Virus Hepatitis B y C: Virología aplicada Conceptos de genotipos, cuasiespecies, mutantes Epidemiología molecular en Argentina Jorge Quarleri Instituto de Investigaciones Biomédicas en Retrovirus y Sida UBA-CONICET

¿Qué los une? Virus envueltos Vías de transmisión Tropismo celular Evolución a cronicidad Polimerasas con errores  MUTANTES  variabilidad Evasión a la respuesta inmune Proteínas virales con capacidad trans-activadora  carcinoma hepático

¿Qué los distingue? Tipo de genoma: RNA (HCV); DNA (HBV) Cinética inicial de replicación: más lenta en HBV Uso de transcripción reversa (sólo HBV) Capacidad de integración al genoma celular (sólo HBV) Tasa de evolución a la cronicidad (85% para HCV y <2% para HBV) Reservorio intracelular (sólo HBV, forma DNAccc) Cura con nuevas terapias (sólo HCV) Vacuna (sólo HBV)

Estructura de los viriones Structures of hepatitis B and C virions. HBV (left) and HCV (right) belong to different viral families but have some similarity in basic ultrastructure. They are similar in size, with diameters of 42 and 50–60 nM, respectively. Both viruses are enveloped, with multiple viral surface proteins in the envelope, and both contain a capsid which is a polymer made up of a single viral core protein. However, the HBV genome resides within the capsid as a partially double-stranded DNA, whereas the HCV genome is present as a single strand of positive-sense RNA. Delaney WE 4th.; Antiviral Res. 2013

Organización genómica Genomic structures of HBV and HCV. HBV (top) and HCV (bottom) have substantially different genomic organization. The partially double-stranded DNA genome of HBV encodes four major overlapping reading frames (ORFs) in a compact 3.2 kilobase genome. The reading frames include the surface antigen ORF (purple), which gives rise to three related surface antigens (large, middle and small); the core and precore reading frames (blue), which give rise to HBcAg and HBeAg, respectively; the polymerase reading frame (green), which gives rise to the polymerase; and the X reading frame (black), which gives rise to the X protein. In contrast, the 9.6 kilobase single-stranded, plus-sense HCV RNA genome is directly translated into a single polyprotein, which is subsequently processed into 10 distinct viral proteins, including the structural proteins core, envelope 1 (E1) and envelope 2 (E2) and the nonstructural (NS) proteins, p7, NS2 protease, NS3/4 protease, NS4B, NS5A and the NS5B polymerase. Delaney WE 4th.; Antiviral Res. 2013

Ciclos de replicación POLIMERASA VIRAL DNAccc Re-infección Replication cycles of HBV and HCV (simplified view). HBV (left) and HCV (right) have substantially different replication strategies. HBV enters hepatocytes in a receptor-mediated process, is uncoated and delivers its DNA to the nucleus. The incomplete double-stranded genome is then completed and repaired to form cccDNA, which is maintained without active replication in the nucleus as a chromatinized episome. Transcription of cccDNA by host RNA polymerases gives rise to viral mRNA molecules which are exported to the cytoplasm and translated; the resulting proteins and pregenomic RNA assemble to form new nucleocapsids, which can either cycle back to the nucleus to amplify the copy number of cccDNA or acquire an envelope and be secreted as progeny virions. While HCV also enters the cell by a receptor-mediated process, its genome is delivered to the cytoplasm, where it is translated and viral proteins assemble to form replication complexes for genome replication. Within the replication complexes, negative-sense RNA strands are copied from the infecting genomes, and from these additional positive-stranded genomes are replicated. Progeny positive RNA strands can be encapsidated, acquire an envelope and leave the cell as progeny virions. Note that no nuclear or DNA form of the HCV genome is generated during infection. Delaney WE 4th.; Antiviral Res. 2013

A C T G Las polimerasas virales se equivocan, y no corrigen RNA polimerasa -RNA dependientes: sintetizan RNA desde un molde RNA (virus RNA tal como HCV) 2. DNA polimerasa RNA-dependiente que sintetizan DNA desde un templado RNA, comúnmente conocida como transcriptasa inversa (HIV, y otros retrovirus; HBV) A C T G Quarleri

Algunas cifras… 3.2-7.9 x 10-5 sustituciones nucleotídicas/ciclo 100 veces > otros DNA virus 1.6-6.2 x 10-5 sustituciones nucleotídicas/ciclo ~ RNA virus ~1010-11 mutaciones / día

¿Depende la variabilidad de HBV y HCV sólo de la pobre fidelidad de las polimerasas virales? Facilitadores Factores virales Polimerasa que comete errores y no los corrige (alta tasa de fijación de mutaciones) Intensa capacidad replicativa (1012 partículas virales/día) En HBV re-infección durante la replicación Recombinación Factores del hospedador En HBV la enzima celular APOBEC contribuye con la hipermutacionalidad Células blanco con vida media larga En HBV, genoma de pequeño tamaño y marcos abiertos de lectura solapados Mutantes defectivas Limitadores Quarleri

CUASIESPECIES VIRALES Población que evoluciona genéticamente como una distribución compleja de variantes que están cercanamente relacionadas pero no son idénticas

Inicial Resistente Bajo fitness Fitness Escape recuperado

Lok A et al, NEJM (2012):366 (3) 216.

MUTANTES Mutaciones puntuales Mutaciones puntuales Deleciones Inserciones Mutaciones puntuales Deleciones Inserciones Gen NS5B NS3/4A  resistencia a drogas NS5A epitopes  evasión Gen polimerasa  resistencia a drogas S  evasión Ac Gen promotor Core  altera expresión HBe preCore  X  capacidad trans-activadora

Supervivencia del más apto (fitness ) MULTIPLICACION VIRAL Drogas antivirales Presión de selección inmune Mutagénesis es replicación dependiente Mutaciones Diversidad genética (cuasiespecies) “Una droga antiviral es una droga que selecciona resistencia” D.Richman (2000) Hepatology 32, 866

Estamentos jerárquicos para la nomenclatura de la heterogeneidad viral Cuasiespecies

Genotipos, subtipos 8%: GENOTIPO 4-8%: SUB-GENOTIPO >25%: GENOTIPO Genetic distance between genotypes for HBV and HCV. HBV and HCV are each divided into multiple genotypes. A comparison of genetic distance between HBV and HCV genotypes is indicated, with nucleotide genetic distance drawn on the same scale. The analysis is based on full-length genome sequences using sequences available in GenBank. Alignments were performed using ClustaX and Maximum likelihood trees were inferered using PAUP v 4b10. Reproduced from (Ray and Howard, 2010), with permission. >25%: GENOTIPO 20-25%: SUB-GENOTIPO Ray SC, Thomas DL. In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases, 2010

Genotipos de HBV Infect Genet Evol. 2013 Jun;16:355-61. doi: 10.1016/j.meegid.2013.03.021. Epub 2013 Mar 26. Hepatitis B virus subgenotyping: history, effects of recombination, misclassifications, and corrections. Shi W1, Zhang Z, Ling C, Zheng W, Zhu C, Carr MJ, Higgins DG. Shi W, et al. (2013) Hepatitis B virus subgenotyping: history, effects of recombination, misclassifications, and corrections. Infect Genet Evol.  Quarleri

Distribución de genotipos de HBV en Argentina Franca et al., 2004, JCM 42 (11), 5015. Pezzano et al., 2011, Clin Microbiol Infect. 2011;17(2):223-31 Quarleri

Genotipos de HCV Hajarizadeh, B. et al. (2013) Epidemiology and natural history of HCV infection Nat. Rev. Gastroenterol. Hepatol. Quarleri

Genotipos: IMPLICANCIAS BIOLÓGICAS Distribución geográfica: si bien algunos están ampliamente distribuidos, otros se encuentran más restringidos a áreas geográficas definidas Pronóstico: mayor chance de evolución a cronicidad y/o cuadros de mayor severidad hepática Tasa de respuesta al tratamiento: Se ajustan tiempos de tratamiento Tasa de replicación viral Quarleri

Muchas gracias Contacto:. quarleri@fmed.uba.ar