstrated high levels of neutralizing antibody to DENV-1 but no neutralizing activity to DENV-4 using FcgRexpressing cells as assay cells.6 Thus, serotype-specific antibodies specifically neutralized the dominant DENV-1 serotype and may have enhanced the growth of the subdominant DENV-4 serotype. In conclusion, this report provides concrete evidence of concurrent DENV infection by multiple DENV serotypes in a DENV patient, and presents evidence on the potential of viremic travelers to introduce more than a single virus serotype to the local mosquito vector population.9,10 Acknowledgements We thank Ms Makiko Ikeda for providing excellent technical assistance for the antibody assay. We also thank Dr Kaw Bing Chua (Temasek Life Sciences Laboratory, Singapore) because this work would not have been possible without the convalescent dengue serum samples he provided. We thank Dr Jeffrey V. Ravetch (Rockfeller University, NY, USA) for generously providing us with the FcgRIIA cDNA and Dr Susheela Tridandapani (Ohio State University College of Medicine, Columbus, OH, USA) for assistance in obtaining the FcgRIIA cDNA. This work was supported in part by the research grant, Research on Emerging and Re-emerging Infectious Diseases (H23-shinkouippan-010; H26-shinkou-jitsuyouka-007) from the Ministry of Health, Labour and Welfare, Japan, the Environment Research and Technology Development Fund (S-8) of the Ministry of the Environment, and a Grant-in-Aid for Young Scientists (B) from JSPS (26870872). Ethical approval: The patient serum sample was de-identified prior to conducting the study. The assay protocol was approved by the institutional Medical Research Ethics Committee of the National Institute of Infectious Diseases, Japan (reference number 210). Conflict of interest: The authors declare that they have no competing interests. References 1. Kutsuna S, Kato Y, Moi ML, Kotaki A, Ota M, Shinohara K, et al. Autochthonous dengue fever, Tokyo, Japan, 2014. Emerg Infect Dis 2015 Jan 22. [Epub ahead of print]. http://dx.doi.org/10.3201/eid2103.141662. 2. Gubler DJ, Kuno G, Sather GE, Waterman SH. A case of natural concurrent human infection with two dengue viruses. Am J Trop Med Hyg 1985;34:170–3.
˜ o-Pino MA, Cropp CB, Farfa´n JA, Vorndam AV, Rodrı´guez-Angulo EM, 3. Loron Rosado-Paredes EP, et al. Common occurrence of concurrent infections by multiple dengue virus serotypes. Am J Trop Med Hyg 1999;61:725–30. 4. Moi ML, Lim CK, Kotaki A, Takasaki T, Kurane I. Detection of higher levels of dengue viremia using FcgR-expressing BHK-21 cells than FcgR-negative cells in secondary infection but not in primary infection. J Infect Dis 2011;203:1405–14. 5. Honma Y. Chikungunya and dengue—Japan: ex Cambodia (Kampong Cham). Archive number 20130829.1909917. ProMED-mail; 2013. 6. Moi ML, Lim CK, Chua KB, Takasaki T, Kurane I. Dengue virus infectionenhancing activity in serum samples with neutralizing activity as determined by using FcgR-expressing cells. PLoS Negl Trop Dis 2012;6:e1536. 7. Ito M, Yamada K, Takasaki T, Pandey B, Nerome R, Tajima S, et al. Phylogenetic analysis of dengue viruses isolated from imported dengue patients: possible aid for determining the countries where infections occurred. J Travel Med 2007;14:233–44. 8. Mady BJ, Erbe DV, Kurane I, Fanger MW, Ennis FA. Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors. J Immunol 1991;147:3139–44. 9. Sukehiro N, Kida N, Umezawa M, Murakami T, Arai N, Jinnai T, et al. First report on invasion of yellow fever mosquito, Aedes aegypti, at Narita International Airport, Japan in August 2012. Jpn J Infect Dis 2013;66:189–94. 10. Kobayashi M, Komagata O, Yonejima M, Maekawa Y, Hirabayashi K, Hayashi T, et al. Retrospective search for dengue vector mosquito Aedes albopictus in areas visited by a German traveler who contracted dengue in Japan. Int J Infect Dis 2014;26:135–7.
Meng Ling Moia Yasuko Honmab Satomi Morib Toshiki Kuzeb Masumi Kanekawab Takayoshi Isodab Naoko Moriwakib Takeshi Hosogaib Akira Kotakia Ichiro Kuranea Masayuki Saijoa Satoru Miyakeb Tomohiko Takasakia,* a National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640 Japan b Narita Airport Quarantine, Furugome, Chiba, Japan *Corresponding author. Received 14 November 2014
Received in revised form 26 January 2015 Accepted 3 February 2015
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid
Letter to the Editor Virological confirmation of concurrent dengue virus serotypes 1 and 4 by virus isolation using Fc-gamma receptor-expressing BHK cells
Keywords: Dengue Concurrent infection Virus isolation Neutralization Virological confirmation
Dengue virus (DENV) is estimated to infect approximately 400 million people annually. In 2014, 160 cases of autochthonous and 173 imported DENV infection cases were confirmed in Japan.1 Concurrent infection by two DENV serotypes is not an uncommon event in dengue hyperendemic areas where several DENV serotypes co-circulate.2 However, neither of the studies by previous investigators has clearly confirmed concurrent infection by virus isolation and separation.2,3 In the present study, we identified and confirmed a case of concurrent DENV-1 and DENV-4 infection in a patient by a method of virus isolation and separation for concurrent infection, using an Fc-gamma receptor (FcgR)expressing BHK cell line.4 The results present evidence of the presence of infectious virus particles of two virus serotypes in a patient.
A serum sample from a dengue patient who returned to Japan from a visit to Cambodia in 2013, was positive for DENV-1 (genotype 1; Genbank Accession number AB873104) and DENV-4 (genotype 1; Genbank Accession number AB873105) virus genome, and negative for anti-DENV IgM and IgG.5 Initial attempts to isolate and separate DENV using non FcgR-expressing Vero and BHK cells resulted in the isolation of DENV-1 only. To isolate DENV-4, the patient’s serum sample was treated with pooled human serum samples with neutralizing activity to DENV-1 but not to DENV-4 (DENV-1 PRNT50 1:10, DENV-4 PRNT50 < 10; as determined by FcgR-expressing BHK cells).6 DENV-1 and DENV-4 strains were isolated and separated, and confirmed by RT-PCR and sequencing of the E-protein gene7 (Table 1). Neutralizing antibodies to one DENV serotype may possess antibody-dependent enhancement (ADE) activity to heterologous serotypes in FcgR-positive cells, resulting in higher levels of progeny virus production.8 ADE activity also hampers serotype cross-reactive neutralizing antibody titers in FcgR-expressing cells but not in FcgR-negative cells.6 In the present study, both neutralization and the ADE mechanism were applied to virus isolation in a concurrent infection using FcgR-expressing cells. The serotype cross-reactive pooled serum samples used in this
Table 1 Virus isolation using FcgR-expressing cells and neutralizing antibodya Passage number
0 1 2
3
Sample number
Patient 1-1* 1-2 1-2-1 1-2-2 1-2-3 1-2-4 1-2-5 1-2-6 1-2-7 1-2-3-1 1-2-3-2 1-2-3-3* 1-2-3-4 1-2-3-5 1-2-3-6 1-2-3-7 1-2-3-8 1-2-3-9 1-2-3-10 1-2-3-11
Isolation method
None (patient serum sample) 10 ml serum sample + 10 ml 1:10 Nt Ab 10 ml serum sample + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml undiluted Nt Ab 1 ml supernatant + 10 ml undiluted Nt Ab 50 ml supernatant only 10 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 1 ml supernatant + 10 ml 1:10 Nt Ab 0.5 ml supernatant + 10 ml 1:10 Nt Ab 0.5 ml supernatant + 10 ml 1:10 Nt Ab 0.1 ml supernatant + 10 ml 1:10 Nt Ab 0.1 ml supernatant + 10 ml 1:10 Nt Ab 10 ml supernatant + 10 ml undiluted Nt Ab 10 ml supernatant + 10 ml undiluted Nt Ab 1 ml supernatant + 10 ml undiluted Nt Ab
Log10 genome copies/ml DENV-1
DENV-4
9.8 7.5 6.0 8.8 4.0 4.4 5.0 7.4 4.9 10.0 -
6.1 7.3 8.6 8.2 8.5 7.9 8.1 8.4 8.8 8.0 8.5 8.2 8.3 7.8 8.1 8.6 8.8 8.0
http://dx.doi.org/10.1016/j.ijid.2015.02.004 1201-9712/ß 2015 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Letter to the Editor / International Journal of Infectious Diseases 33 (2015) 177–178
178 Table 1 (Continued ) Passage number
Sample number
Isolation method
Log10 genome copies/ml
1-2-3-12 1-2-3-13 1-2-3-14 1-2-3-15 1-2-3-16 1-2-3-17
1 ml supernatant + 10 ml undiluted Nt Ab 0.5 ml supernatant + 10 ml undiluted Nt Ab 0.5 ml supernatant + 10 ml undiluted Nt Ab 0.1 ml supernatant + 10 ml undiluted Nt Ab 0.1 ml supernatant + 10 ml undiluted Nt Ab 10 ml supernatant only
DENV-1
DENV-4
-
8.5 7.9 8.5 7.8 7.9 8.7
FcgR, Fc-gamma receptor; DENV, dengue virus. a Use of non-FcgR-expressing Vero cells for virus isolation in the first passage resulted in the isolation of DENV-1 isolate only, as determined by conventional RT-PCR. In the presence of neutralizing antibodies, only DENV-1 was isolated (10 ml supernatant + 10 ml 1:10 Nt Ab, DENV-1 = 6.6 log10 genome copies/ml; 1 ml supernatant + 10 ml 1:10 Nt Ab, DENV-1 = 4.7 log10 genome copies/ml) using sample 1-2, by BHK cells. ‘Nt Ab’ denotes pooled sera with neutralizing activity to DENV-1 but not to DENV-4 using FcgRexpressing cells as assay cells; ‘-’ denotes ‘not detected’; underlined numbers indicate supernatant used for further passage (supernatant with a low DENV-1 titer and a high DENV-4 titer was chosen for further passage); ‘*’ indicates sample used for sequencing and comparison to the DENV-1 and DENV-4 sequences of serum sample. The E-protein sequences of the DENV-1 (sample number 1-1) and DENV-4 (sample number 1-2-3-3) isolates were 100% identical to those DENV-1 and DENV-4 sequences using serum samples.
study demonstrated high levels of neutralizing antibody to DENV-1 but no neutralizing activity to DENV-4 using FcgRexpressing cells as assay cells.6 Thus, serotype-specific antibodies specifically neutralized the dominant DENV-1 serotype and may have enhanced the growth of the subdominant DENV-4 serotype. In conclusion, this report provides concrete evidence of concurrent DENV infection by multiple DENV serotypes in a DENV patient, and presents evidence on the potential of viremic travelers to introduce more than a single virus serotype to the local mosquito vector population.9,10 Acknowledgements We thank Ms Makiko Ikeda for providing excellent technical assistance for the antibody assay. We also thank Dr Kaw Bing Chua (Temasek Life Sciences Laboratory, Singapore) because this work would not have been possible without the convalescent dengue serum samples he provided. We thank Dr Jeffrey V. Ravetch (Rockfeller University, NY, USA) for generously providing us with the FcgRIIA cDNA and Dr Susheela Tridandapani (Ohio State University College of Medicine, Columbus, OH, USA) for assistance in obtaining the FcgRIIA cDNA. This work was supported in part by the research grant, Research on Emerging and Re-emerging Infectious Diseases (H23-shinkouippan-010; H26-shinkou-jitsuyouka-007) from the Ministry of Health, Labour and Welfare, Japan, the Environment Research and Technology Development Fund (S-8) of the Ministry of the Environment, and a Grant-in-Aid for Young Scientists (B) from JSPS (26870872). Ethical approval: The patient serum sample was de-identified prior to conducting the study. The assay protocol was approved by the institutional Medical Research Ethics Committee of the National Institute of Infectious Diseases, Japan (reference number 210). Conflict of interest: The authors declare that they have no competing interests. References 1. Kutsuna S, Kato Y, Moi ML, Kotaki A, Ota M, Shinohara K, et al. Autochthonous dengue fever, Tokyo, Japan, 2014. Emerg Infect Dis 2015 Jan 22. [Epub ahead of print]. http://dx.doi.org/10.3201/eid2103.141662. 2. Gubler DJ, Kuno G, Sather GE, Waterman SH. A case of natural concurrent human infection with two dengue viruses. Am J Trop Med Hyg 1985;34:170–3.
˜ o-Pino MA, Cropp CB, Farfa´n JA, Vorndam AV, Rodrı´guez-Angulo EM, 3. Loron Rosado-Paredes EP, et al. Common occurrence of concurrent infections by multiple dengue virus serotypes. Am J Trop Med Hyg 1999;61:725–30. 4. Moi ML, Lim CK, Kotaki A, Takasaki T, Kurane I. Detection of higher levels of dengue viremia using FcgR-expressing BHK-21 cells than FcgR-negative cells in secondary infection but not in primary infection. J Infect Dis 2011;203:1405–14. 5. Honma Y. Chikungunya and dengue—Japan: ex Cambodia (Kampong Cham). Archive number 20130829.1909917. ProMED-mail; 2013. 6. Moi ML, Lim CK, Chua KB, Takasaki T, Kurane I. Dengue virus infectionenhancing activity in serum samples with neutralizing activity as determined by using FcgR-expressing cells. PLoS Negl Trop Dis 2012;6:e1536. 7. Ito M, Yamada K, Takasaki T, Pandey B, Nerome R, Tajima S, et al. Phylogenetic analysis of dengue viruses isolated from imported dengue patients: possible aid for determining the countries where infections occurred. J Travel Med 2007;14:233–44. 8. Mady BJ, Erbe DV, Kurane I, Fanger MW, Ennis FA. Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fc gamma receptors. J Immunol 1991;147:3139–44. 9. Sukehiro N, Kida N, Umezawa M, Murakami T, Arai N, Jinnai T, et al. First report on invasion of yellow fever mosquito, Aedes aegypti, at Narita International Airport, Japan in August 2012. Jpn J Infect Dis 2013;66:189–94. 10. Kobayashi M, Komagata O, Yonejima M, Maekawa Y, Hirabayashi K, Hayashi T, et al. Retrospective search for dengue vector mosquito Aedes albopictus in areas visited by a German traveler who contracted dengue in Japan. Int J Infect Dis 2014;26:135–7.
Meng Ling Moia Yasuko Honmab Satomi Morib Toshiki Kuzeb Masumi Kanekawab Takayoshi Isodab Naoko Moriwakib Takeshi Hosogaib Akira Kotakia Ichiro Kuranea Masayuki Saijoa Satoru Miyakeb Tomohiko Takasakia,* a National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640 Japan b Narita Airport Quarantine, Furugome, Chiba, Japan *Corresponding author. Received 14 November 2014
Received in revised form 26 January 2015 Accepted 3 February 2015
