- Email: [email protected]
First genome characterization of a novel hepatitis C virus genotype 5 variant
Infection, Genetics and Evolution 39 (2016) 173–175
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
First genome characterization of a novel hepatitis C virus genotype 5 variant Cécile Henquell a,b,⁎, Saydou Yameogo c, Lassana Sangaré c a b c
CHU de Clermont-Ferrand, Laboratoire de Virologie, Centre National de Référence des Entérovirus et Parechovirus – Laboratoire Associé, 58 rue Montalembert, F-63003 Clermont-Ferrand, France Université d'Auvergne, EA-4843, Faculté de Médecine, 28 place Henri Dunant, F-63000 Clermont-Ferrand, France CHU Yalgado, avenue du Capitaine Thomas Sankara, 03 BP7022 Ouagadougou, Burkina Faso
a r t i c l e
i n f o
Article history: Received 28 October 2015 Received in revised form 19 January 2016 Accepted 21 January 2016 Available online 22 January 2016
a b s t r a c t We report a new hepatitis C virus (HCV) genotype 5 variant from a woman living in Burkina Faso. Phylogenetic analysis of the near full-length genome sequence suggests that this isolate HCV5_BF16 could be the first reported strain belonging to a new HCV 5 subtype, distinct from the 5a subtype. © 2016 Elsevier B.V. All rights reserved.
Keywords: HCV Genotype 5 New subtype Near full-length sequence
Hepatitis C virus (HCV) strains exhibit a noteworthy genetic diversity and are classified into 7 distinct genotypes and a further 67 subtypes on the basis of phylogenetic analyses of the whole viral genome sequences (Smith et al., 2014). Over the last few years, owing to advances in sequencing technologies and the investigation of viral diversity, numerous new subtypes have been reported in low- and middle-income countries. HCV genotypes and subtypes vary in their genetic diversity level and their geographical distribution according to transmission routes and human migration. A few epidemic subtypes, notably 1a, 1b and 3a, have a global distribution and account for most infections in high-income countries through infected blood, blood products and injecting drug use. Many other subtypes are considered as endemic strains circulating for long periods of time over large geographical areas in sub-Saharan Africa (genotypes 1, 2 and 4) and Asia (genotypes 3 and 6), leading to highly divergent strains (Messina et al., 2015). As a remarkable exception, genotype 5 comprises a single known subtype 5a, which is responsible for b1% of infections worldwide. Almost all HCV5 infections occur in South Africa, where they account for up to 58% of cases, and in some Eastern African countries (Messina et al., 2015). Small epidemics within restricted geographical areas have been reported elsewhere in regions with very low HCV5 prevalence such as Europe (Jover et al., 2001; Henquell et al., 2004; Verbeeck et al., 2010) and Syria (Antaki et al., 2009), mainly due to blood transfusion and unsafe medical practices (Verbeeck et al., 2010; Henquell et al., 2011).
⁎ Corresponding author at: CHU de Clermont-Ferrand, Laboratoire de Virologie, Centre National de Référence des Entérovirus et Parechovirus – Laboratoire Associé, 58 rue Montalembert, F-63003 Clermont-Ferrand, France. E-mail address: [email protected] (C. Henquell).
http://dx.doi.org/10.1016/j.meegid.2016.01.016 1567-1348/© 2016 Elsevier B.V. All rights reserved.
The isolate (lab code BF16) characterized in this study was amplified from a serum sampled in 2009 for blood testing from a woman living in Burkina Faso. No further epidemiological data are known for this patient. The HCV antibody test (AxSYM® HCV version 3.0, Abbott) was positive and confirmed by the INNO-LIA™ HCV Score test (Innogenetics) in Burkina Faso. Genotyping and further sequencing and molecular analyses were performed in the virology laboratory at the teaching hospital of Clermont-Ferrand, France. The near full-length sequence (nucleotide positions 96-9183, according to the H77 reference strain, accession number NC_004102), was determined by RT-PCR and direct sequencing from overlapping PCR fragments. First, three fragments (5′UTR, E1 and NS5B) were amplified and sequenced. Strain specific primers were then designed and used to obtain the genome sequence (9100 nt) by sequence walking (Supplementary Table 1). The newly characterized sequence has been deposited in GenBank under accession number KT595242. The near-complete coding region of the BF16 sequence (nucleotide position 342-9183, corresponding to 98% of the H77 sequence coding region) was compared with the complete coding region of 115 reference sequences representative of all HCV genotypes and subtypes known to date (Smith et al., 2014). The dataset was completed with five recently published HCV 5a African sequences (Gededzha et al., 2014). Maximum likelihood tree showed that HCV5_BF16 forms a separate cluster within genotype 5, distinct from all other existing subtype 5a strains (Fig. 1, Supplementary Fig. 1 for all bootstrap values). The same phylogenetic analysis performed separately on each HCV gene confirmed this clustering, supported by 100% of bootstrap replications (data not shown). On the basis of the current classification (Smith et al., 2014), confirmed subtypes within the major genotypes differ from other sequences by at least 15% of nucleotide positions over the coding region. Genetic distances were estimated between the BF16 isolate and the seven published HCV
174
C. Henquell et al. / Infection, Genetics and Evolution 39 (2016) 173–175
Fig. 1. Maximum likelihood phylogeny estimated from HCV coding region (ORF). Phylogenetic analysis was obtained with PhyML (http://www.atgc-montpellier.fr/phyml/), using the Maximum Likelihood method based on the General Time Reversible (GTR) model with gamma distribution (+G) and invariable sites (+I) (Guindon et al., 2010). Tree was reconstructed with MEGA5. The sequences analyzed corresponded to the almost full-length coding region (8959 nt length, corresponding to the nt positions 342–9183, according to the H77 reference strain). The HCV_BF16 isolate described in this study is underlined. The other sequences were selected from the HCV reference sequences available on the ICTV website (http://talk. ictvonline.org/links/hcv/hcv-classification.htm) (n = 115) and recently published near-full coding region sequences of HCV 5a isolates (Gededzha et al., 2014) (n = 5). Support value of nodes was estimated by bootstrap (total 1000 replicates). Asterisks indicate 100% value. The scale bar represents 0.2 nucleotide substitutions per site.
5a strains by MEGA5 (Table 1). BF16 coding region sequence showed 23% nucleotide distance from the 5a sequences and 16% at the amino acid level. As recombination between different genotypes and subtypes has been described, in particular a 2/5 recombinant in an African patient (Legrand-Abravanel et al., 2007), potential recombination events across the BF16 genome were assessed using algorithms implemented in RDP4 software (Martin et al., 2015) and, finally, ruled out.
The isolate (lab code BF16) characterized in this study was amplified from a serum sampled in 2009 for blood testing from a woman living in Burkina Faso. No further epidemiological data are known for this patient. The HCV antibody test (AxSYM® HCV version 3.0, Abbott) was positive and confirmed by the INNO-LIA™ HCV Score test (Innogenetics) in Burkina Faso. Genotyping and further sequencing and molecular analyses were performed in the virology laboratory at the teaching hospital of
C. Henquell et al. / Infection, Genetics and Evolution 39 (2016) 173–175 Table 1 Mean genetic distances between the HCV5_BF16 sequence and the other HCV5 published sequences (coding region). Region
Core E1 E2 P7 NS2 NS3 NS4A NS4B NS5A NS5Ba ORF
Length (nt)
573 576 1092 189 651 1893 162 783 1353 1582 8854
Nucleotide
Amino acid
Mean pairwise distance (%) versus
Mean pairwise distance (%) versus
EUH1480
All 5a
EUH1480
All 5a
16.1 24.3 29.5 32.3 28.2 23.0 24.8 24.5 24.7 19.9 24.0
14.6 25.4 27.3 27.5 28.1 21.9 24.3 23.4 24.1 19.6 23.1
13.6 20.3 18.7 33.3 28.1 10.6 16.7 12.3 20.4 13.3 16.5
9.3 19.4 16.1 24.0 25.6 9.1 15.4 11.7 18.9 13.2 16.0
Pairwise distances were calculated with MEGA 5.1 (Tamura et al., 2011). Comparative HCV5a sequences are the two full-length sequences EUH1480 (accession number Y13184) and SA13 (AF064490), and the five near full-length sequences ZADGM2088 (KC767829), ZADGM2582 (KC767830), ZADGM869 (KC767831), ZADGM0518 (KC767832) and ZADGM3013 (KC767834). a Partial sequence.
Clermont-Ferrand, France. The near full-length sequence (nucleotide positions 96-9183, according to the H77 reference strain, accession number NC_004102), was determined by RT-PCR and direct sequencing from overlapping PCR fragments. First, three fragments (5′UTR, E1 and NS5B) were amplified and sequenced. Strain specific primers were then designed and used to obtain the genome sequence (9100 nt) by sequence walking (Supplementary Table 1). The newly characterized sequence has been deposited in GenBank under accession number KT595242. The near-complete coding region of the BF16 sequence (nucleotide position 342-9183, corresponding to 98% of the H77 sequence coding region) was compared with the complete coding region of 115 reference sequences representative of all HCV genotypes and subtypes known to date (Smith et al., 2014). The dataset was completed with five recently published HCV 5a African sequences (Gededzha et al., 2014). Maximum likelihood tree showed that HCV5_BF16 forms a separate cluster within genotype 5, distinct from all other existing subtype 5a strains (Fig. 1, Supplementary Fig. 1 for all bootstrap values). The same phylogenetic analysis performed separately on each HCV gene confirmed this clustering, supported by 100% of bootstrap replications (data not shown). On the basis of the current classification (Smith et al., 2014), confirmed subtypes within the major genotypes differ from other sequences by at least 15% of nucleotide positions over the coding region. Genetic distances were estimated between the BF16 isolate and the seven published HCV 5a strains by MEGA5 (Table 1). BF16 coding region sequence showed 23% nucleotide distance from the 5a sequences and 16% at the amino acid level. As recombination between different genotypes and subtypes has been described, in particular a 2/5 recombinant in an African patient (LegrandAbravanel et al., 2007), potential recombination events across the BF16 genome were assessed using algorithms implemented in RDP4 software (Martin et al., 2015) and, finally, ruled out. Taken together, these results suggest that the HCV5_BF16 isolate belongs to a new subtype within genotype 5. According to the recently updated international consensus criteria, a new subtype assignment is only made when sequence data are provided for three or more nonlinked isolates including at least one complete or nearly complete coding region (Smith et al., 2014). This single sequence has been named
175
HCV5_KT595242 but cannot yet be formally assigned as a new subtype. Further investigations in African countries are needed to complete our knowledge of this new variant of the rarest HCV genotype. In contrast to the other major genotypes, for which origins and spread histories have been reconstructed from molecular evolutionary analyses (Simmonds, 2013), numerous uncertainties remain for HCV5, especially about its original geographical region and its genetic diversification. These data contribute to a better understanding of viral diversity, which is crucial in the development of new pan-genotype antiviral active drugs and the search for a vaccine. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.01.016. Acknowledgments We thank Dr. Kouanda Abdoulaye (Blood bank, CHU Yalgado Ouédraogo) and the staff of the National Center of Blood Transfusion in Ouagadougou, Burkina Faso, for the sample collection. We thank Nathalie Rodde for her technical assistance and Jeffrey Watts for help in preparation of the English text. References Antaki, N., Haddad, M., Kebbewar, K., Abdelwahab, J., Hamed, O., Aaraj, R., Alhaj, N., Haffar, S., Assil, M., Ftayeh, M., Assaad, F., Doghman, D., Ali, T., Nasserelddine, M., Ali, A., Antaki, F., 2009. The unexpected discovery of a focus of hepatitis C virus genotype 5 in a Syrian province. Syrian working group for the study of viral hepatitis. Epidemiol. Infect. 137, 79–84. Gededzha, M.P., Selabe, S.G., Blackard, J.T., Kyaw, T., Mphahlele, M.J., 2014. Near fulllength genome analysis of HCV genotype 5 from South Africa. Infect. Genet. Evol. 21, 118–123. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. Henquell, C., Cartau, C., Abergel, A., Laurichesse, H., Regagnon, C., De Champs, C., Bailly, J.L., Peigue-Lafeuille, H., 2004. High prevalence of hepatitis C virus type 5 in Centre of France evidenced by a prospective study from 1996 to 2002. J. Clin. Microbiol. 42, 3030–3035. Henquell, C., Guglielmini, J., Verbeeck, J., Mahul, A., Thibault, V., Lebray, P., Laperche, S., Trimoulet, P., Foucher, J., Le Guillou-Guillemette, H., Fouchard-Hubert, I., LegrandAbravanel, F., Metivier, S., Gaudy, C., D'Alteroche, L., Rosenberg, A.R., Podevin, P., Plantier, J.C., Riachi, G., Saoudin, H., Coppere, H., André, E., Gournay, J., Feray, C., Vallet, S., Nousbaum, J.B., Baazia, Y., Roulot, D., Alain, S., Loustaud-Ratti, V., Schvoerer, E., Habersetzer, F., Perez-Serra, R.J., Gourari, S., Mirand, A., Odent-Malaure, H., Garraud, O., Izopet, J., Bommelaer, G., Peigue-Lafeuille, H., van Ranst, M., Abergel, A., Bailly, J.L., 2011. Evolutionary history of hepatitis C virus genotype 5a in France, a multicenter ANRS study. Infect. Genet. Evol. 11, 496–503. Jover, R., Pérez-Serra, J., de Vera, F., Alamo, J.M., Muñoz, C., Yago, C., Martínez-Ramírez, R., Vidal, J.V., 2001. Infection by genotype 5a of HCV in a district of Southeast Spain. Am. J. Gastroenterol. 96, 3042–3043. Legrand-Abravanel, F., Claudinon, J., Nicot, F., Dubois, M., Chaput-Regaud, S., SandresSauné, K., Pasquier, C., Izopet, J., 2007. New natural intergenotypic (2/5) recombinant of hepatitis C virus. J. Virol. 81, 4357–4362. Martin, D.P., Murrell, B., Golden, M., Khoosal, A., Muhire, B., 2015. RDP4: detection and analysis of recombination patterns in virus genomes. Virus Evol. 1, 1–5. Messina, J.P., Humphreys, I., Flaxman, A., Brown, A., Cooke, G.S., Pybus, O.G., Barnes, E., 2015. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 61, 77–87. Simmonds, P., 2013. The origin of hepatitis C virus. Curr. Top. Microbiol. Immunol. 369, 1–15. Smith, D.B., Bukh, J., Kuiken, C., Muerhoff, A.S., Rice, C.M., Stapleton, J.T., Simmonds, P., 2014. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 59, 318–327. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Verbeeck, J., Kwanten, L., D'Heygere, F., Beguin, A., Michiels, S., Desombere, I., LerouxRoels, G., Lemey, P., Nevens, F., van Ranst, M., 2010. HCV genotype distribution in Flanders and Brussels (Belgium): unravelling the spread of an uncommon HCV genotype 5a cluster. Eur. J. Clin. Microbiol. Infect. Dis. 29, 1427–1434.
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
First genome characterization of a novel hepatitis C virus genotype 5 variant Cécile Henquell a,b,⁎, Saydou Yameogo c, Lassana Sangaré c a b c
CHU de Clermont-Ferrand, Laboratoire de Virologie, Centre National de Référence des Entérovirus et Parechovirus – Laboratoire Associé, 58 rue Montalembert, F-63003 Clermont-Ferrand, France Université d'Auvergne, EA-4843, Faculté de Médecine, 28 place Henri Dunant, F-63000 Clermont-Ferrand, France CHU Yalgado, avenue du Capitaine Thomas Sankara, 03 BP7022 Ouagadougou, Burkina Faso
a r t i c l e
i n f o
Article history: Received 28 October 2015 Received in revised form 19 January 2016 Accepted 21 January 2016 Available online 22 January 2016
a b s t r a c t We report a new hepatitis C virus (HCV) genotype 5 variant from a woman living in Burkina Faso. Phylogenetic analysis of the near full-length genome sequence suggests that this isolate HCV5_BF16 could be the first reported strain belonging to a new HCV 5 subtype, distinct from the 5a subtype. © 2016 Elsevier B.V. All rights reserved.
Keywords: HCV Genotype 5 New subtype Near full-length sequence
Hepatitis C virus (HCV) strains exhibit a noteworthy genetic diversity and are classified into 7 distinct genotypes and a further 67 subtypes on the basis of phylogenetic analyses of the whole viral genome sequences (Smith et al., 2014). Over the last few years, owing to advances in sequencing technologies and the investigation of viral diversity, numerous new subtypes have been reported in low- and middle-income countries. HCV genotypes and subtypes vary in their genetic diversity level and their geographical distribution according to transmission routes and human migration. A few epidemic subtypes, notably 1a, 1b and 3a, have a global distribution and account for most infections in high-income countries through infected blood, blood products and injecting drug use. Many other subtypes are considered as endemic strains circulating for long periods of time over large geographical areas in sub-Saharan Africa (genotypes 1, 2 and 4) and Asia (genotypes 3 and 6), leading to highly divergent strains (Messina et al., 2015). As a remarkable exception, genotype 5 comprises a single known subtype 5a, which is responsible for b1% of infections worldwide. Almost all HCV5 infections occur in South Africa, where they account for up to 58% of cases, and in some Eastern African countries (Messina et al., 2015). Small epidemics within restricted geographical areas have been reported elsewhere in regions with very low HCV5 prevalence such as Europe (Jover et al., 2001; Henquell et al., 2004; Verbeeck et al., 2010) and Syria (Antaki et al., 2009), mainly due to blood transfusion and unsafe medical practices (Verbeeck et al., 2010; Henquell et al., 2011).
⁎ Corresponding author at: CHU de Clermont-Ferrand, Laboratoire de Virologie, Centre National de Référence des Entérovirus et Parechovirus – Laboratoire Associé, 58 rue Montalembert, F-63003 Clermont-Ferrand, France. E-mail address: [email protected] (C. Henquell).
http://dx.doi.org/10.1016/j.meegid.2016.01.016 1567-1348/© 2016 Elsevier B.V. All rights reserved.
The isolate (lab code BF16) characterized in this study was amplified from a serum sampled in 2009 for blood testing from a woman living in Burkina Faso. No further epidemiological data are known for this patient. The HCV antibody test (AxSYM® HCV version 3.0, Abbott) was positive and confirmed by the INNO-LIA™ HCV Score test (Innogenetics) in Burkina Faso. Genotyping and further sequencing and molecular analyses were performed in the virology laboratory at the teaching hospital of Clermont-Ferrand, France. The near full-length sequence (nucleotide positions 96-9183, according to the H77 reference strain, accession number NC_004102), was determined by RT-PCR and direct sequencing from overlapping PCR fragments. First, three fragments (5′UTR, E1 and NS5B) were amplified and sequenced. Strain specific primers were then designed and used to obtain the genome sequence (9100 nt) by sequence walking (Supplementary Table 1). The newly characterized sequence has been deposited in GenBank under accession number KT595242. The near-complete coding region of the BF16 sequence (nucleotide position 342-9183, corresponding to 98% of the H77 sequence coding region) was compared with the complete coding region of 115 reference sequences representative of all HCV genotypes and subtypes known to date (Smith et al., 2014). The dataset was completed with five recently published HCV 5a African sequences (Gededzha et al., 2014). Maximum likelihood tree showed that HCV5_BF16 forms a separate cluster within genotype 5, distinct from all other existing subtype 5a strains (Fig. 1, Supplementary Fig. 1 for all bootstrap values). The same phylogenetic analysis performed separately on each HCV gene confirmed this clustering, supported by 100% of bootstrap replications (data not shown). On the basis of the current classification (Smith et al., 2014), confirmed subtypes within the major genotypes differ from other sequences by at least 15% of nucleotide positions over the coding region. Genetic distances were estimated between the BF16 isolate and the seven published HCV
174
C. Henquell et al. / Infection, Genetics and Evolution 39 (2016) 173–175
Fig. 1. Maximum likelihood phylogeny estimated from HCV coding region (ORF). Phylogenetic analysis was obtained with PhyML (http://www.atgc-montpellier.fr/phyml/), using the Maximum Likelihood method based on the General Time Reversible (GTR) model with gamma distribution (+G) and invariable sites (+I) (Guindon et al., 2010). Tree was reconstructed with MEGA5. The sequences analyzed corresponded to the almost full-length coding region (8959 nt length, corresponding to the nt positions 342–9183, according to the H77 reference strain). The HCV_BF16 isolate described in this study is underlined. The other sequences were selected from the HCV reference sequences available on the ICTV website (http://talk. ictvonline.org/links/hcv/hcv-classification.htm) (n = 115) and recently published near-full coding region sequences of HCV 5a isolates (Gededzha et al., 2014) (n = 5). Support value of nodes was estimated by bootstrap (total 1000 replicates). Asterisks indicate 100% value. The scale bar represents 0.2 nucleotide substitutions per site.
5a strains by MEGA5 (Table 1). BF16 coding region sequence showed 23% nucleotide distance from the 5a sequences and 16% at the amino acid level. As recombination between different genotypes and subtypes has been described, in particular a 2/5 recombinant in an African patient (Legrand-Abravanel et al., 2007), potential recombination events across the BF16 genome were assessed using algorithms implemented in RDP4 software (Martin et al., 2015) and, finally, ruled out.
The isolate (lab code BF16) characterized in this study was amplified from a serum sampled in 2009 for blood testing from a woman living in Burkina Faso. No further epidemiological data are known for this patient. The HCV antibody test (AxSYM® HCV version 3.0, Abbott) was positive and confirmed by the INNO-LIA™ HCV Score test (Innogenetics) in Burkina Faso. Genotyping and further sequencing and molecular analyses were performed in the virology laboratory at the teaching hospital of
C. Henquell et al. / Infection, Genetics and Evolution 39 (2016) 173–175 Table 1 Mean genetic distances between the HCV5_BF16 sequence and the other HCV5 published sequences (coding region). Region
Core E1 E2 P7 NS2 NS3 NS4A NS4B NS5A NS5Ba ORF
Length (nt)
573 576 1092 189 651 1893 162 783 1353 1582 8854
Nucleotide
Amino acid
Mean pairwise distance (%) versus
Mean pairwise distance (%) versus
EUH1480
All 5a
EUH1480
All 5a
16.1 24.3 29.5 32.3 28.2 23.0 24.8 24.5 24.7 19.9 24.0
14.6 25.4 27.3 27.5 28.1 21.9 24.3 23.4 24.1 19.6 23.1
13.6 20.3 18.7 33.3 28.1 10.6 16.7 12.3 20.4 13.3 16.5
9.3 19.4 16.1 24.0 25.6 9.1 15.4 11.7 18.9 13.2 16.0
Pairwise distances were calculated with MEGA 5.1 (Tamura et al., 2011). Comparative HCV5a sequences are the two full-length sequences EUH1480 (accession number Y13184) and SA13 (AF064490), and the five near full-length sequences ZADGM2088 (KC767829), ZADGM2582 (KC767830), ZADGM869 (KC767831), ZADGM0518 (KC767832) and ZADGM3013 (KC767834). a Partial sequence.
Clermont-Ferrand, France. The near full-length sequence (nucleotide positions 96-9183, according to the H77 reference strain, accession number NC_004102), was determined by RT-PCR and direct sequencing from overlapping PCR fragments. First, three fragments (5′UTR, E1 and NS5B) were amplified and sequenced. Strain specific primers were then designed and used to obtain the genome sequence (9100 nt) by sequence walking (Supplementary Table 1). The newly characterized sequence has been deposited in GenBank under accession number KT595242. The near-complete coding region of the BF16 sequence (nucleotide position 342-9183, corresponding to 98% of the H77 sequence coding region) was compared with the complete coding region of 115 reference sequences representative of all HCV genotypes and subtypes known to date (Smith et al., 2014). The dataset was completed with five recently published HCV 5a African sequences (Gededzha et al., 2014). Maximum likelihood tree showed that HCV5_BF16 forms a separate cluster within genotype 5, distinct from all other existing subtype 5a strains (Fig. 1, Supplementary Fig. 1 for all bootstrap values). The same phylogenetic analysis performed separately on each HCV gene confirmed this clustering, supported by 100% of bootstrap replications (data not shown). On the basis of the current classification (Smith et al., 2014), confirmed subtypes within the major genotypes differ from other sequences by at least 15% of nucleotide positions over the coding region. Genetic distances were estimated between the BF16 isolate and the seven published HCV 5a strains by MEGA5 (Table 1). BF16 coding region sequence showed 23% nucleotide distance from the 5a sequences and 16% at the amino acid level. As recombination between different genotypes and subtypes has been described, in particular a 2/5 recombinant in an African patient (LegrandAbravanel et al., 2007), potential recombination events across the BF16 genome were assessed using algorithms implemented in RDP4 software (Martin et al., 2015) and, finally, ruled out. Taken together, these results suggest that the HCV5_BF16 isolate belongs to a new subtype within genotype 5. According to the recently updated international consensus criteria, a new subtype assignment is only made when sequence data are provided for three or more nonlinked isolates including at least one complete or nearly complete coding region (Smith et al., 2014). This single sequence has been named
175
HCV5_KT595242 but cannot yet be formally assigned as a new subtype. Further investigations in African countries are needed to complete our knowledge of this new variant of the rarest HCV genotype. In contrast to the other major genotypes, for which origins and spread histories have been reconstructed from molecular evolutionary analyses (Simmonds, 2013), numerous uncertainties remain for HCV5, especially about its original geographical region and its genetic diversification. These data contribute to a better understanding of viral diversity, which is crucial in the development of new pan-genotype antiviral active drugs and the search for a vaccine. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.01.016. Acknowledgments We thank Dr. Kouanda Abdoulaye (Blood bank, CHU Yalgado Ouédraogo) and the staff of the National Center of Blood Transfusion in Ouagadougou, Burkina Faso, for the sample collection. We thank Nathalie Rodde for her technical assistance and Jeffrey Watts for help in preparation of the English text. References Antaki, N., Haddad, M., Kebbewar, K., Abdelwahab, J., Hamed, O., Aaraj, R., Alhaj, N., Haffar, S., Assil, M., Ftayeh, M., Assaad, F., Doghman, D., Ali, T., Nasserelddine, M., Ali, A., Antaki, F., 2009. The unexpected discovery of a focus of hepatitis C virus genotype 5 in a Syrian province. Syrian working group for the study of viral hepatitis. Epidemiol. Infect. 137, 79–84. Gededzha, M.P., Selabe, S.G., Blackard, J.T., Kyaw, T., Mphahlele, M.J., 2014. Near fulllength genome analysis of HCV genotype 5 from South Africa. Infect. Genet. Evol. 21, 118–123. Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. Henquell, C., Cartau, C., Abergel, A., Laurichesse, H., Regagnon, C., De Champs, C., Bailly, J.L., Peigue-Lafeuille, H., 2004. High prevalence of hepatitis C virus type 5 in Centre of France evidenced by a prospective study from 1996 to 2002. J. Clin. Microbiol. 42, 3030–3035. Henquell, C., Guglielmini, J., Verbeeck, J., Mahul, A., Thibault, V., Lebray, P., Laperche, S., Trimoulet, P., Foucher, J., Le Guillou-Guillemette, H., Fouchard-Hubert, I., LegrandAbravanel, F., Metivier, S., Gaudy, C., D'Alteroche, L., Rosenberg, A.R., Podevin, P., Plantier, J.C., Riachi, G., Saoudin, H., Coppere, H., André, E., Gournay, J., Feray, C., Vallet, S., Nousbaum, J.B., Baazia, Y., Roulot, D., Alain, S., Loustaud-Ratti, V., Schvoerer, E., Habersetzer, F., Perez-Serra, R.J., Gourari, S., Mirand, A., Odent-Malaure, H., Garraud, O., Izopet, J., Bommelaer, G., Peigue-Lafeuille, H., van Ranst, M., Abergel, A., Bailly, J.L., 2011. Evolutionary history of hepatitis C virus genotype 5a in France, a multicenter ANRS study. Infect. Genet. Evol. 11, 496–503. Jover, R., Pérez-Serra, J., de Vera, F., Alamo, J.M., Muñoz, C., Yago, C., Martínez-Ramírez, R., Vidal, J.V., 2001. Infection by genotype 5a of HCV in a district of Southeast Spain. Am. J. Gastroenterol. 96, 3042–3043. Legrand-Abravanel, F., Claudinon, J., Nicot, F., Dubois, M., Chaput-Regaud, S., SandresSauné, K., Pasquier, C., Izopet, J., 2007. New natural intergenotypic (2/5) recombinant of hepatitis C virus. J. Virol. 81, 4357–4362. Martin, D.P., Murrell, B., Golden, M., Khoosal, A., Muhire, B., 2015. RDP4: detection and analysis of recombination patterns in virus genomes. Virus Evol. 1, 1–5. Messina, J.P., Humphreys, I., Flaxman, A., Brown, A., Cooke, G.S., Pybus, O.G., Barnes, E., 2015. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 61, 77–87. Simmonds, P., 2013. The origin of hepatitis C virus. Curr. Top. Microbiol. Immunol. 369, 1–15. Smith, D.B., Bukh, J., Kuiken, C., Muerhoff, A.S., Rice, C.M., Stapleton, J.T., Simmonds, P., 2014. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 59, 318–327. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Verbeeck, J., Kwanten, L., D'Heygere, F., Beguin, A., Michiels, S., Desombere, I., LerouxRoels, G., Lemey, P., Nevens, F., van Ranst, M., 2010. HCV genotype distribution in Flanders and Brussels (Belgium): unravelling the spread of an uncommon HCV genotype 5a cluster. Eur. J. Clin. Microbiol. Infect. Dis. 29, 1427–1434.