The rubella epidemiological data collected in this study indicate that the high prevalence of rubella in China alternated between different regions and that the endemicity continuously occurred in areas covering eastern, central, and western portions of the country. Overall, the incidence of rubella in western China was significantly higher than that in other regions.
Reported rubella cases were concentrated in those under 15 years of age, most of whom were primary and middle school students. However, in eastern China, the proportion of rubella cases in those less than 15 years of age decreased significantly, and the proportion within the 15- to 39-year-old age group increased. This may be due to the introduction of the rubella vaccine into some provinces in eastern China, such as Shandong, since the 1990s. The immunization strategy involved 2 doses of vaccine, including the first dose for infants at between 8 and 18 months of age and the second dose for 7- or 12-year-olds (30
). If routine vaccine coverage in children is not maintained, immunization of children could alter transmission dynamics and potentially lead to an increase in susceptibility in older age groups (27
). A shift in risk to older age groups has already occurred in Brazil and Costa Rica (4
). This is of concern because older age groups include women of childbearing age, thus raising the potential for an increase in the incidence of CRS.
In 2008, the rubella vaccine was introduced into the national immunization program in China. Measles-rubella vaccine (MR) was introduced for 8-month-old infants, and measles-rubella-mumps vaccine (MMR) was given to infants at 18 to 24 months of age. However, in some circumstances, when rubella vaccines were not available, they were replaced by measles vaccines in order to achieve the measles elimination goal initiated by WHO (19
). Furthermore, seroepidemiological surveys of Chinese females in the cities of Shenzhen (20
), Beijing, and Chongqing (18
) revealed that only about 80% were immunized against rubella, which is too low to provide herd immunity to the population. Considering the shift in the ages at risk and the seroepidemiological data, the development of a routine rubella vaccination program should be a priority, and both children and women of childbearing age should be included.
In 2005, the WHO Regional Committee of the Western Pacific Region (WPR) formally declared regional measles elimination a goal with a target date set for 2012 (1
). Measles and rubella are similar diseases, both characterized by a rash that may be difficult to differentiate clinically (35
). In addition, rubella epidemics usually occur in the spring or early summer, similar to measles, peaking between April and May (13
). Therefore, during the measles elimination campaign in China, particularly in 2009, when the incidence of rubella was higher than that of measles, measles control might have been hampered because large numbers of suspected measles cases, which were in reality rubella cases, may have appeared. Therefore, it is crucial to eliminate rubella during measles eradication campaigns. However, countries wishing to control and eliminate rubella must not only maintain high vaccine coverage but also be supported by high-quality surveillance, including molecular epidemiological studies, in order to obtain information about the circulation of indigenous viruses and the importation of new strains from other parts of the world (29
The data presented in this study demonstrate that 1E rubella virus has been the predominant virus genotype since 2001 and that only 2 provinces had incidences of the genotype 2B virus in 2008. Therefore, the genotype 1E rubella virus is most likely to cause a nationwide epidemic. From 2001 to 2007, multiple transmission chains of genotype 1E rubella viruses were found in different parts of China (34
). Some transmission chains of 1E faded, while other similar sequences continued to circulate in various provinces between 2008 and 2009. This might be related to the large-scale use of rubella vaccine during the nationwide immunization program in 2008, which may have interrupted some transmission chains. Some genotype 1E strains still survive and circulate.
Chinese genotype 1E rubella virus isolates were grouped into 2 clusters. Cluster 1 isolates were collected from 2001 to 2009, revealing the long-term circulation of this cluster in China since its emergence in 1997 (based on Bayesian MCMC analysis). Cluster 2 consisted of isolates from Hainan Province from between 2007 and 2008 (and may have emerged in 2006, based on Bayesian MCMC analysis), suggesting that it may recently have been introduced into Hainan Province.
Cluster 1 strains within the 1E genotype predominate in China and have not been found elsewhere, although sequences of many other 1E strains from other countries are available for comparison. This indicates that cluster 1 is unique to China. These viruses may have arisen from mutations and random genetic drifts that conferred a selective advantage to this lineage following its emergence in 1997.
Various methods based on Bayesian statistics that allow evolutionary rates, molecular phylogeny, and population dynamics to be coestimated in a single analysis starting from a nucleotide sequence alignment have recently been developed. The phylogeny and times of divergence of the rubella virus lineages were inferred using partial E1 fragment sequences sampled at different times and a relaxed molecular clock model, which incorporates the time-dependent nature of the evolutionary process by assuming independent rates in different branches, rather than a strict clock.
Recently developed methods based on coalescent theory for inferring the demographic history of a population on the basis of the gene sequences of a sample have allowed the reconstruction of the history of epidemics due to highly variable RNA viruses (6
). BSP, a nonparametric piecewise-constant model of population size, can fit several models, which solved the problem that coalescent methods usually require the assumption of a demographic model that is a mathematical description of the changes in effective population size, and information about the demographic behavior of a study was seldom obtained in advance. The BSP approach makes it possible to reconstruct novel and complex demographic scenarios (10
). The greater sensitivity of the nonparametric BSP method showed that the rubella virus population size remained constant until 2007, when a decline in the effective number of infections occurred with the introduction of rubella vaccine into the national immunization program in 2008.
, RNA virus nucleotide misincorporation rates per site ranged from 10−3
, due to the intrinsic error rates of the RNA polymerase and the lack of proofreading (2
). Genetic mutations are the basis of RNA virus evolution, as they allow the virus population to rapidly adapt to new environments and escape host antiviral responses (7
). As an RNA virus, rubella virus has the potential to continually mutate, so close monitoring of the genetic variations of wild-type rubella virus strains is necessary. In this study, we estimated that the mean mutation rate of genotype 1E rubella viruses was 1.65 × 10−3
substitutions per site per year, based on the 739-nt window of the E1 gene, which is lower than the rates of the measles virus (0.78 × 10−2
), mumps viruses (1.86 × 10−2
), and coxsackievirus group A16 (0.91 × 10−2
). The effects of strong negative selection can be seen in the 16-fold lower evolutionary rate in the 1st and 2nd codon positions of the partial E1 genome sequence, frequently causing amino acid changes, than in the 3rd codon position, rarely resulting in amino acid substitution. The evolution rate of rubella virus is relatively low and may be due to the high degree of conservation in both nucleotide and amino acid sequences of rubella virus (11
). Although the 739-nt region within the E1 glycoprotein for diagnostic applications contains important functional domains, including a hemagglutination-inhibiting and -neutralizing epitope, and antigenic sites, negative or positive selection pressures may differ considerably across a viral genome, and further studies on other genes or the whole genome are required to confirm this finding.
To our best knowledge, genotype 1E viruses were first identified in 1995 in France (26
) and then in 1997 in the United States, Canada, the Caribbean, and Italy (23
). In addition to North America and Europe, 1E viruses have now been isolated in South America, Africa, and Asia (28
). Global genotype 1E viruses investigated in this study share the recent common ancestor that originated in 1995, the same year of the first isolation in France, suggesting that genotype 1E viruses first appeared in Europe. However, analysis of additional sequences is needed to determine the origin and evolution of genotype 1E rubella viruses with greater accuracy.
A shift in the predominant genotype from genotypes 1F and 2B to genotype 1E was found with the 2001 rubella epidemic in China. Subsequently, 1E rubella viruses continually circulated in China for more than 9 years. Epidemiological data show that a rubella epidemic occurred in 2008. The transmission dynamics of endemic viruses may change due to vaccine introduction in 2007, and these may be contained or even eliminated. Ongoing molecular epidemiological surveillance of circulating rubella viruses is necessary.
Because humans are the only reservoir of rubella virus, the transmission of rubella virus is influenced by various factors relating to the amount and virulence of the viruses, the number of unimmunized hosts, and their interaction. So, it can be postulated that the more recent decrease in the effective number of the rubella virus infections (those actually transmitted) might be due to a decrease in the virus population that is related to routine vaccination with rubella vaccine in the national immunization program and the decrease in the immunity gap related to susceptibility to the infection.
In conclusion, after the first isolation of 1E genotype rubella virus (cluster 1) in 2001 in China, it continuously circulated throughout the epidemic of 2008, but unlike the last rubella outbreak in 2001, no genotypic shift occurred. In addition, although the circulation of the virus has not been interrupted following the introduction of the rubella vaccine into the national immunization program in 2008, some lineages within the 1E genotype disappeared. Cluster 2 viruses within the 1E genotype were found only in Hainan Province, which is most likely the result of importation from other regions due to high rates of migration and tourism. The most recent common ancestor of global genotype 1E rubella virus may be traced back to February 1995 (range, January 1995 to April 1995). The tMRCA estimates for Chinese clusters were dated to May 1997 (cluster 1) and March 2006 (cluster 2). Estimated by the BSP method, the effective number of infections remained constant until 2007. Although a rubella epidemic occurred in the epidemiology in 2008, the epidemic started a decline that led to a decrease in the effective population size with the introduction of rubella vaccine into the national immunization program in the same year.