Regional Meeting on Sentinel Surveillance of , Bacterial Meningitis and Pneumonia 09-11 December 2013 - Cancun, Mexico

Experience of the Rotavirus Regional Reference Laboratory

José Paulo Gagliardi Leite, Ph.D

Laboratory of Comparative and Environmental Virology Owaldo Cruz Institute – Oswaldo Cruz Foundation – MoH Training of Human Resources (1) Training of Human Resources (2)

Technical Visits / Local Technical Capacity

Nicaragua Paraguay Chile Bolivia Dominican Republic

Haiti = Fiocruz

Individual Training at Fiocruz: 06

Quality Control = EQA panel from WHO - Global Reference Laboratory – CDC

Work with Regional Rotavirus LabNet

NT strains

Genotyping Regional Samples

Quality Control Tests

WHO - Rotavirus Technical Working Group Rotavirus Genotype Distribution, Rotavirus Genotype Distribution, AFR, 2009-2012 AMR, 2009-2012

WHO, Serhan F - Updated on 30 Aug 2013 Untypeable strains

Mixed infections

Sequencing

VP4 , VP7, VP6 and NSP4 VP6

SEQUENCIACIÓN WHO – Global Reference Laboratory - CDC

One Step for VP7/VP4 + Sequencing “There is nothing permanent except change” Heraclitus

Webster et al. Microbiol. Rev., 56: 152 - 179, 1992 Research,

Development of New Methodologies & Protocols Tulio Fumian qPCR Rotavirus – NSP3 Norovirus - GII

Human & Immunotherapeutics, in press

Real-time RT-PCR assays to differentiate wild-type group A rotavirus strains from Rotarix® and RotaTeq® strains in stool samples

Rashi Gautam, Mathew D. Esona, Slavica Mijatovic- Rustempasic, Ka Ian Tam, Jon R. Gentsch and Michael D. Bowen. Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA WHO - Global Reference Laboratory MECHANISMS FOR ROTAVIRUS EVOLUTION (1)

Ponctual Mutation(s)

Rearrangements MECHANISMS FOR ROTAVIRUS EVOLUTION (2) Reassortments • Reassortment of RV-A strains - P and G antigen combinations - Intragenogroup reassortment of all genes

• Role of animal RV-A in diversity - Examples of interspecies infections - Human-animal RV-A reassortants

116E (P8[11]G9) India (10 HU/1 BO)

AU-1, Ro-5829 (P3[9]G3) Eg2295 (P[14]G8) Japan, Israel (11 FE) Egypt (BO/LA/HU?)

Br1054 (P[8]G5) Brazil (8 HU/3 PO ?) reassortments Is G and P typing enough to follow the variability of group A rotavirus strains after vaccine introduction? Wa-like

DS-1-like

AU-1-like

• Payne DC, Edwards KM, Bowen MD, Keckley E, Peters J, Esona MD, Teel EN, Kent D, Parashar UD, Gentsch JR (2010) Sibling Transmission of Vaccine-Derived Rotavirus (RotaTeq) Associated With Rotavirus Gastroenteritis. Pediatrics 125(2): e438-e441.

• Donato CM, Ch'ng LS, Boniface KF, Crawford NW, Buttery JP, Lyon M, Bishop RF, Kirkwood CD (2012) Identification of Strains of RotaTeq Rotavirus Vaccine in Infants With Gastroenteritis Following Routine Vaccination. Journal of Infectious Diseases206(3): 377-383.

• Bucardo F, Rippinger CM, Svensson L, Patton JT. Vaccine-derived NSP2 segment in from vaccinated children with gastroenteritis in Nicaragua. Infect Genet Evol. 2012;12:1282-94. RV1 = 1 DOSE

Reassortment events are possible between the human RVA derived vaccine RV1 and wild type strains and represent examples of this evolutionary process. Intra-genotypic reassortment Rotarix® vaccine x ES15221-08 (VP1)

Southeast VP2

unvaccinated child 42 months VP1 from Rotarix RVA Genotypes by Brazilian Region: 2009-2013 G2...... G3, G9, G12, P6 Northeastern Southeastern

Southern Genetic background of G12P[9] and G12P[8] rotavirus A strains detected in Brazil. Mariela Martínez Gómez*b, Hugo Reis Resquea, Eduardo de Mello Volotãob, Marcelle Figueira Marques da Silvab, Tatiana Lundgren Roseb, Mark Zellerc, Elisabeth Heylenc, Jelle Matthijnssensc, José Paulo Gagliardi Leiteb

Genotypes Strain Origin VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5 RVA/USA/Rotarix-A41CB052A/1988/G1P1A[8] Human G1 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/BRA/RJ12419/2006/G12P[8] Human G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/BRA/BA20142/2011/G12P[8] Human G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/BRA/BA20144/2011/G12P[8] Human G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZAF/3176WC/2009/G12P[6] Human G12 P[6] I1 R1 C1 M1 A1 N1 T1 E1 H1

Wa-like RVA/Human-wt/BGD/Matlab13/2003/G12P[6] Human G12 P[6] I1 R1 C1 M1 A1 N1 T2 E1 H1 RVA/Human-wt/ARG/6653/2008/G12P[8] Human G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/ZAF/3133WC/2009/G12P[4] Human G12 P[4] I1 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Porcine-wt/IND/RU172/2002/G12P[7] Porcine G12 P[7] I5 R1 C1 M1 A1 N1 T1 E1 H1 RVA/Human-wt/BGD/N26/2002/G12P[6] Human G12 P[6] I2 R2 C2 M2 A2 N1 T2 E6 H2 RVA/Human-wt/BGD/RV161/2000/G12P[6] Human G12 P[6] I2 R2 C2 M2 A2 N2 T2 E1 H2 RVA/Human-wt/BGD/RV176/2000/G12P[6] Human G12 P[6] I2 R2 C2 M2 A2 N2 T2 E6 H2 DS-1-like RVA/Human-tc/PHL/L26/1987/G12P[4] Human G12 P[4] I2 R2 C2 M1/M2 A2 N1 T2 E2 H1 RVA/Human-tc/THA/T152/1998/G12P[9] Human G12 P[9] I3 R3 C3 M3 A12 N3 T3 E3 H6 RVA/Human-wt/BRA/PE15776/2008/G12P[9] Human G12 P[9] I3 R3 C3 M3 A3 N3 T3 E3 H6 RVA/Human-wt/BRA/PE18974/2010/G12P[9] Human G12 P[9] I3 R3 C3 M3 A3 N3 T3 E3 H6

AU-1-like RVA/Human-wt/ARG/Arg720/1999/G12P[9] Human G12 P[9] A12 RVA/Human-wt/PRY/Py1135ASR07/2007/G12P[9] Human G12 P[9] E3 H6 G12P[9] and G12P[8] Brazilian strains revealed distinct genetic backgrounds: AU-1-like and Wa-like, respectively BRAZILIAN RVA STRAINS GENETIC BACKGROUND Genotypes Strain Origin VP7 VP4 VP6 VP1 VP2 VP3 NSP1 NSP2 NSP3 NSP4 NSP5 G3P[8] Human G3 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 (2005/2009/2011) (vaccinated/non-vaccinated children) G9P[8] (2001/2003/2004/2005/2006/2011) (vaccinated /non- Human G9 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 Wa-like vaccinated children) G12P[8] Human G12 P[8] I1 R1 C1 M1 A1 N1 T1 E1 H1 (2006/2011) (vaccinated/non-vaccinated)

G12P[9] Human G12 P[9] I3 R3 C3 M3 A3 N3 T3 E3 H6 (2008/2010) (vaccinated) AU-1-like G2P[4] (2005/2006/2007/2008/2009/2010/2011) (vaccinated/non- Human G2 P[4] I2 R2 C2 M2 A2 N2 T2 E2 H2 DS-1-like vaccinated children)

There is no evidence of interference by the RV1 vaccine in the evolution of the 11 genes from Brazilian strains G3P[8], G9P[8] G12P[8]; G12P[9]; G2P[4] genotypes

G3P[8], G9P[8], G12P[8] = Wa-like

G12P[9] = AU-1-like

G2 = DS-1-like

Evolutionary analysis of G1P[8] Rotavirus A strains in Brazil between 1986-2013

Structural Proteins Non-structural Proteins

There is no evidence of interference by the RV1 vaccine in the evolution of the 11 genes from Brazilian strains G1P[8] genotype over a 27-year period. P[8] lineages chronology in Brazil

RV1

RV1: P[8]-1 Most recent strains: P[8]-3

Silva et al., 2013 P[8]-3.2

P[8]-3.4

P[8]-3.6

P[8]-3.1 P[8]-3.3

P[8]-3.5 Conclusions: A clear shift in RVA genotypes distribution was observed between 2006 – 2013 in different countries.

G and P genotyping still important to characterize RVA strains.

There is no evidence of interference of P[8]-3 sub-lineage in the effectiveness of RV1.

There is no evidence of interference by the RV1 vaccine in the evolution of the 11 genes from Brazilian strains G1, G2, G3, G9, and G12.

Epidemiological information – vaccination data / status – is very important, as well as age, severity of infection etc..

G1P[8]: differentiation between wild type virus and RV1, and if necessary sequencing of 11 genes (reassortments)

G1-4P[8]: differentiation between wild type virus and RV5, and if necessary sequencing of 11 genes (reassortments)

Emerging and unusual binary combination genotypes: G12, G8, G10, P9, G3/G4/G9 P[6[/[4]/[9]: sequencing of 11 genes (animal x human origin)

RVA gene constellation: sequencing X sequencing analysis (Bioinformatics' Support) Laboratory of Comparative and Environmental Virology

6 4 1 5 2 3

8

7 9

Rotavirus Group: Alexandre (1), Tatiana (2), Rosane (3), Eduardo (4), Mariela (5), Hugo (6), Yasmin (7), Carol (8), Marcelle (9). VPSRMA- Fiocruz/MS

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