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Journal of Medical Virology 82:1527–1536 (2010)

Occult Hepatitis B in the Genotype H-Infected Nahuas and Huichol Native Mexican Population Sonia Roman,1 Yasuhito Tanaka,2 Anis Khan,2 Fuat Kurbanov,2 Hideaki Kato,2 Masashi Mizokami,3 and Arturo Panduro1* 1

Department of Molecular Biology in Medicine, Civil Hospital of Guadalajara, Fray Antonio Alcalde, Health Sciences Centre, University of Guadalajara, Guadalajara, Jalisco, Mexico 2 Department of Virology and Liver Unit, Nagoya City University, Graduate School of Medical Sciences, Nagoya, Aichi, Japan 3 Research Centre for Hepatitis and Immunology, International Medical Centre of Japan Kounodai Hospital, Kounodai, Ichikawa, Japan

Mexico is considered to be a low endemic country for HBV infection. However, a high anti-HBc against a low hepatitis B surface antigen (HBsAg) seroprevalence is the reported characteristic of native Mexicans. HBV diagnosis and genotype distribution was examined in native populations (Nahuas and Huichol, n ¼ 306), and compared to a non-native population (Mestizos, n ¼ 17). Overall, 6% of the natives were positive for HBsAg and 33% had detectable anti-HBc. HBsAg prevalence was lower in Nahuas compared to Huichols (1.4% vs. 9.4%, P < 0.002). Occult hepatitis B was detected in 14.2% (41/289) of natives, who either tested positive (5.88%, 17/289 HBsAg-negative) or negative for anti-HBc marker (8%, 24/289 HBsAgnegative). Age-adjusted anti-HBc seroprevalence and HBsAg quantitation revealed a sub-optimal sensitivity of conventional immunoassays. Nahuas had HBV/H and Huichol had HBV/A as the predominant genotypes followed by genotypes D, C, B, A, and D, G and H, respectively. A less variable HBV/H was characteristic in Mestizos, compared to a much variable HBV/H identified among the Nahuas. In conclusion, these findings indicate a high HBV endemicity among native Mexican groups where occult B infection is common. The different distribution of HBV genotypes among natives suggests multiple reservoirs of HBV from which these genotypes spread into the local communities. High anti-HBc seroprevalence against a low HBsAg prevalence rate may be due to the limited sensitivity of the immunoassays for the detection of HBsAg that are available in Mexico and/or unknown immunogenetic characteristics of native Mexicans. J. Med. Virol. 82:1527–1536, 2010. ß 2010 Wiley-Liss, Inc. KEY WORDS: Hepatitis B virus; occult hepatitis B infection; HBV genotypes; native Mexicans; Mexico

ß 2010 WILEY-LISS, INC.

INTRODUCTION Occult hepatitis B has been defined as a condition where hepatitis B virus (HBV) genomes (HBV-DNA) are detectable in the serum or liver tissue of hepatitis B surface antigen (HBsAg)-negative subjects that are either positive or negative for serological markers of previous HBV infection [Brechot et al., 2001; Raimondo et al., 2007]. This condition is common among infected individuals belonging to highly endemic regions [Torbenson and Thomas, 2002; Marrero and Lok, 2004] or to high risk groups such as chronic hepatitis C virus carriers, hepatocarcinoma [Cacciola et al., 1999; Pollicino et al., 2007; Raimondo et al., 2007; Adachi et al., 2008; Miura et al., 2008], hemodialysis [Minuk et al., 2004], human immunodeficiency virus-infected patients, [Torres-Barranda et al., 2006; Raimondo et al., 2007] and to a lesser degree, among the general population or voluntary blood donors [Raimondo et al., 2008; Zaaijer et al., 2008]. Occult hepatitis B infection has important clinical implications since carriers may be a source of HBV transmission in cases of blood transfusion, organ transplantation (mainly in orthotopic liver transplantation), reactivation of HBV infection as a consequence of immunosuppressive conditions or as a risk factor for Conflict of interest statement: All authors hereby declare not to have an association or affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript that may affect the reporting of the work submitted. Grant sponsor: Consejo Nacional de Ciencia y Tecnologia, Mexico (Conacyt 2004-01-025; to A. P.); Grant sponsor: Ministry of Health, Labour and Welfare of Japan (Grant-in-Aid; to M. M.). *Correspondence to: Arturo Panduro, Department of Molecular Biology in Medicine, Hospital Civil de Guadalajara ‘‘Fray Antonio Alcalde’’, PO Box 2-500, 44280 Guadalajara, Jalisco, Mexico. E-mail: [email protected], [email protected] Accepted 9 May 2010 DOI 10.1002/jmv.21846 Published online in Wiley InterScience (www.interscience.wiley.com)

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the development of hepatocarcinoma [Torbenson and Thomas, 2002; Raimondo et al., 2008]. The frequency of occult hepatitis infection varies geographically and could depend upon viral genotype, the implementation of a vaccination program or differences in the sensitivity of the screening methods to detect serological markers of particular HBV genotypes [Torbenson and Thomas, 2002; Allain, 2004; Marrero and Lok, 2004; Raimondo et al., 2007, 2008]. HBV genotypes have distinct geographical distributions [Miyakawa and Mizokami, 2003] and correlate with the severity of liver disease. HBV genotypes F and H are the most common genotypes in Latin American countries. HBV genotypes H and G are more common in Mexico than in the rest of the world [Sanchez et al., 2002, 2007; Tanaka et al., 2008]. The HBsAg seroprevalence in the general Mexican population is reported to have been low and steady since 1974 to date [Roman et al., 2009]. Higher seroprevalences were reported in geographical regions where native Mexicans are the most represented local population [Roman et al., 2009]. An antiHBc prevalence rate higher than 50% has been reported among the native Mexican population despite a low HBsAg seroprevalence (0.1%) rate [Alvarez Mun˜ oz et al., 1989; Cisneros-Castolo et al., 2001]. Mexico, as are the majority of Latin American countries, is characterized by the presence of the Mestizos and native ethnic groups within the country’s population. The genetic background of the Mexican Mestizo is an admixture of the specific indigenous settlers of Mesoamerica and the Spaniards that arrived in the early 1500s and the African slaves brought over to the Americas [Aceves et al., 2006; Bedoya et al., 2006; Wang et al., 2008]. While 90% of the Mexican population is comprised of Mestizos living in urban areas, the remaining 10% (close to 12 million people) are still conserved in their original native lifestyle and inhabit the rural areas [INEGI, 2005; CDI, 2008]. The identification of the HBV genotype has become important because of its increasingly recognized clinical implication in the manifestation and outcome of the infection [Kidd-Ljunggren et al., 2002; Schaefer, 2005]. In some cases, genotyping can be used as a tool to study the origin and migration of HBV genotypes associated with a particular native population [Khan et al., 2008]. Studies on the molecular epidemiology of HBV genotypes are scarce in Mexico because of the diverse ethnic populations living in different geographical areas of the country. In this study, we have identified a high HBV endemicity and a high rate of occult hepatitis B infection in two different populations of native Mexicans living in the west of the country, along with the heterogeneous distribution of HBV genotypes. MATERIALS AND METHODS Ethnic Background and Geographic Location of Study Participants A cross-sectional study was performed in three populations with distinct geographical locations and J. Med. Virol. DOI 10.1002/jmv

ethnic backgrounds. One group was natives from the Nahuas community that are descendents of the Aztec population that settled in Tenochitlan (now Mexico City and the surrounding regions) before the arrival of the Spanish conquistadors. The Nahuas participants came from the State of Guerrero to settle in Autlan, Jalisco, which is located in Western Mexico, and were tested between May and August 2005. Secondly, the Huichol are natives that belong to an ancient population living in isolated communities within the Western mountains of Nayarit. This group was tested between 2003 and 2005. A third group included the Mestizos from Mexico City diagnosed with chronic hepatitis and HBsAg positive. In both native groups, the research team explained the nature of the study to the community members. Only those who voluntarily agreed to participate were included in the study. The ethnic identity of both native groups was accessed following the definitions recommended by the National Institute of Anthropology and History as described previously [Aceves et al., 2006]. The study protocol was approved by the Ethical Committee of the Civil Hospital of Guadalajara and written informed consent was obtained from each participant. Serum Samples A total of 306 serum samples were collected from the natives (Nahuas, n ¼ 147; Huichol, n ¼ 159) and 17 serum samples were collected from the Mestizos. After the isolation of serum fraction, the samples were kept frozen at 808C until assaying. Molecular, immunological, and biochemical tests were performed at the Department of Molecular Biology in Medicine as described below. Clinical History and Demographical Data Clinical history and demographical data including age, gender, occupation, civil status, education level, and risk factors related to HBV infection (such as blood donations, surgery, blood transfusions, hemodialysis, acupuncture, number of sex partners, and household conditions) were collected from all of the participants by a structured questionnaire. Biochemical Tests Liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured in serum using an enzymatic method (Human, Wiesbaden, Germany) in an automatic analyzer. Abnormal cutoff values for ALT and AST enzymes were 40 and 50 UI/ml, respectively. Serology All samples were screened to detect HBsAg and antiHBc (total immunoglobulin M and immunoglobulin G) by using the third-generation microparticle enzyme immunoassays (IMx HBsAg (V2) and IMx CORE (Abbott Laboratories, Chicago, IL) on the IMx analyzer.

HBV Genotypes and Occult Hepatitis in Mexicans

Both assays were used as per the manufacturer’s recommendations without modifications. Specimens reactive to HBsAg had a signal to cutoff (S/CO) ratio equal to or greater than 1.000, and those reactive to antiHBc were within the range of 50.00–99.90 percentage of inhibition (% INH). If the available number of serum samples was sufficient, HBV-DNA positive cases were retested by the quantitative HBsAg and anti-HBc detection chemiluminescent immunoassay (CLIA) (Lumipulse, Fujirebio, Toyko, Japan) in comparison to the IMx results. The sensitivity threshold of this method is 50 mIU/ml of HBsAg. Some of the samples were tested using a novel high-sensitivity chemiluminescent immunoenzyme assay (CLEIA) for the detection of HBsAg. The CLEIA prototype differs from the conventional CLIA method in that the antibody reagent is a combination of three monoclonal antibodies that recognize the determinant ‘‘a’’ as the constructive epitope, amino acids 111–130, and amino acids 31–50, respectively. The tracer reagent is a combination of two monoclonal antibodies, each of which recognize the determinant ‘‘a’’ as the constructive epitope in the different region of the solid antibody and amino acids 51–69, respectively. The use of detergents added to the reaction buffer and an improved labeled tracer contributes to the enhancement of the signal and detection limit. The CLEIA prototype has an analytical sensitivity of 0.05 mIU/ml and a cutoff point of 0.22 mIU/ml for the detection of HBsAg [Matsubara et al., 2009]. HBV-DNA Isolation and Amplification Viral DNA was extracted from 100 mL of serum with the use of the QIAamp DNA Blood mini kit (Qiagen Sciences, GmbH, Hilden, Germany) in accordance with the manufacturer’s instructions. To obtain maximal sensitivity, a nested PCR protocol to amplify a 432 or 218-nt fragment in the small-S region using previously reported primers [Sanchez et al., 2002] was followed to detect HBV-DNA. Standard precautions to avoid crosscontamination were exercised both through the extraction and amplification procedures. A negative control serum was also included to each run to ensure specificity. To confirm the positive samples, amplification of the HBV precore/core region was carried out with at least one of the three primer sets: sense 50 -GAGACCACCGTGAACGCCC-30 (1611–1629) antisense 50 GGAGTGCGAATCCACACT-CC-30 (2298–2279), and either antisense 50 -ATAGC-TTGCCTGAGAGC-30 or antisense 50 -CTCCACAG-AAGCTCCAAATTCTTTA-30 (1942–1918) [Khan et al., 2008]. Additionally, the HBV-DNA samples that were positive with the first set of partial-S region primers [Sanchez et al., 2002] were further tested with genotype H-specific primers which were designed to improve the specificity of the amplification reactions (H1: 50 -CTA CAG CAT GGG AGC ACC TCT CTC MAC GGC-30 , position 2844–2873; H2: 50 -GTG GAT CKG GTG GCG AGG TTG TCA GAA TGC-30 , position 3127–3080);

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numbering is according to the HBV genotype H reference strain [Genbank accession number AY090454] under standard PCR conditions. Since we have observed that HBV genotype H is predominant in Mexico, this procedure is performed routinely for quality control purposes before direct sequencing. Furthermore, samples that amplified with only one set of PCR primers and/ or were non-repeatable were considered to be negative. Complete genomes of HBV/H strains were amplified as two partially overlapping fragments and subsequently amplified by use of overlapping primers as described elsewhere [Khan et al., 2008]. Sequence and Phylogenetic Analyses PCR products were directly sequenced in both directions with corresponding amplification primers by using the BigDye Terminator Cycle Sequencing Kit and the thermal cycler GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA). Sequences were determined by using an automated fluorescent DNA analyzer (ABI 3100, Applied Biosystems). Sequencing data were analyzed through the use of ABI Sequencing Analysis version 5.3) (Applied Biosystems). Sequence alignment was performed with ClustalX software, or MEGA 4.0 (http://www. megasoftware.net) and confirmed by visual inspection. Genetic distances were estimated using the six-parameter method and NJ phylogenetic trees were built. To confirm the reliability of the phylogenetic tree analysis, bootstrap resampling was carried out 1000 times. Complete HBV genomes were assembled using GENETYX-MAC version 11.0 (Software Development Co. Ltd., Tokyo, Japan). Phylogenetic analysis was performed on samples which compared the best and high scoring matched by a homology search with BLASTN (http: www.ddbj.nig. ac.jp) and also with partial S gene sequences retrieved from DDBJ/EMBL/GenBank corresponding to the accession numbers mentioned in the phylogenetic trees. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB516356-AB516389 and AB516391-AB516395. Statistical Analyses Data for continuous variables are given as mean  standard deviation. The Chi-square test was performed between prevalence of HBV markers and each study group. Values of P < 0.05 were considered to be significant. RESULTS Demographics and Main Risk Factors The demographic and clinical data are shown in Table I. Both native populations live in rural mountain areas in one-room houses with a high household density. The participants were mostly young adults and married. J. Med. Virol. DOI 10.1002/jmv

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TABLE I. Demographics and Clinical Data of Native Mexicans Subject data Gender M/F Age (mean  SD) Age group (years) 10–20 21–30 31–40 41–50 51–60 >60 Civil status Married Single Other Occupation Peasant Housewife Other Education level None or 6 years Main risk factors Blood donors Surgery Transfusions Hemodialysis Acupuncture Multiple sex partners (3) MSM High household density (4 ppr) HBsAg positive Anti-HBc positive HBV-DNA positive AST (IU/L) (mean  SD) ALT (IU/L) (mean  SD)

Nahuas (n ¼ 147)

Huicholes (n ¼ 159)

44/103 29  12

45/114 41  15

36 (24) 54 (37) 36 (24) 10 ( 7) 11 (8) —

10 (6) 35 (22) 42 (26) 31 (20) 20 (13) 21 (13)

121 (82) 22 (15) 4 (3)

143 (90) 13 (8) 3 (2)

49 (60) 88 (33) 10 (7)

87 (55) 64 (40) 8 (5)

116 (79) 31 (21)

135 (85) 24 (15)

9 (6) 26 (18) 8 (5) 0 (0) 0 (0) 13 (9) 0 (0) 147 (100) 2 (1.4) 55 (37) 25 (17) 34  17 36  28

6 (4) 17 (11) 7 (4) 0 (0) 0 (0) 15 (9.5) 2 (1.2) 159 (100) 15 (9.4) 45 (28) 25 (16) 20  11 13  9

Men work as peasants and women are housewives and family caretakers and the practice of polygamy is common in the Huichol communities. The main risk factors in these populations are the high household density, low income, and reported multiple sexual partners. Other risk factors, such as blood donation and antecedents of surgery were found to a lesser degree in comparison to inhabitants living in urban areas [Valdespino et al., 2007; Roman et al., 2009]. Only two men from the Huichol group reported having sexual relationships with men. None of the participants had clinical manifestations of liver disease. All liver function tests were within the normal limits. Alcohol consumption is common in both populations. By contrast, the Mestizos from Mexico City were HBsAg/ anti-HBc positive and had a clinical diagnosis of chronic viral hepatitis.

In this study, occult hepatitis B infection was defined as undetectable HBsAg in serum, either positive or negative to anti-HBc, whereas HBV-DNA is positively detected by PCR in at least two different HBV genomic regions. Anti-HBc Titers and Age-Adjusted Prevalence The anti-HBc titers were reported as a percent inhibition as indicated in the assay, and accordingly the results were divided into four categories: 0–30% (very low), 31–49% (low), 50–69% (moderate), and 70– 100% (high). In the Huichol, 72% of the cases had very low-to-low negative titers, against 63% identified in the Nahuas samples. Conversely, in the Huichol, only 28% had moderate to high positive titers compared to 37% identified in the Nahuas (data not shown). These findings are related to the prevalence of anti-HBc stratified by age, as shown in Figure 1. Both groups where exposed to the infection at an early age. However, the prevalence of anti-HBc between the ages of 21 and 40 is lower in the Huichol compared to the Nahuas (P < 0.02) and tends to decrease in both groups as age increases. These differences may suggest that HBV infection is rapidly cleared due to differences in age or genetic background between both native groups. Detection of HBsAg in HBV-DNA Positive Samples The Huichol with the higher HBV seroprevalence rate had HBsAg seropositive samples slightly above the IMx detection limit (S/CO near 1.00), suggesting that in this population there may be a rapid host response to eliminate the virus. In the Nahuas cases, those that were HBV-DNA positive/HBsAg negative were under the S/CO ratio of 1 (Table II). Among the 50 HBV-DNA cases, 9 HBsAg positive samples were consistent with both methods. However, among the 41 occult cases, serum samples MEX007N and MEX018N (Nahuas), and MEX315H (Huichol) were negative by MEIA (IMx system) but positive by the quantitative method

Prevalence of HBV Infection The overall prevalence of HBV markers is shown in Table I. The Huichol had a significantly higher HBsAg prevalence compared to the Nahuas (9.4%, 15/159 vs. 1.4%, 2/147) (P < 0.002). Both groups have been highly exposed to the HBV infection, since the anti-HBc marker was prevalent in 33% (100/306) of the cases. J. Med. Virol. DOI 10.1002/jmv

Fig. 1. Age-adjusted prevalence of anti-HBc among the Nahuas and Huichol native Mexicans.

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TABLE II. Immunoassay Sensitivity of HBV-DNA Positive Samples HBsAg assays

PCR assays

Sample ID

IMx-USA S/CO

CLEIA-Japan (mIU/ml)

HBV-DNA

MEX001N MEX002N MEX003N MEX004N MEX005N MEX006N MEX007N MEX008N MEX009N MEX010N MEX011N MEX012N MEX013N MEX014N MEX015N MEX016N MEX018N MEX019N MEX020N MEX021N MEX022N MEX023N MEX024N MEX025N MEX026N MEX301H MEX302H MEX303H MEX304H MEX305H MEX306H MEX307H MEX308H MEX309H MEX310H MEX311H MEX312H MEX313H MEX314H MEX315H MEX316H MEX317H MEX318H MEX319H MEX320H MEX321H MEX322H MEX323H MEX324H MEX325H

157.48 0.57 0.32 0.35 0.36 0.32 0.39 185.58 0.32 0.48 0.43 0.43 0.41 0.35 0.60 0.37 0.93 0.71 0.54 0.49 0.45 0.42 0.37 0.33 0.31 0.96 1.20 0.48 1.10 1.10 0.48 0.93 1.30 0.54 0.41 0.46 0.54 0.55 0.59 0.60 0.60 0.66 0.75 0.77 0.80 0.84 1.03 0.94 1.37 1.40

11,640,997 Neg Neg Nt Neg Neg 0.34 3,258,397 Neg Nt Nt Nt Nt Nt Nt Nt 0.23 Neg Nt Neg Neg Neg Neg Neg Neg Neg Nt Nt Nt Nt Nt Neg Nt Nt Nt Neg Neg Neg Nt 4.98 Nt Nt Nt Neg Nt Nt 1.51 Neg Nt 1.91

þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ

Genotype sequencing H A H H H H B H C D D H H C H H Ns Ns Ns Ns Ns Ns Ns Ns Ns A H D G A A A D A Ns Ns Ns Ns Ns Ns Ns Ns Ns Ns Ns Ns Ns Ns Ns Ns

Accession number AB516385 AB516356 AB516357 AB516358 AB516359 AB516360 AB516361 AB516362 AB516363 AB516364 AB516365 AB516366 AB516367 AB516368 AB516369 AB516370

AB516379 AB516386 — AB516380 AB516381 AB516382 AB516383 — AB516384

S/CO, signal/cutoff index; CLEIA, chemiluminescent immunoenzyme assay; Neg, negative; Nt, not tested; Ns, not sequenced; N series, Nahua cases; H series, Huichol cases.

(CLEIA) with 0.34, 0.23, and 4.98 mIU/ml of HBsAg, respectively. Phylogenetic Analysis and Genetic Variability The 41 DNA-HVB positive/occult cases (23 Nahuas and 18 Huichol) were detected by at least two of three distinct sets of primers to amplify sequences within the

small S region (nt55-475) and polymerase/preS1-coding region (nt2844-3127) or core region (nt1611-2298). Twenty-five samples were partially sequenced, 16 Nahuas and 9 Huichol. Among the 16 Nahuas cases, 14 sequences were from the small S region only, 1 was from the core region, and 1 was both from the S and C regions (the accession numbers are listed in Table II). Regarding the nine Huichol cases, six sequences were small S region alone, and three were from the core region J. Med. Virol. DOI 10.1002/jmv

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substitutions, that were common among three and five individual cases, respectively. However, these substitutions were not detected in other HBV genotype H-sequences retrieved from the GenBank, including previous Mexican isolates. Other unique substitutions were L15V, L21W, K24R, P45A, L61S, T63I, and T68I. However, the amino acid substitutions at positions 18, 19, 23, 30, 34, 44, and 57 are shared with HBV genotype F isolates retrieved from the GenBank (Fig. 4). In comparison to the three complete HBV genomes obtained from the Mestizo group in this study (MEX904M, MEX912M, and MEX914M), a close homology was observed with strains previously isolated from Nicaragua (AY090454 and AY090457) and the USA (AY090460), respectively (data not shown). DISCUSSION Fig. 2. Distribution of HBV genotypes among the Nahuas and Huichol native Mexicans.

(Table II). The remaining sequences that tested DNAHBV positive were not able to be sequenced due to the lack of serum after running several tests to confirm positivity. A high degree of diversity in the HBV genotypes was observed in the native groups as shown by the phylogenetic analysis. The predominant genotype in the native populations was genotype H in 44% (11/25), followed by genotype A in 24% (6/25), then D in 16% (4/25), C in 8% (2/25), and B with G in 4.1% (1/25) each; whereas in the Mestizo group, only genotype H was detected (Fig. 2). In the Huichol, located in Western Mexico, genotype A was predominant in 55.5% (5/9) of the cases, followed by genotype D in 22% (2/9), and genotypes G and H in 11.1% (1/9) each. In contrast, the Nahuas from the state of Guerrero, predominantly had genotype H in 62.5% of the cases (10/16), followed by genotypes C and D, 12.5% (2/16) each, and then genotypes A and B, 6.5% (1/16) each. Interestingly, partial-S sequences of HBV genotype H isolates from the Mestizo clustered separately from the Nahuas genotype H isolates (Fig. 3A). However, within the respective clusters, the isolates revealed high homology. To exclude the possibility of PCR-related contamination among the Mestizo samples (control group), certain samples were additionally sequenced in the preC/C region. The phylogenetic tree constructed on the preC/C region revealed a distinct topography of the selected samples within the cluster, together with other isolates of the same group (Fig. 3B). Furthermore, when the respective S gene-nucleotide alignment and deduced amino acid sequences from the HBV Nahuas and Mestizo isolates were compared, the former showed numerous and repetitive amino acid substitutions compared to the latter. The Mestizo isolates had a higher homology to the reference sequence MEX3C and cases MEX910M and MEX917M only had three and one amino acid substitutions, respectively. In contrast, the Nahuas isolates had N40S and N52S J. Med. Virol. DOI 10.1002/jmv

This is the first HBV molecular epidemiological study carried out among the native Mexican population. An overall seroprevalence of HBsAg and anti-HBc was 6% and 33%, respectively in the study cohort which is in accordance with the previous HBsAg seroprevalence studies that had shown a high anti-HBc and low HBsAg seroprevalence in the native Mexican population [Alvarez Mun˜ oz et al., 1989; Cisneros-Castolo et al., 2001]. Therefore, this may suggest that occult hepatitis B could be a common finding in some or perhaps most of the Mexican native communities as found in this study with an occult hepatitis B rate in 14.2% (41/289) of cases. Occult hepatitis B infection was detected in both Nahuas and Huichol groups either in those positive to anti-HBc (7%) or without any serological marker (8%). Individuals representing Nahuas and Huichol in this study neither had an alteration of liver function enzymes nor any clinical manifestation of acute or chronic liver disease. Both native groups had been exposed to HBV infection as indicated by the presence of the anti-HBc marker. However, in the Huichol, the HBsAg seropositivity rate was significantly higher (P < 0.002) and the anti-HBc prevalence rate between the ages of 21 and 40 was lower compared to the Nahuas (P < 0.02). However, studies on the expression of HLA phenotypes have not been documented in these populations which could explain the possible differences in a typical host immune response for each native group. Different factors that could be associated with the occult course of hepatitis infection should be considered, for example time and recovery from infection as a result of host immune response, low viral load detected by a highly sensitive molecular method, differences in sensitivity of the HBsAg and anti-HBc immunoassay as well as variability of the HBV genome [Candotti et al., 2008; Miura et al., 2008; Raimondo et al., 2008; Zahn et al., 2008]. After the arrival of the Spaniards, several epidemics caused a high mortality rate that reduced the native population size by almost 90%, which generated natural selection [Sanchez-Albornoz 1974; Powell, 1975]. Therefore, the high prevalence of occult hepatitis B infection in this study may be due to the fact that native

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Fig. 3. A: Phylogenetic tree of the partial S region sequences detected among native Mexicans (N, Nahuas, H, Huichol, and M, Mestizo). B: Phylogenetic tree of the partial C sequences detected among native Mexicans (N, Nahuas, H, Huichol, and M, Mestizo).

Mexicans have been undisturbed by medical intervention, such as passive/active immunization or antiviral drugs, which suggests that the humoral and cellular immune pressure on the HBV envelope protein may be one of the major mechanisms generating occult hepatitis B infection in Mexico. In the 41 occult B cases, HBV-DNA detectable by nested PCR of HBV genome was associated with HBsAg and anti-HBc (49% of the cases) titers close to the S/CO value of the immunoassay. This could be due to low viral load and the low concentration of the HBsAg protein,

since differences in the sensitivity of the immunological test were evident in three cases. There have been warnings against the limited sensitivity of different HBsAg detection assays to detect escape mutants [Gerlich, 2004; Ly et al., 2006]. Furthermore, immunological assays for HBsAg detection have primarily been developed on the basis of genotype A and D strains genomic reference (WHO IS and reference panel 2004) [WHO, 2004]. This is why differences in sensitivity have been shown in the detection of genotype B and C strains [Mizuochi et al., 2005]. However, there is no study with J. Med. Virol. DOI 10.1002/jmv

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Fig. 4. Deduced amino acid sequences of HBsAg cytosolic loop of HBV genotype H Mestizo and Nahuas isolates. *Reference sequence AF369535: HBV Mexican strain, genotype H, isolate MEX3C [Sanchez et al., 2002].

reference to genotypes H and F, which are predominant in Latin American countries including Mexico [Sanchez et al., 2002; Gutierrez et al., 2004; Norder et al., 2004; Devesa and Pujol, 2007; Mello et al., 2007]. A heterogeneous distribution of HBV genotypes was identified between the native and Mestizo groups. HBV genotype H was the main genotype in the Nahuas population as well as in the Mestizo group from Mexico City, which indicates that these two communities may have shared a common epidemic network. Recent mitochondrial DNA studies from individuals from Mexico City indicate a native Indian origin rather than Spanish or Black African mitochondrial DNA [Price et al., 2007; Wang et al., 2008]. Historical evidence regarding the Nahuas population and the Mestizos of the same origin with a common ancestral background and different from those from South America, may help to understand the diversity in HBV genome variability between genotypes H and F that predominate in Mexico and South America, respectively [Sanchez-Albornoz, 1974; Powell, 1975; Orozco, 1983; Rivas-Salmon, 2003]. Conversely, genotype A was mainly detected in the Huichol group. There is a possibility that genotype A was present in Mexico at the time of the arrival of the Spaniards or that this genotype was introduced in this region through black slaves during that period. Similar speculations may be made in relation to the other native populations from Latin America where genotype A predominates over genotype F [Quintero et al., 2002]. The presence of the genotypes B and C may be associated with different migration events of people from Asia [Horai et al., 1993]. During the colonial period and more J. Med. Virol. DOI 10.1002/jmv

recently in the last century, people from the Philippines, China, and Japan have migrated to Mexico [Israel, 1975]. With the arrival of the Spaniards to the New World, there were two main groups of native populations in Central Mexico: the sedentary or pacific groups living mainly in Central Mexico and the nomads that remained in the mountain regions. The Nahuas were from the former group and the Huichol were from the latter [Powell, 1975]. The exposure to the non-H genotypes found in this population could have been through the black slaves who primarily worked in the gold and silver mines, and who then escaped to the mountains and mixed perhaps within the Huichols. Risk factors of HBV transmission could have been due to sexual intercourse or even anthropophagy which was common at that time. The 218-nt stretch of the small-S region sequenced in this study encodes a short segment of the aminoterminal transmembrane domain (TMD-I) and the complete cytosolic loop (CYL-I) between amino acid residues 24–80. Mutational analysis studies have shown that substitutions at cysteine residues 48, 65, and 69 cause defective sub-viral particles and abolish the secretion of HBsAg particles [Mangold and Streeck, 1993]. However, these specific substitutions were not detected in the Nahuas sequences which could explain occult hepatitis B infection. However, the novel amino acid substitutions that were prevalent in this segment demonstrates that HBV genotype H sequences have a higher genetic variability compared to other genotype H sequences in GenBank. Future studies will rely on full

HBV Genotypes and Occult Hepatitis in Mexicans

sequenced HBV/H genomes from occult hepatitis B cases obtained from this population in order to associate clinical findings with the genetic variability of this genotype. In conclusion, this study confirms the existence of high endemicity regions in Mexico and affirms that occult hepatitis B infection is common in native Mexican populations. Genotype H is the most predominant genotype, followed by genotypes A and D, and that the genotype distribution is heterogeneous in the populations studied. A high anti-HBc prevalence against a low HBsAg prevalence rate may be related to the limited sensitivity of the immunoassays available in Mexico and/or to unknown immunogenetic characteristics of native Mexicans. This is a significant warning that HBV infection may be underestimated due to differences in genotype and/or the genetic variability of the HBV that predominates in the country. Further comparative studies are required to confirm the degree of sensitivity of different HBsAg immunoassays using serological panels from different communities living in Mexico, in order to estimate the exact burden of HBV infection in the country and to establish sensitizing infection-control strategies. ACKNOWLEDGMENTS S. R. and A. P. extend their gratitude to Eloy Zepeda and Guadalupe Huezo for their helpful contributions during the field study. A. P. received a scholarship from the National Council of Science and Technology in Mexico for his sabbatical year in Japan. S. R. received a scholarship from the University of Guadalajara for her postdoctoral in Japan. REFERENCES Aceves D, Ruiz B, Nun˜ o P, Roman S, Zepeda E, Panduro A. 2006. Heterogeneity of apolipoprotein E polymorphism in different Mexican populations. Hum Biol 78:65–75. Adachi S, Shibuya A, Miura Y, Takeuchi A, Nakazawa T, Saigenji K. 2008. Impact of occult hepatitis B virus infection and prior hepatitis B virus infection on development of hepatocellular carcinoma in patients with liver cirrhosis due to hepatitis C virus. Scan J Gastroenterol 43:849–856. Allain JP. 2004. Occult hepatitis B virus infection. Transfus Clin Biol 11:18–25. Alvarez Mun˜ oz MT, Bustamante Calvillo ML, Martı´nez Garcı´a MC, Moreno Altamirano L, Guiscafre-Gallardo H, Guiscafre JP, Mun˜ oz O. 1989. Seroepidemiology of the hepatitis B and delta in the southeast of Chiapas, Mexico. Arch Inves Med (Mex) 20:189–195. Bedoya G, Montoya P, Garcı´a J, Soto I, Bourgeois S, Carvajal L, Labuda D, Alvarez V, Ospina J, Hedrick PW, Ruiz-Linares A. 2006. Admixture dynamics in Hispanics: a shift in the nuclear genetic ancestry of a South American population isolate. Proc Natl Acad Sci 103:7234–7239. Brechot C, Thiers V, Kremsdorf D, Nalpas B, Pol S, Paterlini-Berchot P. 2001. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely ‘‘occult’’? Hepatol 34:194–203. Cacciola I, Pollicino T, Squadrito G, Cerenzia G, Orlando ME, Raimondo G. 1999. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med 341:22–26. Candotti D, Grabarczyk P, Ghiazza P, Roig R, Casamitjana N, Ludicone P, Schmidt M, Bird A, Crookes R, Brojer E, Miceli M, Amiri A, Li C, Allain JP. 2008. Characterization of occult hepatitis B virus from blood donors carrying genotype A2 or genotype D strains. Hepatology 49:537–547.

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