Molecular characterization of the hepatitis B virus in autochthonous and endogenous populations in the Western Brazilian Amazon

Introduction: Hepatitis B virus (HBV) infection is a serious public health issue worldwide. Hepatitis B virus is classified into eight genotypes, varying from A to H, with distinct geographical distributions. In Brazil, the most frequent genotypes are A, D, and F. Methods: This study aimed to characterize the HBV genotypes in cases of hepatitis B virus and hepatitis D virus (HDV) co-infections in an endemic area in the Western Brazilian Amazon. We analyzed 86 serum samples reactive for HBsAg from indigenous and non-indigenous populations obtained from previous serological surveys. Results: Of the 86 reactive serum samples, 39 were found to be HBV-DNA-positive by semi-nested PCR. The genotypes were established by sequencing the amplified S gene region. We obtained 20 sequences classified into three genotypes: A, D, and F. Genotype A was the most frequent (60%), followed by D (35%) and F (5%). Conclusions: The distribution of the HBV genotypes reflected the pattern of historical occupation of the region.

Hepatitis B represents one of the most prevalent viral infections in the world.There are at least 2 billion people with serological evidence of hepatitis B virus (HBV) infection; 350 million of these are patients with chronic disease, with higher risk of death from cirrhosis and hepatocellular carcinoma 1 .
Hepatitis B virus belongs to the genus Orthohepadnavirus of the Hepadnaviridae family.It is composed of a circular double-stranded DNA particle where the negative strand is complete and the positive one is incomplete at the 5' end.It is an enveloped virus, 42nm in diameter, and with 3,200 nucleotides 2 .It can be classified phenotypically into four subtypes, which in turn may be further classified according to the antigenic determinant of the surface antigen (HBsAg): adw, ayw, adr, and ayr [2][3] .HBV can also be classified by genotype into eight strains, ranging from A to H 4 .Their structural and functional differences are associated with clinical severity, treatment failure, and possible interference with vaccine response.These genotypes can vary up to 8% within its genome 5 .
The different genoty pes have a varied distribution; genotype A has a universal distribution, being the predominant genotype in Europe, North America, Africa, and India.Genotypes B and C are predominant in Eastern and Southeast Asia and Australia.Genotype D is mainly found in the Middle East and the Mediterranean.Genotype E seems to be predominant in West Africa, whereas genotype G is distributed throughout the United States, Mexico, and France.Genotype F is mainly found in Central and South America and Alaska.Finally, genotype H is unique to Central America and the United States [5][6][7] .
The Brazilian Amazon region is characterized as one of the regions of the world with a high occurrence of HBV-associated disease and its sequelae 8 .The State of Amazonas, the Brazilian Amazon, the Juruá, the Solimões, and the Purus

METHODS
Dias ALB et al -HBV genotype in the Western Brazilian Amazon river basins are considered areas of high endemicity of HBV and Delta (HDV) infections [9][10] .Among the indigenous population of the Western Amazon, serum epidemiological studies reported prevalence rates of chronic carriers above 20% 8,11 .
In the Brazilian Amazon, genotype A has been shown to be the most commonly found genotype, followed by genotypes D and F 12 .However, genotype F is generally the most prevalent genotype in isolated indigenous communities 13 .
This study aimed to perform phylogenetic analyses to characterize the HBV genotype isolates from HBsAg-reactive subjects and to identify these in a population basis prevalence study done in an endemic area of HBV infection in the Western Brazilian Amazon.Our results may reveal differences concerning HBV distribution.The analyses were performed on the nucleotide sequences of the S regions of surface genes.

Specimens
We conducted a molecular epidemiological study of genotypic characterization of HBV in individuals positive for the HBsAg antigen in urban, rural, and indigenous communities from the county of Lábrea in the province of the Amazonas (Figure 1).
The selected samples were collected during the course of crosssectional serological surveys of HBV infection, including the general asymptomatic population from urban and rural villages of Lábrea County, from February 2006 to July 2008.
A total of 1,510 samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for HBV (HBsAg) and HDV markers (anti-HD IgG), using a commercial kit (DiaSorin, S.p.A., Saluggia, Italy).The test procedures followed the manufacturer's recommendations and were performed automatically at the laboratory facilities of the Virology Unit of Tropical Medicine Foundation Doutor Heitor Vieira Dourado (FMT-HVD).

DNA extraction
Viral DNA was extracted from the serum samples using the Qiamp DNA Blood Mini Kit (Qiagen Sciences, Maryland, USA), according to the procedures recommended by the manufacturer.A single change was made in the protocol of increasing the time of digestion with proteinase K and the Assay Lyses buffer (AL) to 4h.
For the amplification of DNA, 5.0μL from the sample was used for the first reaction, and 1.0μL of the PCR product was used for the second reaction to a mixture containing: 5.0μL 10× PCR buffer, 2.0μL of dNTP mix (10mM), 2.0μL MgCl 2 (50mM), 2.0μL of each primer, and 0.4μL of platinum Taq DNA polymerase (5U/μL) (Invitrogen, San Diego, CA, USA), resulting in a final volume of 50μL.The cycle conditions used initially were 94°C for 5min for denaturing, followed by 35 cycles of 94°C for 30s, 52.6°C for 2min, 72°C for 30s, and a final extension of 72°C for 7min in a Mastercycler Gradient thermocycler (Eppendorf, Hamburg, Germany).

Sequencing
The samples were purified with a Wizard SV Gel kit and a PCR Clean-Up System (Promega, Madison WI, USA), sequenced with forward and reverse primers using a Big Dye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions.The reactions were performed in an automatic Sequencer ABI PRISM 3130 XL Genetic Analyzer (Applied Biosystems).

Sequence analysis
To confirm the virus type, the sequences obtained were analyzed using the GenBank BLAST tool.Subsequently, the sequences were edited and aligned using the BioEdit Sequence Alignment Edit version 7.0.9.0 15 .Thirty sequences obtained from GenBank, corresponding to the eight genotypes of HBV, were used for the alignment.

Genotype identification
We performed a phylogenetic analysis comparing the different genotypes of HBV obtained from the eight GenBank sequences (X75666, X75662, X75657, X75667, AF160501, AY090460, X69798, D00329).The phylogenetic analysis and molecular evolution were performed using the Molecular Evolutionary Genetics Analysis (MEGA) Program version 4.0.2. 16.The Neighbor-Joining (NJ) model was used for the construction of phylogenetic trees.The confidence level was obtained using the nonparametric bootstrap method based on 1,000 replicates.The genotypes were classified by similarity analysis of sequences obtained in the study and sequences

DISCUSSION
corresponding to genotypes A to H obtained from GenBank.The HBV of non-human primates (accession number GenBank AF046996) was used as the outgroup.

Ethical considerations
This study was reviewed and approved by the Research Ethical Commission of FMT-HVD, Manaus, Amazonas, Brazil HBV DNA by semi-nested PCR was positive in 45.3% (39/86).This percentage was 37.5% (21/56) for those co-infected with HDV.
Twenty-two samples were submitted to sequencing of the S region of the surface gene and were compared with the eight sequences obtained from GenBank.
Among the samples from indigenous people, 44.4% (4/9) had genotype A, and 55.6% (5/9) had genotype D. Of the non-indigenous samples, genotype A was prevalent with 72.7% incidence (8/11), followed by genotype D with 18.2% (2/11) and genotype F with 9.1% (1/11).The results of this study contribute to the information regarding the distribution of HBV genotypes in the Western Brazilian Amazon, as molecular epidemiology studies of HBV in that region are still scarce.As the geographical distribution of HBV genotypes is very diverse 5 , the molecular characterization of HBV genotypes in a given region can reveal aspects of the origin of the virus in populations of that region.
The samples evaluated in this study showed a predominance of genotypes A and D, whereas genotype F was only found in one sample.Studies on the distribution of HBV genotypes in Brazil and the Amazon also show that in the northern region, genotype A is the most common, followed by genotype D and genotype F 12,[17][18][19] .However, other studies performed in the Brazilian Amazon revealed that genotype F is more prevalent [20][21][22] .
We believe that this difference could be associated with the type of samples evaluated; while our samples were from a populationbased prevalence study, including asymptomatic individuals, the majority of the studies' published samples were usually derived from patients with established chronic disease or from patients with fulminant hepatitis 21,23 .
In the past decade, genotype F was identified at a higher frequency in Amerindians 13 .This genotype is frequent in isolated tribes in the Amazon with no contact with non-indigenous people 24 .The same study showed that contact with non-indigenous people favored the introduction of genotypes A and D into these populations.
W hen analyzing samples previously characterized by a monoclonal antibody technique compared with primers for genotype F, we identified genotype A as the most frequent, followed by D and F, which suggested that there was an over-estimation of genotype F in other studies because they used monoclonal antibodies to identify the serotypes 18 .
The study region was first reached in the late nineteenth century during a collection of forest products and then again in the middle of the twentieth century during the rubber boom, with a great migration of people from the northeast region of Brazil into this region [25][26][27][28] .
It has been reported that about 50% of HBV circulating in the country is genotype A, from an African subtype, possibly related to the introduction of Africans in Brazil by the slave trade 29 .Besides the influence of African origin, the influence of European colonization and the presence of Lebanese peddlers in the Amazon region during the period of rubber exploitation 30 may explain the strong presence of genotypes A and D that were found in our samples.
We found only a positivity of 45.3% (39/86) of viral DNA in our sample, whereas other studies showed higher positivity rates, such as 76% 19,31 .Our results may be influenced by the large number of individuals co-infected with HDV at 65.1% (56/86), which spontaneously suppresses HBV [32][33] , as well as problems in handling the samples during field work in precarious places such as the rural Amazon.
This study defines the HBV genotype profile in the region and suggests that HBV distribution is possibly heterogeneous in the Amazon.The detected HBV genetic profile is, in fact, associated with the historic human occupation of the study region.

FIGURE 1 -
FIGURE 1 -Map of the study area.

FIGURE 2 -
FIGURE 2 -Neighbor-Joining phylogenetic tree from hepatitis B virus DNA (S gene) representing the genotypes found.