Transmission Pattern of Measles Virus Circulating in China During 1993-2021: Genotyping Evidence Supports That China Is Approaching Measles Elimination.

BACKGROUND: To provide useful insights into measles elimination progress in China, measles surveillance data were reviewed, and the transmission patterns of measles viruses circulating in China during 1993-2021 were analyzed. METHODS: Measles incidence data from the National Notifiable Disease Reporting System of the China Center for Disease Control and Prevention were analyzed. A total of 17 570 strains were obtained from 30 of 31 provinces in mainland China during 1993-2021. The recommended genotyping window was amplified. Genotyping analysis was conducted for comparison with the reference strains. Phylogenetic analyses were performed to identify genetic relationships among different lineages within the genotypes. RESULTS: With high coverage of routine immunization and intensive supplementary immunization activities, measles incidence has shown a downward trend since 1993, despite 2 resurgences, reaching a historic low level in 2020-2021 (average 0.5 per million). During 1993-2021, 9 genotypes including domestic genotype H1; imported genotypes B3, D4, D8, D9, D11, G3, and H2; and vaccine-associated genotype A were identified. Among them, the genotype H1 strain circulated endemically in China for more than 25 years; the last strain was detected in Yunnan Province in September 2019. Multiple imported genotypes have been identified since 2009 showing different transmission patterns. Since April 2020, no imported strains have been detected, while vaccine-associated genotype A continues to be detected. CONCLUSIONS: The evidence of low incidence during 2020-2021 and virological surveillance data in this study confirm that China is currently approaching measles elimination.

Nationwide distribution of varicella-zoster virus clades in China.

BACKGROUND: In 2010, a universal nomenclature for varicella-zoster virus (VZV) clades was established, which is very useful in the monitoring of viral evolution, recombination, spread and genetic diversity. Currently, information about VZV clades has been disclosed worldwide, however, there are limited data regarding the characterization of circulating VZV clades in China, even where varicella remains widely epidemic. METHODS: From 2008 to 2012, clinical samples with varicella or zoster were collected in General Hospital in eight provinces and analyzed by PCR, restriction endonuclease digestion and sequencing. The viral clades were determined by analysis of five single nucleotide polymorphisms (SNPs) within the 447-bp fragment of open reading frame (ORF) 22, and the restriction fragment length polymorphisms (RFLPs) of ORF 38 (PstI), ORF 54 (BglI) and ORF 62 (SmaI) were evaluated to understand genetic diversity of VZV and determinate varicella vaccine adverse event (VVAE). RESULTS: Seventy-seven varicella and 11 zoster samples were identified as being positive for VZV. The five SNPs profile showed that the majority of VZV strains belonged to clade 2, but clade 5 and clade 4 strains were also found in Guangdong. The RFLPs analysis of the DNA fragments of ORF 38, 54 and 62 showed that 85 of these samples were characterized as PstI + BglI + SamI-, and the remaining three VZV strains from varicella patients were characterized as PstI-BglI + SamI+ which is the genetic profile of VVAEs. CONCLUSIONS: The study suggested that the predominant clade 2 VZVs had been continually circulating since at least the 1950s in China. Nearly all VZV strains except VVAEs possessed the genetic profile of PstI + BglI + Sam-. However, the other clades were also found to be co-circulating with clade 2, especially in the border regions. These results highlighted the need for the constant and broad use of virologic surveillance to provide an important genetic baseline for varicella control and vaccination programs in China.

Functional and structural characterization of neutralizing epitopes of measles virus hemagglutinin protein.

Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.

Emergence and continuous evolution of genotype 1E rubella viruses in China.

In China, rubella vaccination was introduced into the national immunization program in 2008, and a rubella epidemic occurred in the same year. In order to know whether changes in the genotypic distribution of rubella viruses have occurred in the postvaccination era, we investigate in detail the epidemiological profile of rubella in China and estimate the evolutionary rate, molecular clock phylogeny, and demographic history of the predominant rubella virus genotypes circulating in China using Bayesian Markov chain Monte Carlo phylodynamic analyses. 1E was found to be the predominant rubella virus genotype since its initial isolation in China in 2001, and no genotypic shift has occurred since then. The results suggest that the global 1E genotype may have diverged in 1995 and that it has evolved at a mutation rate of 1.65 × 10(-3) per site per year. The Chinese 1E rubella virus isolates were grouped into either cluster 1 or cluster 2, which likely originated in 1997 and 2006, respectively. Cluster 1 viruses were found in all provinces examined in this study and had a mutation rate of 1.90 × 10(-3) per site per year. The effective number of infections remained constant until 2007, and along with the introduction of rubella vaccine into the national immunization program, although the circulation of cluster 1 viruses has not been interrupted, some viral lineages have disappeared, and the epidemic started a decline that led to a decrease in the effective population size. Cluster 2 viruses were found only in Hainan Province, likely because of importation.

Hepatitis B virus with a proposed genotype I was found in Sichuan Province, China.

To date, eight hepatitis B virus (HBV) genotypes, A-H, have been designated, and two additional genotypes, I and J, have also been proposed. A serological survey targeting children in difficult-to-reach vaccination areas was carried out in remote counties of Sichuan Province, China. HBV genotypes and serotypes were also determined from HBsAg-positive serum samples by direct sequencing. Phylogenetic analysis showed two strains isolated from the Yi ethnic children clustered with the proposed genotype I. The pairwise genome genetic distance was 7.5% between genotypes I and C, and ranged from 8.4% to 15.2% between genotype I and other genotypes, except genotype C. Grouping Scan analyses of the two strains revealed apparent recombination events between an unknown genotype and genotype C. Two out of four HBV strains isolated from the Yi ethnic children were confirmed to be genotype I, suggesting widespread circulation and common infection with genotype I HBV in the local Yi population. High prevalence of HBsAg and low hepatitis B vaccination coverage indicated that additional efforts are needed to control HBV infection in those areas.

Rubella virus genotypes in the People's Republic of China between 1979 and 2007: a shift in endemic viruses during the 2001 Rubella Epidemic.

The incidence of rubella cases in China from 1991 to 2007 was reviewed, and the nucleotide sequences from 123 rubella viruses collected during 1999 to 2007 and 4 viral sequences previously reported from 1979 to 1984 were phylogenetically analyzed. Rubella vaccination was not included in national immunization programs in China before 2007. Changes in endemic viruses were compared with incidences of rubella epidemics. The results showed that rubella epidemics occur approximately every 6 to 8 years (1993/1994, 2001, and 2007), and a shift of disease burden to susceptible young adults was observed. The Chinese rubella virus sequences were categorized into 5 of the 13 rubella virus genotypes, 1a, 1E, 1F, 2A, and 2B; cocirculations of these different genotypes were found in China. In Anhui province, a shift in the predominant genotype from 1F and 2B to 1E coincided with the 2001 rubella epidemic. This shift may have occurred throughout China during 2001 to 2007. This study investigated the genotype distribution of rubella viruses in China over a 28-year period to establish an important genetic baseline in China during its prevaccination era.

A novel nucleotide insertion in S gene of hepatitis B virus in a chronic carrier.

Hepatitis B virus DNA was extracted from serum of a chronic carrier and polymerase chain reaction was performed on S gene. Direct sequencing showed a variant HBsAg with additional 4-amino acid insertion, and clone sequencing confirmed the mixture of variant HBsAg and wildtype HBsAg. Of 16 clones with 12-nucleotide insertion, 15 clones had identical AGAACAACACAA insertion between nucleotide 494 and nucleotide 495, and one clone had GGAACAACTCAA insertion in the same position plus 3-nucleotide deletion from nucleotide 491 to nucleotide 493. S114T, C121Y, T126S/A, Q129K, G130R, T131N, M133T, G145R, N146D substitution and premature stop codon were also found in those clones. However, the origin of HBV with 4-amino acid insertion in HBsAg was unclear.

Genetic characterization of measles viruses in China, 2004.

Genetic characterization of wild-type measles virus was studied using nucleotide sequencing of the C-terminal region of the N protein gene and phylogenetic analysis on 59 isolates from 16 provinces of China in 2004. The results showed that all of the isolates belonged to genotype H1. 51 isolates were belonged to cluster 1 and 8 isolates were cluster 2 and Viruses from both clusters were distributed throughout China without distinct geographic pattern. The nucleotide sequence and predicted amino acid homologies of the 59 H1 strains were 96.5%-100% and 95.7%-100%, respectively. The report showed that the transmission pattern of genotype H1 viruses in China in 2004 was consistent with ongoing endemic transmission of multiple lineages of a single, endemic genotype. Multiple transmission pathways leaded to multiple lineages within endemic genotype.

Molecular epidemiology of measles viruses in China, 1995-2003.

This report describes the genetic characterization of 297 wild-type measles viruses that were isolated in 24 provinces of China between 1995 and 2003. Phylogenetic analysis of the N gene sequences showed that all of the isolates belonged to genotype H1 except 3 isolates, which were genotype A. The nucleotide sequence and predicted amino acid homologies of the 294-genotype H1 strains were 94.7%-100% and 93.3%-100%, respectively. The genotype H1 isolates were divided into 2 clusters, which differed by approximately 2.9% at the nucleotide level. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Even though other measles genotypes have been detected in countries that border China, this report shows that genotype H1 is widely distributed throughout the country and that China has a single, endemic genotype. This important baseline data will help to monitor the progress of measles control in China.

Mumps Epidemiology and Mumps Virus Genotypes Circulating in Mainland China during 2013-2015.

With the implementation of mumps virus (MuV) vaccination in the expanded program on immunization (EPI) in mainland China since 2008, the incidence of mumps has decreased, and the natural epidemic pattern of mumps has slightly changed during 2013-2015. The two epidemic peaks (April-July and November-December) became less obvious than those observed from 2004 to 2012. Children and adolescents younger than 15, particularly in the five-to-nine-year-old age group, remain the target group and should be the focus of high-quality immunization activities in mainland China. However, it was also found that the incidence and reported cases of mumps decreased in each age group during 2013-2015, particularly in the five-to-nine-year-old and ten-to-fourteen-year-old age groups. The proportion of mumps cases among adults in some provinces also increased. Unlike the changes in the epidemiological characteristics of mumps affected by vaccination, the data of MuV virology surveillance indicated that most of the MuV transmission chains have not yet been effectively interrupted, and MuV remains a natural epidemic pattern in mainland China. In the MuV virology surveillance, 194 MuV strains during 2013-2015 were isolated from 10 of 31 provinces in mainland China. Based on the phylogenetic analysis of the small hydrophobic (SH) gene, both genotype F (99.0%) and G (1.0%) were identified, and genotype F was still the predominant genotype continuously circulating in mainland China. Representative genotype F and G strains isolated in China from 1995 to 2012 were selected for further analysis. The results indicated that there were multiple transmission chains within genotype F, with no obvious geographical or time differences. The high genetic diversity of genotype F strains could be a result of the continuous transmission and evolution of the MuV in mainland China. Genotype G was also detected in four provinces in mainland China. Because of the limited epidemiological data, it was uncertain whether the genotype G MuV strains found in 2011 and 2013 were imported from other countries. Therefore, combined high-quality epidemiological and virological surveillance is necessary for mumps control; it can also be used to observe the changes in epidemiological characteristics and viral transmission of mumps over time after mumps-containing vaccine (MuCV) implementation and to provide a comprehensive epidemiological and genetic baseline for mumps elimination in mainland China.

Evolutionary analysis of mumps viruses of genotype F collected in mainland China in 2001-2015.

Mumps incidence in mainland China remains at a high level. Genotype F has been the predominant genotype of mumps virus (MuV) in the last 20 years in mainland China. To better understand the genetic characteristics of MuV in China, the sequences of the Small Hydrophobic (SH), Hemagglutinin-Neuraminidase (HN) and Fusion (F) genes of MuVs of genotype F collected during 2001-2015 were determined. The evolutionary rates of the HN and F genes were similar (0.5 × 10-3 substitutions/site/year) whereas the SH gene evolutionary rate was three times faster. The most recent common ancestor of genotype F was traced back to 1980. Four lineages were identified within HN and F MuV sequences. A phylogeographic analysis indicated that the genotype F viruses originally spread from the Liaoning and Shandong provinces followed by a spread to the South and East of China. This study provides important genetic baseline data for the development of prevention and control measures of mumps.

Evolutionary analysis of rubella viruses in mainland China during 2010-2012: endemic circulation of genotype 1E and introductions of genotype 2B.

Rubella remains a significant burden in mainland China. In this report, 667 viruses collected in 24 of 31 provinces of mainland China during 2010-2012 were sequenced and analyzed, significantly extending previous reports on limited numbers of viruses collected before 2010. Only viruses of genotypes 1E and 2B were found. Genotype 1E viruses were found in all 24 provinces. Genotype 1E viruses were likely introduced into mainland China around 1997 and endemic transmission of primarily one lineage became established. Viruses reported here from 2010-2012 are largely in a single cluster within this lineage. Genotype 2B viruses were rarely detected in China prior to 2010. This report documents a previously undetected 2B lineage, which likely became endemic in eastern provinces of China between 2010 and 2012. Bayesian analyses were performed to estimate the evolutionary rates and dates of appearance of the genotype 1E and 2B viral linages in China. A skyline plot of viral population diversity did not provide evidence of reduction of diversity as a result of vaccination, but should be useful as a baseline for such reductions as vaccination programs for rubella become widespread in mainland China.

Epidemiologic and genetic characteristics of mumps viruses isolated in China from 1995 to 2010.

The epidemiologic and genetic characteristics of mumps viruses detected in China from 1995 to 2010 were analyzed in this study. Mumps remains endemic in China with a high overall incidence rate. The incidence of mumps in Western China was higher than that in other regions of the country. Each year, most of mumps cases occurred between April and July, but a small peak also occurred in November and December. Mumps cases primarily affected the under 15 year old age group. Virologic data demonstrated that genotype F was the predominant circulating genotype throughout China for at least 15 years and no other genotype was detected between 1995 and 2010. Analysis of sequence data from the small hydrophobic (SH) gene indicated that multiple transmission chains of genotype F were found in various provinces of China, with no apparent chronologic and geographic restriction. This is the first report describing the epidemiology of mumps and genetic characterization of mumps viruses at the national level in China.

Genetic characterization of the hemagglutinin genes of wild-type measles virus circulating in china, 1993-2009.

BACKGROUND: China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies. PRINCIPAL FINDINGS: Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993-2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10(-3) substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure. CONCLUSIONS: Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.

[Analysis on mutation of S gene and P gene of hepatitis B virus in two counties of Sichuan Province].

OBJECTIVE: To analyze HBV S gene/P gene mutation in 2 counties (districts) of Sichuan province. METHODS: HBV DNA were extracted from sera positive both for HBsAg and HBeAg. After PCR and nucleotide sequencing, nucleotide/amino acid mutation in S and P gene were compared and analyzed. RESULTS: Of 47 serum samples from patients with chronic HBV infection, amino acid mutation in 'a' determinant occurred in 12 samples (25.53%,12/47), correlating with T126A, I126T/S, P127T, T131N, M133L, M133T and T140I; high proportion of mutation clustered in first loop of 'a' determinant (92.86%,13/14), rtV207I mutation in C domain of reverse transcription occured in one sample. CONCLUSION: Naturally occurring mutation in 'a' determinant clustered predominantly in the first loop and usually associated with altered antigenicity, posing a potential threat to successfully vaccinated individuals; Lamivudine-resistant mutant might occur in patient even without nucleotide analogue treatment.

[D9 measles virus was first isolated from an imported measles case in Sichuan Province of China].

OBJECTIVE: To isolate and identify the genotype and molecular characteristic of an imported D9 measles virus. METHOD: To isolate the virus with Vero/SLAM cell line. And RT-PCR (reverse transcription-polymerase chain reaction) were performed to amplify the 450 nucleotide acids of carboxyl terminal of N (nucleoprotein, N) protein. The phylogenetic tree was constructed and the homology similarity was analyzed. RESULTS: Sichuan isolate MVi/Sichuan.CHN/07.09/1 was clustered within the same genotype group with WHO D9 genotype reference strain, the homology of nucleotide acid between Sichuan isolate and WHO D9 genotype reference strain was 96.9%. The homology of nucleotide acid and amino acid between Sichuan isolate and 2008 D9 genotype representative strain were 99.8%-100% and 99.3%-100% respectively. Compared with the Chinese measles vaccine strain, the homology of nucleotide acid and amino acid of Shanghai-191 were 92.3% and 90.7% respectively. Compared with the endemic H1 genotype representative measles strain, the homology of nucleotide acid and amino acid were 90.8% and 92.1% respectively. CONCLUSION: The imported virus was D9 genotype measles virus.

[A seroepidemiologic analysis of hepatitis B in Sichuan province].

OBJECTIVE: To understand the changes of hepatitis B infection rates, before and after the hepatitis B vaccine was included into EPI, and to evaluate the effect of immunization which would lead to the development of a more appropriate hepatitis B control strategy. METHODS: Seroepidemiologic method, with multi-section random sampling method were chosen. 14 sites from 8 counties were involved. 2-4 ml of the vein blood was drawn from all the individuals engaged in the study including 3806 samples. HBsAg, anti-HBs, anti-HBc of the samples were tested with ELISA. RESULTS: Standardized positive rates of HBsAg and HBsAb were found as 7.05% and 29.77% respectively with the overall infection rate of HBV as 40.30%. The hepatitis B vaccine coverage of the children under 15 years was 70.73% and the positive rates for both HBsAg and anti-HBs were 2.62% and 56.68%, respectively. The coverage of hepatitis B vaccine among children under 3 years was 83.44% and the positive rates of both HBsAg and anti-HBs were 1.47% and 67.69% respectively, hepatitis B vaccine coverage of children under 3 years was 85.77%, with positive rates of HBsAg and anti-HBs as 1.78% and 75.44% respectively. CONCLUSION: Results from our study revealed that since the introduction of hepatitis B vaccination, the prevalence rates of HBsAg and HBV infection had an obvious decline, especially in children aged under 15 years of old, suggesting that some changes had occurred in the epidemic characteristics of hepatitis B in Sichuan.

[Genetic characterization of Chinese rubella virus isolates from 2003 to 2007].

57 rubella virus strains were isolated using Vero cell line or Vero/SLAM cell line from patients' throat swabs during rubella outbreaks and sporadics in 10 provinces of China from 2003 to 2007. Fragments of 1107 nucleotides of E1 genes of the isolates were amplified by RT-PCR, the PCR products were directly sequenced and analyzed. The phylogenetic analysis based on 739 nucleotides showed that out of 57 Chinese rubella virus strains, 55 belong to a distinguish branch of 1E genotype when comparing with 1E genotype rubella strains from other countries, and the other 2 Chinese rubella virus strains belong to 2B genotype. Most of the nucleotide mutations of 57 rubella viruses were silent mutations, and the amino acid sequences were highly conserved. Except one amino acid change (Thr212 --> Ser212) in two rubella viruses at the hemagglutination inhibition and neutralization epitopes, there had no change found at the important antigenic epitope sites of the other rubella viruses. 1E genotype rubella viruses isolated from 10 provinces of China from 2003 to 2007, and two imported 2B genotype rubella viruses from Vietnam suggested that 1E genotype was the predominant genotype in this period of time. The rubella virus genotypes circulated during 2003 to 2007 were different from that circulating during 1979 to 1984 and 1999 to 2002, the rubella prevailed in recent years was mainly caused by 1E genotype rubella viruses with multi-transmission routes.