Hepatitis B virus (HBV), which belongs to the family of
The high degree of genomic heterogeneity categorizes HBV into 10 genotypes (A-J), and an intergroup difference of around 7.5% is observed. All genotypes, except E and G, are classified further into 25 different subgenotypes, with a difference of around 4% being observed (Kramvis et al. 2005; Kostaki et al. 2018). HBV-A and HBV-D are present around the globe, whereas HBV-A is mainly seen in Europe and Africa, and HBV-D in the Middle East and Europe. HBV-B and HBV-C are generally found in Oceania and Eastern Asia, HBV-E in both Central and Western Africa. HBV-F and HBV-H are found in Alaska and Latin America only. HBV-D is considered to be pandemic. HBV-D1 is dominant in Australia, Europe, Indonesia, North Africa, and Western Asia, whereas HBV-D2 is seen in Albania, Japan, Malaysia, North-Eastern Europe, Russia, and United Kingdom (Tallo et al. 2008; Bissinger et al. 2015; Kostaki et al. 2018). A recent study in Brazil showed that due to Italian colonization, the dominance of genotype D/D3 is observed (Paoli et al. 2018). A similar study was performed before in the same region, and once again, genotype D/D3 was found to be the most relevant genotype (Chacha et al. 2017).
The prevalence of HBV can be classified into three regions; low (< 2%), middle (2–7%), and high (> 8%) endemities. Turkey is categorized as middle endemicity with a prevalence rate of 0.8–5.7%, while the Turkish Republic of Northern Cyprus (TRNC) falls into a low category with a rate of 1.2% (Arikan et al. 2016; Ozguler and Sayan 2018). However, Cyprus is an island located in the Eastern Mediterranean, to the south of Turkey. Since 1974, there have been two communities living separately on the island: Turkish and Greek Cypriots. The exact population of North Cyprus is not known as the population number is dynamic, this is due to sex worker and immigrant trafficking that occurs along with constant international student and tourist travel. However, there is no data regarding HBV dynamics for South Cyprus. The South of the island is subject to more immigrations and human trafficking (Kaptanoglu et al. 2013; U.S. Department of State Publication 2018).
Cyprus had an estimated population of 1 193 635 in 2011. Around 352 000 were believed to live in North Cyprus, but the number has climbed up to half a million. Half of these are Cypriot-born children or Turkish settlers. Around 230 000 of those are classified as native-born TRNC citizens. The exact population remains unknown as North Cyprus has a dynamic society of students and tourists who regularly visit the island (Christou 2018; World Population Review 2018). Immunisation against HBV in the TRNC was rare in the late 80s, and the program was first introduced in the country in July 1998 (Kurugol et al. 2009). Between 2014 and 2018, 3149 HBsAg positive Turkish citizens were living in the TRNC, where 98.16% were of Turkish origin and 1.84% was Turkish Cypriots (KKTC Sağlık Bakanlığı 2019). Previous studies have concluded that the overall HBsAg positivity rate for the TRNC is 1.2% (Arikan et al. 2016).
There is a high demand for genotype information and investigation regarding HBV infected individuals. This importance rules to be informed regarding molecular and epidemiological specifications. As with the aim of this importance, we have aimed to analyze the dispersion of genotype/subgenotype/serotype together with
Demographic characteristics of the patients.
Characteristics | Patient group | Study group |
---|---|---|
Patients, n | 170 | 108 |
Gender, M/F, n (%) | 106 (63) / 64 (37) | 68 (63) / 40 (37) |
Age, years (mean ± SD) | 49 ± 31 | 41.5 ± 23.5 |
Nationality Turkish | 122 (71) | 83 (77) |
Turkish Cypriot | 48 (29) | 25 (23) |
HBsAg value, S/Co* (mean ± SD) | 3882.5 ± 3712.5 | 3882.5 ± 3712.5 |
Abbreviations: M – male; F – female; *S/Co: Sample/Cut-off. HBsAg value was obtained using Abbott Architect i1000SR/i2000SR systems (Abbott, USA).
Sequences obtained were subsequently analyzed using a special online tool, the Geno2pheno (Centre of Advanced European Studies and Research, Bonn, Germany) drug resistance platform. The following target region and amino acid positions were analyzed for the determination of antiviral drug-associated potential vaccine-escape mutations (ADAPVEM) regions 161, 164, 172, 173, 175, 176, 182, and 193–196; HBIg selected escape mutation regions 118, 120, 123, 124, 129, 133, 134, 144, and 145; vaccine escape mutation regions 120, 126, 133, 143–145, and 193; Hepatits B misdiagnosis mutation regions 120, 131, 133, and 143; immune-selected mutation regions 100, 101, 105, 109, 110, 114, 117, 119, 120, 123, 127, 128, 130–134, 140, and 143– 145. The target region and amino acid position for the determination of HBV
HBV genotype/subgenotypes were also phylogenetically analyzed using the Neighbour-Joining method. Primarily, the sample sequences and reference sequences were all aligned. A phylogenetic tree was created using CLC Sequence Viewer 8.0 (CLC bio A/S, Qiagen, Denmark). A bootstrap value of 1000 was chosen.
Three HBsAg glycoproteins share an
HBV
Mutation characteristic | Mutation pattern | Nucleos(t)ide analogue | Patient, n (%) |
---|---|---|---|
Primary resistance mutation | rtM204I | LAM, LDT, L-FMAU, FTC | 2 (1.85) |
rtI233V | ADV | 1 (0.92) | |
Total* | – | – | 3 (2.78) |
Partial resistance mutation | rtL80I | LAM, LDT | 1 (0.92) |
rtL180M | LAM, LDT, L–FMAU, FTC | 2 (1.85) | |
Total* | – | – | 3 (2.78) |
Compensatory mutation | rtL91I | LDT | 7 (6.48) |
rtQ149K | ADV | 7 (6.48) | |
rtV214A | LAM, L-FMAU, FTC, TDF | 2 (1.85) | |
rtQ215H/P/S | LAM, L-FMAU, FTC, TDF | 18 (16.67) | |
rtN238D | ADV | 6 (5.56) | |
Total* | – | – | 29 (26.85) |
ADAPVEM | rtM204I/sW196L | LAM, LDT | 2 (1.85) |
Total* | – | – | 2 (1.85) |
Abbreviations: LAM – lamivudine; LDT – telbivudine; L-FMAU – clevudine; FTC – emtricitabine;
TDF – tenofovir; ADV – adefovir; ETV – entecavir;
ADAPVEM – antiviral drug-associated potential vaccine escape mutant.
Total: the number of total included
HBsAg escape mutations in the study patients.
HBsAg escape mutation category | Mutation pattern | Patient, n (%) | Combined pattern | Patient, n (%) |
---|---|---|---|---|
HBIg escape | sP120T, sQ129H, sM133I, sY134N, sD144E, sC147S | 7 (6.48) | sM133I + sD144E | 1 (0.92) |
Vaccine escape | sP120S, sQ129H, sS143L, sD144E, sC147S, sS193L | 9 (8.34) | sT126S + sS193L | 1 (0.92) |
HBsAg misdiagnosis | sP120T, sP120S, sR122K, sT131I, sM133I, sC147S | 10 (9.25) | – | – |
Immune escape | sQ101H, sG119R, sP120T, sT123N, sT131N, sY134F, sD144E | 9 (8.34) | sG119R + sT123N | 1 (0.92) |
Total* | – | 17 (15.74) | – | 3 (2.78) |
The total number of patients which HBsAg mutation was detected.
HBV genotypes, subgenotypes, and HBsAg serotypes of the samples.
HBV genotype | HBV subgenotype | Patients, n (%) | HBsAg serotype, n (%)* | Nationality |
---|---|---|---|---|
D | D1 | 106 (98) | TR, TRNC | |
D2 | 1 (1) | TRNC | ||
E | – | 1 (1) | – | TR |
Total | – | 108 (100) | 97 (100) | – |
Abbreviations: TR – Turkey, TRNC – Turkish Republic of Northern Cyprus.
Genotype E strain and short sequences (n = 11) were not included in serotype analysis.
In our former research the following HBV genotypes were found, namely: D/D1; 70.6%, D/D2; 5.9%, D/D3; 1.5%, A/A1; 7.4%, A/A2; 2.9%, and E; 11.8% (Sayıner and Abacıoglu 2010; Arikan et al. 2016). However, in this study, D/D1 was found in 98% of the samples, and D/D2 and E only in 1% of the samples examined, respectively (Table IV). The most often detected genotype was D/D1 for both patient groups. This is the only similarity with our previous works and the main dissimilarity we observed in this study was that D/D2 was found in a Turkish Cypriot, and a Turkish person was found to have genotype E, which has not been previously observed (Arikan et al. 2016).
Regarding other than Mediterranean region, high rates of genotype D have also been observed in the Middle East, South Asia, and North-East Europe (Sunbul et al. 2014; Zehender et al. 2014). Our results support these findings as the majority of the students, sex workers, and labor workers mostly travel and immigrate to the TRNC from Europe, Turkey, and Africa. Therefore, we can state that genotype D was introduced into the society via migrations in the past several decades from these regions, while other genotypes such as the serotype E could also be observed in the future (Zehender et al. 2014). In 2018, the article by Velkov et al. (2018) has been published that presents the global genotype distribution of HBV, assessing 125 countries and over 900 publications. Their findings indicated that genotype D was dominant in Eastern Europe, the majority of Asia and North Africa. HBV genotype distribution shows a similar pattern among the countries in the same region but varies amongst different parts of the world.
Large population migrations can modify public health dynamics. High frequency of genotypes A-D was observed in North America following migrations from Asia and Europe. A similar situation was observed in the Caribbean where genotypes A and D were found as a result of migrations from the African continent (Velkov et al. 2018; Al-Sadeq et al. 2019). We can see that migrations mainly from Turkey for working and living, and other parts of the Middle East for other purposes such as studying have caused genotype D to be dominant and new genotypes such as E introduced to the TRNC.
In this study, a total of 3/108 (2.78%) primary, 3/108 (2.78%) partial and 29/108 (26.85%) compensatory mutations were observed in the
The
In the study by Al-Sadeq et al. (2019) performed in the Middle East and North Africa region,
In conclusion, HBV-D/D1 was the dominant strain, and
One of the limitations of this study is a sample size, as larger samples will generate more significant results. The lack of prior work is another limitation as there is only one previous study, and additional work will uncover significant results in the future. Another limitation is that information is not available about the HBV infection status or phase of the patients as they were not follow-up patients.