José M. Echevarría

Genetic Diversity of Hepatitis B Virus Strains Derived Worldwide: Genotypes, Subgenotypes, and HBsAg Subtypes

Sequences of 234 complete genomes and 631 hepatitis B surface antigen genes were used to assess the worldwide diversity of hepatitis B virus (HBV). Apart from the described two subgenotypes each for A and F, also B, C, and D divided into four subgenotypes each in the analysis of complete genomes supported by significant bootstrap values. The subgenotypes of B and C differed in their geographical distribution, with B1 dominating in Japan, B2 in China and Vietnam, B3 confined to Indonesia, and B4 confined to Vietnam, all strains specifying subtype ayw1. Subgenotype C1 was common in Japan, Korea, and China; C2 in China, South-East Asia, and Bangladesh, and C3 in the Oceania comprising strains specifying adrq–, and C4 specifying ayw3 is encountered in Aborigines from Australia. This pattern of defined geographical distribution was less evident for D1–D4, where the subgenotypes were widely spread in Europe, Africa, and Asia, possibly due to their divergence having occurred a longer time ago than for genotypes B and C, with D4 being the first split and still the dominating subgenotype of D in the Oceania. The genetic diversity of HBV and the geographical distribution of its subgenotypes provide a tool to reconstruct the evolutionary history of HBV and may help to complement genetic data in the understanding of the evolution and past migrations of man.

Diagnostic Problems Caused by HBsAg Mutants – A Consensus Report of an Expert Meeting

A panel of 16 experts from 9 countries convened on April 14 at Schloss Reinhartshausen near Wiesbaden in Germany, to discuss the diagnostic significance of mutants, variants and genotypes of hepatitis B virus (HBV). Since the description of Australia antigen in 1965 and the subsequent observation that it was the envelope of the HBV and now designated hepatitis B surface antigen (HBsAg), this lipoprotein has been a mainstay in the diagnosis of HBV infections. HBsAg tests are used routinely in the diagnosis of acute and chronic liver disease, the screening of blood or organ donors and the surveillance of persons at risk to acquire or to transmit HBV. Current immunoassays for HBsAg are very specific and sensitive (both 199%) and are usually able to detect !0.5 ng HBsAg/ml serum. Their performance is validated in extensive trials before licensing and their detection limit is assayed with an International Standard for HBsAg. An immanent problem of virology is the variability of viruses. Due to the low fidelity of the viral nucleic acid polymerases and the high replication rates, virtually all nucleotide positions of a viral genome can be mutated within a relatively short time. However, the viability of the virus and its adaptation to the host allow the selection and outgrowth of only a limited number of mutants. The survival strategy of HBV in the population is mainly based on induction of immune tolerance and persistence of high viremia. In this state of infection the existing viral genome is favored whereas mutants are less fit and selected against or may be subject to an immune reaction and preferably eliminated. In fact, most of the HBsAg carriers in Germany have a very similar S gene sequence. However, under selective pressure the virus can express many different viable HBsAg mutants. Summary of the Presentations

Infections of the Nervous System Caused by Varicella-Zoster Virus: A Review

Varicella-zoster virus (VZV) is a cause of neurologic disease among humans. Both primary infection and recurrence may lead to neurologic infection and disease. Neurologic syndromes associated with acute VZV infection are caused by abnormal immune responses, the most frequent manifestation being cerebellar ataxia. Those associated with recurrences are often due to the direct effect of viral replication in the nervous tissue. VZV reaches the nervous system either through the bloodstream or by direct spread from sensory ganglia where it remains latent. Postherpetic neuralgia, acute encephalitis, aseptic meningitis and myelitis are the most frequent diseases and have been recorded both in association with herpes zoster and in the absence of a cutaneous rash. Early diagnosis may be established by detecting virus-specific DNA sequences in the cerebrospinal fluid (CSF) after amplification by the polymerase chain reaction and then confirmed by detection of intrathecally produced, specific IgG antibody. Virus isolation from CSF and antibody testing in serum are unsuitable for diagnosis. Early acyclovir therapy is recommended in immuno-compromised patients and those with serious disease.

Chronic Hepatitis E in HIV Patients: Rapid Progression to Cirrhosis and Response to Oral Ribavirin

Chronic hepatitis E virus infection with rapid progression to cirrhosis is reported in 2 human immunodeficiency virus (HIV)-infected patients with severe immunosuppression. Monotherapy with ribavirin led to temporary viral response and marked improvement of liver damage. Chronic hepatitis E should be regarded as another opportunistic event within HIV infection.

Follow-up of the prevalence of hepatitis C virus genotypes in Spain during a nine-year period (1996-2004)

BACKGROUND Recent data suggest that the prevalence of genotype 4 HCV strains among Spanish carriers is increasing. OBJECTIVE To assess changes in the prevalence of HCV genotypes in Spain during the last nine years. METHODS HCV RNA was amplified by the polymerase chain reaction from 3161 serum samples from unselected, anti-HCV-positive individuals, and the HCV genotype was identified by a reverse hybridisation assay (line probe assay, LiPA). Samples came from 17 different regions of Spain and were obtained between January, 1996 and December, 2004. RESULTS The overall prevalence of HCV genotypes was: 1b, 41.3%; 1a, 24.1%; 3, 19.6%; 4, 11.6%; 2, 3.1%; and 5, 0.3%. The prevalence of genotypes 1a, 3 and 4 increased significantly among patients born after 1950, and that of genotype 1b decreased among them. These significant differences in regard to age were also observed among patients lacking notified high-risk factors. A main switch-up in prevalence of genotypes 1a and 3 was found when patients born in 1941-1950 were compared with those born in 1951-1960, but the same finding in genotype 4 was delayed to patients born in 1961-1970. CONCLUSIONS Two separate epidemics of HCV seem to have occurred in Spain during the last 30 years. The former one involved the spread of HCV genotypes 1a and 3. The second was more recent, and involved the spread of genotype 4.

Epidemiology of viruses causing chronic hepatitis among populations from the Amazon Basin and related ecosystems

On the last twenty years, viral hepatitis has emerged as a serious problem in almost all the Amerindian communities studied in the Amazon Basin and in other Amazon-related ecological systems from the North and Center of South America. Studies performed on communities from Bolivia, Brazil, Colombia, Peru and Venezuela have shown a high endemicity of the hepatitis B virus (HBV) infection all over the region, which is frequently associated to a high prevalence of infection by hepatitis D virus among the chronic HBV carriers. Circulation of both agents responds mainly to horizontal virus transmission during childhood through mechanisms that are not fully understood. By contrast, infection by hepatitis C virus (HCV), which is present in all the urban areas of South America, is still very uncommon among them. At the moment, there is not data enough to evaluate properly the true incidence that such endemicity may have on the health of the populations affected. Since viral transmission might be operated by mechanisms that could not be acting in other areas of the World, it seems essential to investigate such mechanisms and to prevent the introduction of HCV into these populations, which consequences for health could be very serious.

Multicenter Proficiency Testing of Nucleic Acid Amplification Methods for the Detection of Enteroviruses

ABSTRACT A multicenter study of molecular detection of enteroviruses was conducted using a proficiency panel. Of 70 data sets, 46 (66%) reported correct results for samples containing at least 1 50% infective dose per ml and for negative samples. Variation in performance between laboratories demonstrates the need for ongoing quality control.

Prevalencia de las infecciones por virus de las hepatitis B, C, D y E en Bolivia

En Bolivia no se han realizado estudios especificos sobre los virus de la hepatitis, por lo que su prevalencia y patrones de circulacion son practicamente desconocidos. De 1992 a 1996 se realizo un estudio seroepidemiologico con el fin de adquirir una primera vision de conjunto sobre las prevalencias de las infecciones por virus de la hepatitis B (VHB), C (VHC), D (VHD) y E (VHE) en distintas poblaciones de Bolivia. Sobre la base de los datos obtenidos en otros lugares de America Latina, se presto atencion especial al estudio de las comunidades autoctonas de la region amazonica. En las zonas rurales del altiplano andino, la infeccion por VHB presento una prevalencia general que corresponderia a una situacion de endemia media o baja (11,2%) y no se encontro ningun portador de anticuerpos contra VHC o VHD. En dos poblaciones de alto riesgo de la ciudad de Cochabamba (ninos sin hogar y trabajadoras del sexo), la prevalencia de infeccion por VHB fue similar (11,6%) y podria considerarse baja en comparacion con la de otras poblaciones analogas de nucleos urbanos en America Latina. La correspondiente al VHC (un caso positivo, 0,5%) seria parecida a la descrita en esas mismas poblaciones, si bien el escaso numero de muestras estudiadas no permite extraer conclusiones mas firmes. En concordancia con observaciones anteriores de comunidades similares de zonas tropicales de Suramerica, en las poblaciones autoctonas de la Amazonia boliviana la infeccion por VHB es sumamente endemica (prevalencia general de 74,0%), pero no se ha detectado la circulacion de VHC. Se sabe que la transmision de VHB es horizontal y tiene lugar desde edades muy tempranas, pero se desconocen los mecanismos de esa actividad. A los 10 anos de edad, mas de la mitad de la poblacion ya ha experimentado la infeccion natural que, 10 anos mas tarde, se habra difundido a practicamente toda la poblacion. La tasa muy baja de individuos positivos al HbsAg (1,6%), la ausencia de ADN virico en las muestras con reactividad aislada a anti-HBc y la alta prevalencia de anti-HBs entre los individuos que presentan marcadores de infeccion natural (92,4%) excluyen la participacion de la transmision vertical en el mantenimiento de la endemia. Hasta el momento, no se ha documentado ningun brote de infeccion por VHD en estas comunidades, pero la alta endemia de infeccion por VHB alerta sobre el riesgo de posibles brotes en el futuro. Los resultados obtenidos con las pruebas de anticuerpos contra VHE sugieren que este virus circula ampliamente en Bolivia y que podria haber producido brotes recientes en el departamento de Cochabamba. Se recomienda vacunar contra VHB en las poblaciones endemicas como medida de corto plazo; buscar activamente en todo el pais brotes y casos esporadicos de hepatitis E y continuar realizando estudios que permitan evaluar las repercusiones sanitarias de la situacion documentada en este estudio.In Bolivia, no studies have been carried out specifically on hepatitis viruses. Thus, their prevalence and circulation patterns are virtually unknown. A seroepidemiologic study was performed from 1992 to 1996 to generate a preliminary idea of the overall prevalence of infection from hepatitis B, C, D, and E viruses (HBV, HCV, HDV, and HEV, respectively) in different Bolivian population groups. Prompted by the data obtained in other areas of Latin America, the study focused on indigenous communities in the Amazon region. In rural areas of the high Andean plateau, HBV infection showed an overall prevalence compatible with medium to low endemicity (11.2%), and no carriers of HCV or HDV antibodies were found. In two high-risk groups in the city of Cochabamba (homeless children and sexual workers), the prevalence of HBV infection was similar (11.6%) and could be considered low by comparison to that of similar population groups in Latin American urban centers. The prevalence of HCV (one positive case, or 0.5%) was similar to that found in similar population groups, although the small number of samples precludes drawing more definite conclusions. As has been noted previously with similar communities in tropical areas of South America, HBV infection is highly endemic in indigenous populations of the Bolivian Amazon (with an overall prevalence of 74.0%), but circulation of HCV has not been detected. It is a well-known fact that HBV is horizontally transmitted and that transmission can take place very early in life, but the mechanisms involved are unknown. By 10 years of age, more than half the population has already had the natural infection that, in approximately 10 more years will have affected virtually the entire population. The very low rate of positivity to HBsAg (1.6%), the absence of viral DNA in samples showing isolated positivity to anti-HBc, and the high prevalence of anti-HBs among individuals who show markers for natural infection (92.4%) suggest vertical transmission plays no role in persistent endemicity. So far, no outbreak of HDV infection has been documented in these communities, but the high endemicity shown by HBV points to the possibility of future outbreaks. Results obtained with tests for the detection of antibodies against HEV suggest that this virus is circulating widely in Bolivia and that it could have caused recent outbreaks in Cochabamba state. Vaccination against HBV in endemic populations is recommended as a short-term measure. Also recommended are actively searching for outbreaks and sporadic cases of hepatitis E in the entire country and performing additional research that will help in assessing the public health consequences of the situation described in this article.

Detection of hepatitis B virus variants resistant to lamivudine and famciclovir among randomly selected chronic carriers from Spain

BACKGROUND Treatment for chronic hepatitis B with lamivudine is often hampered by the emergence of point mutations in the YMDD motif of the HBV DNA polymerase gene that confer drug resistance. This usually occurs after several months of therapy, but early detection of lamivudine-resistant mutants has been reported among patients in South Korea. Data from Japan and France suggest that naturally occurring, lamivudine-resistant hepatitis B virus (HBV) variants can be found among chronic carriers who have never received lamivudine treatment. Famciclovir can be used as an alternative when lamivudine-resistant variants emerge, though the substitute treatment may also give rise to the emergence and selection of drug-resistant variants. METHODS The presence of mutations related with lamivudine and famciclovir resistance was studied in serum samples from 79 randomly selected Spanish HBV carriers, using a line probe assay (LiPA) on HBV genome fragments amplified by polymerase chain reaction. Data concerning antiviral therapy prior to sampling were available for these patients. RESULTS Mutations related with resistance to either drug were detected in ten patients. Three of them (3.8% of the 79 carriers studied) had no record of prior lamivudine or famciclovir treatment at the time of sampling. Wild-type strains together with either the rtM204I (M552I) or rtV207I (V555I) point mutation were found in two of these cases, and the rtV207I mutation alone was detected in the third. CONCLUSIONS These findings seem to indicate that lamivudine and famciclovir-resistant variants circulate among Spanish HBV carriers. Since it is expected that antiviral therapy will be ineffective when drug-resistant variants are present before the beginning of treatment, it could be beneficial to test for these variants as an additional routine procedure when designing antiviral therapy on an individual basis.

Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS® HBsAg assay

The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS® HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty‐seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS® HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118–120, 125–127, and 143–145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (<25 S/CO units) was, in addition, recorded in samples containing mutants 143L (2 samples) and 115N + 120Q + 131K + 144A (1 sample). Testing samples at the 1/40 dilution by the modified assay did not produce, in contrast, false negative results, and reactivity below 25 S/CO units was recorded only in three cases. These results confirm that the capability of immunoassays to detect the presence of natural HBsAg mutants in clinical samples may be improved significantly by introducing changes in their design, and show that such improvement has been achieved successfully with the new VITROS® HBsAg ES Assay. J. Med. Virol. 80:598–602, 2008.