Evolution of rubella viruses following long term endemic circulation Zhen Zhu / Wenbo Xu National Measles/Rubella Laboratory, CCDC Regional Reference Laboratory for Measles/Rubella, WPRO Accelerating Progress towards Measles/Rubella Control and Elimination Goals Hotel Royal, Geneva, Switzerland, 21-23 June 2016

Outline Rubella epidemiological status Rubella virological surveillance in China Characterization of the full-length genomic sequences of Chinese rubella viruses Challenge

China rubella cases during 2004-2015 Rubella vaccine was introduced into EPI Rubella was formally integrated into case-based measles surveillance Rubella incidence showed a gradually decreasing trend since 2008, and reached a lowest level in 2015. Epidemic rubella cycles in China is about every 7 to 8 years, and it may change due to the introduction of the RCV.

The age group with the rubella infection changed significantly The reported rubella cases were mainly concentrated in those under 15 years of age during 2004-2013, and the proportion of this age group decreased in 2014 and 2015, and only about 42.75% in 2015. The proportion of 15-39 age group increased gradually, and about 53.66% in 2015.

Rubella virus isolates collected in China LabNet 1999~2015：1737 from 29 provinces 1E:1027；1F:15；2B: 691；2A: 4

The prevalence trends of RV genotypes in China during 1999 - 2015 Genotype 1E, instead of genotype 1F, became the predominant genotype during 2001-2013; 1F viruses were not found after 2002; Genotype 2B viruses have been sporadically detected before 2010, while the detection rate of genotype 2B gradually increased since 2011 and become the predominant during 2014-2015.

Evolution of genotype 1E rubella virus between 2001 and 2015 Almost all Chinese 1E viruses from mainland China grouped into a single lineage. Two closely related clusters (Cluster A and Cluster B) could be identified within the Chinese lineage； Cluster B seems to have been replaced by Cluster A in recent years；

Genotype 1E rubella virus in 2015 Cluster A (2002-2015) Cluster B (2001-2009) Heilongjiang Hunan Tianjin 4 genotype 1E rubella virus strains in 2015 belong to three different transmission chains Rubella virological surveillance should be strengthened.

The prevalence of genotype 2B rubella virus in China between 2000 and 2015 2B-lineage3 virus first detected in 2011 share a high similarity with viruses from the Vietnam and Japan outbreaks At least four lineages（Lineage 1-4）were found during 2000-2015 in China; 2B-Lineage 3 viruses group to a Cluster C, which has been found to have a wide geographic distribution from 2006 to the present. Outbreaks could be considered as a multi-country epidemic 2B-lineage3 become epidemic in China since 2011

The detection rate of genotype 2B increased since 2011 9 provinces, 12.6% 10 provinces, 32.9% 9 provinces, 43.8% Since 2011, 2B-lineage3 virus has been found in 23 provinces； Genotype 2B becomes the predominant in China. The genotype replacement occurred again after 2001. 13 provinces，63.6% 15 provinces，98.7%

Circulating pattern of genotype 2B in China during 2000-2015 2011 2012 2013 2014 2015 2008 2000 2006 2008 Unpublished data

Representative RV strains were selected for whole genome sequencing

Genetic Characterization of Genotype 1F Virus To date, genotype 1F has been found only in China; Both sequences were found to be 9,761 nts in length with 1-nt deletion occurred in nt 6,476 within the IR. No amino acid variations were found at positions where functional domains of the proteins were identified. 42 nucleotide and 7 amino acids specific variations were identified in 1F viruses； Genotype 1F could be traced back to 1987, and it disappeared after 2002. NSP: 33 nts SP:9 nts NSP: 7 aa 1987

Genetic Characterization of Genotype 1E and 2B Viruses Variability at coding positions in different domains Average genetic distance in different domains E1 Most nucleotide substitutions are transitional and occur at the 3rd codon positions; the highest nucleotide variability was found in HVR-II (nt 997-1031) in p150，The most conserved region was the MT domain E1 was the most conserved among the three SP proteins Unpublished data

Specific nucleotides (nt) and amino acids (aa) substitutions in Chinese genotype 1E RVs C1021T G3709A W5521G C5902T Y8623A R4900C R1424T C2413T C2893T M3538T Y4753G R5236C Y7303A C3691T R5428T M3712G C5431T (G/S/V)462C T2796C Unpublished data

Specific nucleotide (nt) and amino acids (aa) substitutions of 1E-ClusterA and 1E-ClusterB before and after BRDII vaccination in 2008 NSP：44 NT variations SP：19 NT variations 1E-A NSP：4 AA variations (Y454F, G462C, S725G, R930C) SP：3 AA variations (P32S, I8M, L117P) NSP：116 NT variations SP：58 NT variations 1E-B NSP：9 AA variations (H470Q, G682A, H716Q, R739H, T798V, P881L, L1017S, T1019P, N1093K) SP：6 AA variations (P89S, P101S, M253T, V57L, A337T, I380F) No amino acid variations were found among the important functional domains. Unpublished data

Specific nucleotides (nt) and amino acids (aa) substitutions in Chinese genotype 2B RVs 56 nt variations，16 aa variations 20 nt varations，4 aa variations 15 nt variations ，2 aa variations No amino acid variations were found among the important functional domains. Unpublished data

Challenges Though the important genetic baseline data had been established in China, surveillance gap still existed (Xinjiang and Tibet）. Continuous virological surveillance should be carried out in all provinces. The shipment of rubella viruses in some of the provinces was delayed because of the airport transportation certification issue. For some provinces, need to timely transport the viruses strains to national lab. Real-time RT-PCR method as routine detection method has been introduced into labnet, and routine quality control should be also included. Rubella virus surveillance should be strengthened during the measles elimination stage.

Acknowledgements US CDC(Joe Icenogle; Paul Rota; Emily Abernathy; Pierre Rivailler; Min-hsin Chen, and rubella team), WHO HQ, WPRO, WHO China NIP of China, IVDC, China CDC, MOH All the staffs from measles/rubella LabNet and EPI in China

Thank you!