Patterns of hepatitis B virus S gene escape mutants and reverse transcriptase mutations among genotype D isolates in Jordan

Introduction

The infection by hepatitis B virus (HBV) is considered a major cause of hepatic-related morbidity and mortality globally with an estimated 257 million people living with chronic infection and approximately 887,000 HBV-related deaths by the end of 2015 (World Health Organization, 2017).

The distinguished features of HBV among other human viruses can be summarized as follows: HBV genome is a partially double-stranded circular DNA of extremely small size (about 3,300 bases) with overlapping open reading frames. In addition, HBV replication cycle is peculiar in respect of an intermediate step involving an error-prone reverse transcriptase (RT), and is manifested in a faster evolution compared to other DNA viruses (Fares & Holmes, 2002; Zhou & Holmes, 2007). The high evolutionary rate of HBV have resulted in its diversification in the human population with emergence of several genotypes (A–J), based on inter-genotype sequence variation of more than 8% (Okamoto et al., 1988; Schaefer, 2007; Stuyver et al., 2000; Tatematsu et al., 2009; Yu et al., 2010). The HBV genotypes are further split into subgenotypes designated by numbers based on intra-genotype sequence variation of 4–8% (Norder et al., 2004). Moreover, recombination events have expanded the genetic diversity of HBV with evidence of substantial influence on shaping the evolutionary history of the virus (Simmonds & Midgley, 2005).

The unique features of HBV genome, particularly for the wide spread of overlapping open reading frames, have implications on treatment and prevention of the virus spread (Liang, 2009). This is particularly evident as follows: mutations arising in the RT region as a result of selective pressure by antiviral drugs can end up in the emergence of virus escape mutants in the adjacent S region with subsequent lack of response to HBV vaccine (Caligiuri et al., 2016; Croagh, Desmond & Bell, 2015). Among the most commonly described mutations in the RT region are mapped at the codons I169, L180, A181, T184, S202, M204, N236 and M250 (Locarnini & Yuen, 2010). These mutations can arise due to inadequate HBV therapy particularly with drugs that have low genetic barrier for resistance (lamivudine; 3TC) with some mutations having enhanced replication and competence effect on the virus (Bock et al., 2002; Locarnini & Yuen, 2010; Luber, 2005).

It is now confirmed by several reports (Lin et al., 2005; Luongo et al., 2015; Ye, Shang & Li, 2015), that transmission of HBV can occur despite successful vaccination as evidenced by the persistence of hepatitis B surface antibody (anti-HBs) in the patients. In addition, mutant viruses may evade serologic detection by the currently used enzyme immunoassays (Sheldon & Soriano, 2008; Teo & Locarnini, 2010). This can end up in the occurrence of occult hepatitis B infection (Purdy, 2007). On the other hand, a few studies indicated that the public health concerns of escape mutants might not be as previously feared (Leong, Lin & Nguyen, 2016). The prototypic and most stable vaccine-escape mutant is G145R (Hsu et al., 2004; Purdy, 2007). Among other commonly detected vaccine-escape mutants are: P120Q, Q129H, F134Y/L, S143L and D144A/E (Coppola et al., 2015).

In Jordan, the sero-prevalence of chronic HBV was estimated to range between 1.4% and 3.5% indicating a low-intermediate endemicity of the virus (Batayneh & Bdour, 2002; Hamoudi, Ghazzawi & Hamoudi, 2016; Hayajneh, Masaadeh & Hayajneh, 2010). Few studies investigated the risk factors for HBV acquisition in the country and the results pointed to horizontal familial transmission, unhygienic dental care, long-term hemodialysis and living abroad for at least 1 year as the risk factors most frequently associated with the spread of the virus (Al Hijazat & Ajlouni, 2008; Hayajneh, Masaadeh & Hayajneh, 2010). Similar to countries in the Middle East and North Africa (MENA) region, genotype D appeared to be predominating HBV genotypes in Jordan despite the low number of studies investigating this question (Hamoudi, Ghazzawi & Hamoudi, 2016; Lin & Kao, 2015; Masaadeh, Hayajneh & Alqudah, 2008).

Phylogenetic inference can be used to investigate the proportion of domestic spread of viruses and to test hypotheses related to variables that are associated with higher likelihood of transmission (Lin et al., 2005; Pybus & Rambaut, 2009; Sallam et al., 2017).

The aim of the current study was to investigate the patterns of HBV S gene escape mutants and the antiviral drug resistance mutations among HBV isolates in Jordan. In addition, we aimed to investigate possible risk factors and proportion of local transmission of the virus in the country.

Methods

Results

Possible domestic transmission of HBV in Jordan

To estimate the proportion of HBV sequences that are possibly linked in transmission clusters indicating domestic transmission, ML analysis was conducted which revealed that the proportion of phylogenetic clustering was 29.7% (11 of the 37 Jordanian sequences; Fig. 1). The clustering sequences were distributed among a dyad (two sequences) and a network of 10 sequences (with nine Jordanian sequences and a single Israeli sequence). The non-clustering Jordanian sequences were present in supported monophyletic clades together with sequences that were collected in Iran, Turkey, Syria and Egypt (Fig. 1).

Discussion

The epidemiological and antiviral drug resistance data of HBV in Jordan are limited. The results of our study are strengthened by the good coverage of sampling from different regions in the country (Fig. 2). Genotyping results showed that subgenotype D1 was found exclusively, which is consistent with previous studies from Jordan and the MENA region where this sub-genotype predominates (Abdelnabi et al., 2014; Al Baqlani et al., 2014; Asaad et al., 2015; Ciccozzi et al., 2014; El-Mowafy et al., 2017; Hamoudi, Ghazzawi & Hamoudi, 2016; Ziaee et al., 2016). The finding that all HBV isolates in our study were exclusively of genotype D is possibly related to origins of infections in the MENA in which genotype D predominates. This is supported by the finding of intermingling of sequences from Iran, Turkey, Syria and Saudi Arabia (Fig. 1).

In the current study, we investigated the patterns of HBsAg escape mutants and HBV RT mutations for the first time in Jordan to the best of our knowledge. The prevalence of HBsAg escape mutants in Jordan was 18.9%, with the R122K mutation which affects HBV detection being the most prevalent type. This is comparable to a study done in Iran, which showed that the frequency of 14% major hydrophilic region mutations (MHR), with the most frequent ones of P120T/S and R122K/T (Moradi et al., 2012). In Turkey, the most frequent mutations observed were T143M and K122R, whereas in Egypt 14.8% presented with mutations in the MHR, and eight different mutations were discovered: R122K, S143L, L109P, S114P, S117N, P127S, P127T and Y134F (Zeid, Ramadan & Shemis, 2016). Previous studies have shown the significance of R122 as a serologic determinant for HBV subtype, with subsequent effect on the antigenicity and immunogenicity of the virus (Ashton-Rickardt & Murray, 1989; Hou et al., 2001; Wu et al., 2012).

The mutations in the HBV RT have been shown to affect the management of HBV infection (Caligiuri et al., 2016; Terrault et al., 2016). For instance, the patients in our study sample who developed HBV RT mutations showed complete or partial resistance to treatment by lamivudine, telbivudine and entecavir. In the three patients with lamivudine resistance mutations, partial or complete resistance to entecavir was detected which is likely related to selection of HBV entecavir mutants in those patients. This finding is in line with recent findings by Geipel et al. (2015).

The most common resistance mutations in Jordan were 180M and 204V, which were always present as co-mutations. Other identified mutations were 173L and 184A mutations, and they were co-mutated with 180M, 204V. M204I/V mutations are frequently accompanied by compensatory mutations in other domains such as 59 rtV173L, rtL180M, rtT184S/G, 58 rtI169T, rtS202I, rtL80V/I and rtQ215S which enhanced the replication efficiency of rt204I/V mutants without significantly affecting lamivudine resistance, by compensating the decrease in efficiency due to resistance-associated changes (Bartholomeusz & Locarnini, 2006a, 2006b; Caligiuri et al., 2016; Zoulim & Locarnini, 2009). For example, dual rtL180M and M204V/I mutants were frequently found in patients in Italy, with three patients having triple mutation of rtV173L, rtL180M and M204V (Quiros-Roldan et al., 2008). Upon comparing the mutant strains with wild HBV strains, patients with rtM204V/I more frequently presented with severe acute hepatitis B and lower serum HBV DNA values (Coppola et al., 2013).

A recent meta-analysis has shown that the incidence of spontaneous primary and secondary mutations among untreated chronic hepatitis B patients was 4.9% for primary mutations of rtM204V/I, while the natural incidence of secondary rtL180M mutations was 2.7% (Zhang et al., 2015). Previous literature suggests that lamivudine is the main cause of YMDD (tyrosine-methionine-aspartateaspartate) mutations (M204I/V) within the catalytic sites (C domain) in HBV P-ORF (Caligiuri et al., 2016; Ji et al., 2011). In Turkey, Japan and China, the a high rates of spontaneous YMDD mutation may increase the unresponsiveness to lamivudine, leading to prolonged therapy time and extra cost (Zhang et al., 2015), for which we need to perform further studies to investigate the probability of development of antiviral resistant mutants worldwide, and to study the effects of the infection with mutant viruses on the clinical course of the disease.

Upon conducting ML analysis to investigate the possibility of domestic transmission of HBV in Jordan, the proportion of phylogenetic clustering was 29.7%. Horizontal transmission is the main mode of transmission of HBV in Jordan (André, 2000). Clustering of chronic HBV infections within the family is common in areas of where HBV is endemic, and horizontal transmission during early childhood is a major route of HBV transmission (Dumpis et al., 2001; Lin et al., 2005; Zampino et al., 2002). The high proportion of clustering among the study subjects highlights the need for intervention measures by public health control strategies to stop the chains of forward transmission of the virus.

Several caveats of our study should be addressed including the low sampling coverage and the short length of RT sequences that were used to conduct the transmission cluster analysis. The study was closed in 2012, which highlights the need for a follow up study to assess the recent trends of genotype and risk factors for HBV infection in the country. Another caveat of our study is the lack of clinical and serological data from the study subjects.

Conclusions

To conclude, we investigated the genotype distribution, risk factors, RT and HBsAg vaccine escape mutations and the transmission of HBV in Jordan over a large area of the country. Based on the findings of our study, we recommend RT mutation analysis before the initiation of therapy in patients with chronic HBV infection in Jordan. Lamivudine monotherapy is strongly discouraged due to high risk of resistance development. In addition, the relatively high proportion of phylogenetic clustering highlights the need for public health control measures to prevent forward transmission of the virus in the country and regionally.

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