The hepatitis B virus (HBV) is responsible for acute and chronic in liver infection. HBV infection has a high impact on public health worldwide1 due to the potential risk of progression to liver cirrhosis and hepatocellular carcinoma (HCC).2,3 The HBV genome is made up of partially double DNA strand with approximately 3,200 base pairs (bp) organized into four transcription units or open reading frames (ORFs), known as P (polymerase), S (surfaces), C (core), and X (protein X).4,5

The HBV replication strategy is preceded by a reverse transcription step; in this step, the lack of polymerase review activity produces high rates of nucleotide substitutions,6 which allow classification into 10 genotypes of A through J and 24 subgenotypes (A1-A3, B1-B5, C1-C6, D1-D6 and F1-F4).7,8 The geographic distribution of genotypes and subgenotypes is related to the origin of the isolates.9 In Brazil, genotypes A (HBV-A), D (HBV-D) and F (HBV-F) are predominant, which reflects the miscegenation of the Brazilian population.10-12

The western Amazon basin is considered an endemic area for HBV infection13-16 with a low to high prevalence distribution.17-19 In the Brazilian occidental Amazon, a significant rate of HBV infection in indigenous peoples was discovered in the state of Rondônia, which borders Bolivia.16

Rondônia state in northern Brazil is known for having the second highest number of cases of hepatitis B,19 poor access to health services, risky cultural habits, and a migratory movement of Indigenous people between villages, all favoring the endemicity of HBV infection and hepatitis Delta virus (HDV) coinfection. The viral characteristics of HBV (genotypes, viral load, specific mutations) and coinfection with HDV are determinants of the course of infection, but are unknown in the region.3,20-23

Therefore, the present study has the main objective of evaluating genotypic and subgenotypic diversity and observing the presence of mutations in the HBV S gene in chronic carriers infected and coinfected with circulating HDV in the Indigenous population living in the border region between Brazil and Bolivia in the western Brazilian Amazon.

The study area encompasses the municipalities of Guajará Mirim and Nova Mamoré, state of Rondônia/Brazil, on the border of Guayaramerin/Beni Bolivia (Fig. 1). The study was accompanied by a clinical and laboratory outpatient reference center for Viral Hepatitis, which is located at the Tropical Medicine Research Center - CEPEM. DNA-HBV molecular tests were performed by the research team at FIOCRUZ-RO Molecular Virology Laboratory.

Fig. 1.

Map of Rondônia highlighting the cities of Guajará Mirim and Nova Mamoré from where the study participants were recruted. Legend: The blue tones distinguish the cities of Guajará Mirim and Nova Mamoré.

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Fig. 2.

Representation of type infection HBV stratified by genotypes. Legend: HBV-individual infection with hepatitis B; HBV+HDV- coinfection between HBV and HDV viruses; HBV-A (genotype A); HBV-D (genotype D); HBV-F (genotype D); Graph A: Represents the 27 indigenous individuals with individual HBV infection, 10 (37%) of which are DNA-HBV negative, 17 DNA-HBV positive characterized in genotypes HBV-A (11%), HBV-D (26%) and HBV- F (26%). Graph B: Represents the 14 indigenous people coinfected with HDV, 4 (29%) being DNA-HBV negative, 10 DNA-HBV positives characterized in genotypes HBV-A (28%), HBV-D (29%) and HBV-F (14%). Among the coinfected individuals, 8 were RNA-HDV positive, characterized by the genotype HDV-3, were coinfected with 25% DNA-HBV negative and 75% DNA-HBV positive, and were characterized as HDV-A (12.50%), HDV-D (50%), and HDV-F (12.50%). The mean age among those coinfected with genotype D was 27 years, 28.5 years among the DNA-HBV negative in nested PCR, 39 years for HBV-F and 41 years among those coinfected with HBV-A.

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HBV molecular surveillance in indigenous populations remains scarce in the region, although genotypic diversity of HBV has already been observed in indigenous and non-indigenous populations in northern Brazil.14,26-28

It was observed in this study that native HBV carriers had a time of infection that ranged from two to 24 years. It is noteworthy that among chronic carriers 49% (20/41) had at least one member of their family infected with HBV. However, due to unavailability of infection information among 41% (17/41) of participants, analyses on the risk of intrafamilial transmission were limited. A study in Brazil's southern region found a similar frequency of HBV infection among family members,29 corroborating the findings in this population.

Regarding sex, there were more chronic male patients (66%; n = 41) than females (34%; n = 41), with most participants being individuals less than 40 years old (71%; n = 29). It was observed that unvaccinated individuals had longer duration of infection than vaccinated individuals (Table 2). Among the 27 male indigenous people, only 12 were vaccinated; that is, 45% (n = 27) had an average of 11 years of HBV infection. The longer duration of infection (14.57 years) among unvaccinated female indigenous people may reflect the low vaccination coverage at the beginning of the 21st century, when indigenous populations had difficult access to villages thus not having access to health care, reflected by the low adherence to the vaccine schedule against hepatitis B.

The molecular characterization of genotypes can reveal the viral origin resulting from the historical process in populations in some regions. The HBV-F genotype is native to America, as it was frequently observed in samples from indigenous populations of Latin America (Argentina, Brazil, Bolivia, Venezuela, and Colombia) and is considered an indigenous genotype.10,26,30-32 However, in the region of the present study, bordering Brazil and Bolivia, a different genotypic pattern was observed, in which the phylogenetic evaluation classified the HBV-D genotype in 41% (11/27), with HBV-genotype F (33%) being less frequent in this population.

HBV-F was classified into two subgenotypes, HBV-F2 with 77.7% (7/9) and HBV-F4 with 23.3% (2/9). In Brazil, HBV-F2 is considered native,10 while the HBV-F4 subgenotype, despite having been identified in Brazil, has a greater distribution between the countries of Argentina and Bolivia.12,32,34,35 The presence of these subgenotypes in the region may be due to the colonization process of the region, marked by an intense migratory flow. The movement of indigenous people between riverine villages might be a factor favoring virus spread at the border, given that the sequences of the HBV-F2 and F4 subgenotypes identified were from indigenous people residing in the riverine villages Baía da Coca, Sotério, Sagarana, Rio Negro Ocaia and Ricardo Franco, located on the border with Bolivia.

In Brazil, the genotypic diversity of HBV is closely linked to the process of population and historical displacement in the region where these peoples reside. According to Mello et al.33 miscegenation in recent years was a determining factor for the genotypic distribution of HBV. This diversity of genotypes has been more evident in native populations, as these peoples have greater contact with other populations,27,33 which represents a characteristic of the Wari peoples since the process of population36 in the state of Rondônia. In Argentina, the predominance of four different HBV subgenotypes circulating in a native population was demonstrated for the first time, the Mbyá-Guarani (F4, F1, A2, and D3), from which the historical process of displacement to Brazil was related.37 Regarding genotypic evaluation, it was not possible to infer whether the Wari population has been infected by non-F genotypes for a long time or whether transmission occurred in recent times, as the size of the fragment obtained through the PCR technique was not sufficient for this type of inference, even though the fragment obtained belongs to a specific region for determining HBV genotypes and subgenotypes.

In the present study, the HBV-D genotype sequences were classified into HBV-D2 and HBV-D3 subgenotypes (formerly HBV-D3 and D6). HBV-D has been suggested as a genotype of European origin38-40 and represents approximately 24.4% of HBV variants in the Amazon.10 Spitz et al.41 suggested that HBV-D3 subgenotype sequences isolated in the Brazilian Amazon were introduced in the country several times because they are not related to European sequences. Unlike other strains, the HBV-D3 subgenotype characterized in other regions of Brazil is related to European sequences.42 However, Spitz et al.,43 in a detailed assessment of the evolutionary history of HBV-D in the Americas through different time scales, based on spatiotemporal reconstruction analyses, suggested time from the most recent common ancestor to the coincident HBV-D3 subgenotype with different migratory movements. Our isolates had a meaningful relationship with European HBV-D3, although most formed a specific cluster between sequences from Canada and Indonesia (Fig. 3). The idea of introducing these subgenotypes still in a period of migratory movements suggested by Spitz et al.43 is reinforced when we observe the findings of the present study based on the historical context of the region and the phylogenetic relationship of our isolates with sequences of European origin and non-European origin.

When evaluating the presence of mutations identified in HBV gene S, it was found that four sequences were from indigenous people with occult infection, and they had mutations V106A, I110, P120T, T118A, and A128 V. Salpini et al.44 observed that the P120T and T118A substitutions correlated with HBV-DNA reactivation in immunosuppressed patients and their absence in chronic carriers, concluding that chronic patients with such mutations are a minority. In the present study, it was not possible to determine whether these mutations were generated from cccDNA immunosuppressive therapy due to the size of the sequence generated for genomic analyses.

In contrast, in the present study, mutations P120T and T118A were observed in chronic carriers, and only one of the four individuals with occult HBV infection was treated. Study refers to position P120 as critical point for prediction of cirrhosis. Another mutation found at position C69 was significantly related in the literature to patients with hepatocarcinoma or cirrhosis.45 Stop codon substitution (C69*) was observed in the HBV genotype F sequence isolated in the present study with hepatitis Delta virus coinfection. This study participant had a history of advanced liver disease with a cirrhosis profile. This corroborates findings that correlate the C69* stop mutation with cirrhosis described by Veazjalali et al.46 in a homogeneous population infected with the HBV-D genotype.

In conclusion, the molecular characterization identified the HBV-D genotype as the most frequent in natives of the region, different from what other studies had shown, as the HBV-D genotype is frequently found in populations in the southern region of Brazil. The presence of vaccine escape mutations in the circulating HBsAg antigenic region may indicate changes in HBsAg antigenicity favoring HBV immune escape in the native population. In addition, the findings can contribute to the evaluation of immunization programs and assistance to vulnerable populations, favoring the prevention of viral hepatitis.