Background

Measles virus (MV), an enveloped virus with a single-stranded, negative sense RNA genome, is a member of the genus Morbillivirus within the family Paramyxoviridae. MV is highly contagious and causes a disease characterized by high fever, cough, coryza, conjunctivitis and appearance of a maculopapular rash [1]. Despite widespread use of a safe and effective vaccine, it is estimated that MV still causes close to half a million deaths per year and is a major cause of child mortality, mostly in develo** countries [2]. However, measles has been eliminated in countries that have maintained high vaccine coverage rates, and four of six WHO regions now have measles elimination goals [3, 4].

MV is a monotypic virus, but genetic variability exists among wild type strains [5]. Molecular epidemiological studies have provided an important tool for map** transmission routes, documenting the elimination of endemic virus strains, and differentiating vaccine from wild-type strains [6–9]. The protocols and nomenclature for genetic characterization of wild-type measles viruses have been standardized by the World Health Organization (WHO) [10]. The WHO currently recognizes 23 genotypes of MV [10–14] based on sequence analysis of the 450 nucleotides coding for the 150 amino acids at the carboxyl-terminus of the nucleoprotein (N) and the coding region of the hemagglutinin (H) gene [10, 11]. WHO recommends that genetic analysis of MV isolates should be conducted during all phases of measles control [15].

China, which successfully eradicated wild-type poliomyelitis virus in 1994, now has established a goal for measles elimination by 2012. One of the strategies to achieve elimination includes strengthening measles surveillance. An accelerated measles control program and surveillance activities were initiated in China during 1997 and 1998, respectively. To improve surveillance, a laboratory network was started in 2001, and is currently composed of one national measles laboratory, 31 provincial measles labs and 331 prefecture labs. Measles laboratory surveillance includes serologic confirmation of suspected measles cases and genetic characterization of wild-type viruses.

Analysis of wild-type MV circulating in China during 1993–1995 and 1998–1999 led to the initial identification of a novel genotype, H1 [16, 17]. Genotype H1 viruses were isolated in Hunan, Shandong, Hebei, Bei**g, Hainan, and Anhui Provinces and were linked to cases detected in the USA in 1997 and 1998 [7]. Epidemiological data from the exported cases suggested that the H1 viruses have a very wide distribution in China including Hong Kong and Guangzhou [7, 17]. However, continued sampling of measles virus strains from different locations within China is needed for a more complete understanding of the distribution of genotypes. This report greatly expands our knowledge of wild-type MVs in China. We describe the genetic characterization of nearly 300 wild-type MVs circulating in China between 1995 and 2003. Viral isolates were obtained from 24 of 31 provinces. The results showed widespread distribution of genotype H1 viruses.

Results and Discussion

To support further progress in measles control the Ministry of Health of China issued a Plan for Accelerated Measles Control in 1998 and National Measles Surveillance Plan in 2004. The current National Measles Surveillance Plan divides the provinces into 2 groups based on average annual measles incidence [18]. Provinces in Group A have an average measles incidence <6/100,000 and a measles elimination and outbreak prevention goal. Provinces in Group B have an average measles incidence >6/100,000 and a measles control goal. Viral isolates were obtained from 17/18 Group A provinces and 7/13 Group B provinces. The majority of the isolates (84%) were from the Group A provinces because the laboratory network is not as well established in the Group B provinces. Many of the laboratories in Group B provinces lack the necessary equipment and supplies to obtain samples for viral isolation. Table 1 lists the number of isolates obtained from each province in China and the location of the provinces is shown in Figure 1. During 1995–2003, the incidence of measles in China was <8/100,000, with fewer than 250 measles deaths reported each year (Figure 2).

Table 1 Number of wild-type measles viruses analyzed between 1995 and 2003 by province. Epidemiologic classification of each province is shown.
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Figure 1
figure 1

The geographic distribution of Chinese measles isolates from 1995 to 2003. No isolates were received from provinces in white.

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Figure 2
figure 2

Average number of measles cases (blue bars) and reported death (black bars) and average measles incidence (red line) between 1991 and 2003 in China. Number of reported deaths for each year is indicated above each black bar.

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The sequences of the 450 nucleotides coding for the COOH-terminus of the nucleoprotein were derived from all of the 297 viral isolates listed in Table 1 and 191 representative sequences were used for phylogenetic analysis. All of the isolates were placed in genotype H1 except 3 isolates, which were in genotype A (Figure 3, 4). The grou** of the sequences within the genotype H1 was supported by bootstrap analysis (data not shown). The ranges of nucleotide sequence and amino acid homologies among the 294 contemporary H1 isolates were 94.7%–100% and 93.3%–100%, respectively.

Figure 3
figure 3

Schematic phylogenetic tree of the N gene sequences of 191 wild-type measles isolates from China compared to the WHO reference sequences for each genotype. For simplicity, strain names are not shown. Sequences from viruses isolated in China from 1995–2003 are indicated by red branches and dots, and WHO reference strains and Shanghai-191 vaccine strain are indicated by blue branches and dots. Positions of the reference strains for genotypes H1 and H2 are indicated by arrows.

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Figure 4
figure 4

Phylogenetic tree of the N gene sequences of 188 wild-type measles isolates from China in genotype H1. The WHO reference strains and another strain on the intermediate cluster are shown in red. Cluster 1 is shown in black, while cluster 2 is shown in blue. WHO strain name is indicated for each sequence.

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Three of the viral isolates, MVi/Henan.PRC/7.99/1, MVi/Hunan.PRC/15.96/10, MVi/** of measles virus in Canada: 1979–2002. J Infect Dis 2004,189(Suppl 1):S171-176. 10.1086/377716" href="/article/10.1186/1743-422X-4-14#ref-CR28" id="ref-link-section-d267949410e2882">28–30]. Since WPRO, including China, has recently initiated a program to eliminate measles by 2012, this report provides important baseline virologic information for China. Hopefully, virologic surveillance in China will be able to document the interruption of transmission of genotype H1 viruses and, in conjunction with epidemiological surveillance, will help to identify the sources of imported virus.

Conclusion

This study reports virologic surveillance data obtained in China during a period when measles control activities were greatly accelerated. The results confirmed that genotype H1 is the endemic genotype throughout China. The virologic data were consistent with endemic measles in that multiple chains of transmission were evident. The H1 viruses were very diverse and formed two major clusters, which were distributed throughout China with no apparent geographic restriction. This important baseline data will contribute to the development of improved measles control programs in China.

Methods

Specimens collection and virus isolation

Throat swab and urine samples were obtained from serologically confirmed measles cases. Clinical specimens were inoculated onto B95a cells [31], and the cells were observed for cytopathic effect (CPE). Inoculated cells were blind-passaged up to three times before being discarded. Cells were harvested when the CPE was maximal. Virus isolations were performed by 24 provincial laboratories in China and the viral isolates were shipped to the National Measles Laboratory, in Bei**g for genetic analysis.

RNA Extraction and RT-PCR

Viral RNA was extracted from infected cell lysates using Trizol reagent according to the manufacturer's directions. RNA pellets were dried and resuspended in 50 μl of sterile distilled water and stored at -70 C until amplification by RT-PCR. RT-PCR was performed using previously described methods [6, 20]. Primers MV63 (5'CCT CGG CCT CTC GCA CCT AGT 3') and MV60 primers (5'GCT ATG CCA TGG GAG TAG GAG TGG 3') were used to amplify a 676 bp fragment of the N gene including the 450 bp fragment recommended for genoty**.

Sequence analysis

The sequences of the PCR products were derived by automated sequencing with primers MV60 and MV63 and the BigDye terminator v2.0 chemistry using reaction conditions that were recommended by the manufacturer (ABI 373, ABI 3100, Perkin Elmer-Applied Biosystems). Sequence proof reading and editing was conducted with Sequencer™ (Gene Codes Corporation). Sequence data were analyzed by using version 7.0 of Bioedit and phylogenetic analyses were performed using Bioedit and Mega ver3.1. The robustness of the grou**s was assessed using bootstrap resampling of 1000 replicates and the trees were visualized with Mega programs. 191 representative nucleotide sequences were deposited in GenBank under accession numbers DQ356683–DQ356873.