Measles was listed as a reportable disease since 1985 and information regarding measles infections before 1985 is incomplete. From 1985 to 1992, four major measles outbreaks occurred in Taiwan (Figure 1) in 1985 (2,219 cases), 1988 (1,386 cases), 1989 (1,060 cases) and 1992 (303 cases). The epidemic in 1985 resulted in 97 deaths with reported case fatality rate of 4.4%, whereas the epidemic of 1988-89 was associated with 12 deaths and a case fatality rate of 0.5%. The high mortality rates associated with the outbreaks in 1985 and 1989 were likely due to under reporting of the cases. In an outbreak investigation among elementary and junior high schools in northeastern Taiwan in 1989, the reporting of measles by physicians was only 6.1% [16]. Beginning in 1993, the annual number of reported measles cases was below 100, the annual number of laboratory confirmed measles cases averaged 8.6, with a range of 0-33 cases. There were no laboratory confirmed cases for years of 1995, 1996, 1999, and 2004 (Figure 1).

Genetic analysis was conducted on 74 measles cases (Table 1) and 4 vaccine reactions that occurred between 1992 and 2008. Genotype information on MeVs that circulated in Taiwan before 1992 is not available. The six genotypes were detected among the 74 measles cases included D3, D5, D9, G2, H1, and H2 (Table 1). Four sequences from genotype A were obtained from patients who presented with rash following vaccination.

Genotype D3 was detected in five cases, and three of these (1 in 2002, 2 in 2003) were travelers from the Philippines (Figure 2). All of the genotype D3 sequences were placed within the same cluster as the reference strain, Illinois. USA/89.1, and more divergent from an earlier D3 sequence, MVs/Taipei.TWN/94 (AJ250068) [17], which was closely related to the Japanese strains (D87485, D87486, and D87490) detected in 1989.

Genotype D5 was detected in nine cases in Taiwan. Genotype D5 has been reported to be circulating in Thailand and had been associated with cases in Japan and imported cases in the USA [18,19]. The source of virus for three of the cases (one each in 1992, 2000, and 2002) was not identified. The other six cases had sequences that were more closely related to the Bangkok reference strain than to the Palau reference strain (Figure 2). Of these six cases with genotype D5 detected, one case was imported from Thailand, one from Germany, and the four more recent cases in 2007-2008 were imported from Japan.

The genotype D9 sequence from 2003 was obtained from a 25-year-old woman who had traveled to Japan for 10 days before onset of clinicalsymptoms. Three other D9 sequences were obtained in 2008, one was from 4 year old girl who traveled to the Philippines 13 days before rash appeared. The other two cases from 2008 were from the same household including a 25 year old man who had traveled to Thailand 10 days before rash onset and then transmitted virus to his 10 months old niece. Genotype D9 was initially detected in East Timor and Java, Indonesia [20,21] and has recently been associated with outbreaks in Japan [22].

Genotype G2 was first identified in 1997 in Indonesia and Malaysia [23,24]. The three genotype G2 viruses detected in Taiwan in 1997 shared identical sequences and were closely related to the sequence of the reference strain for genotype G2, MVi/Amsterdam.NET/49.97 (AF171232), which was isolated from secondary cases of measles associated with an index case from Indonesia. Though the source of the virus was not identified, a linkage to Indonesia or Malaysia is speculated (Figure 2).

Genotype H1 was detected in 50 of the 74 samples from cases occurring during 1992-2008. In 1992, genotype H1 sequences belonged to two lineages within what has been described as genotype H1, cluster 1 [25] (Figure 3). Two H1 sequences obtained in 1993 were identical to one of the sequences detected during the previous year (MVs/Keelung.TWN/18.92/3). Four identical genotype H1 sequences were obtained from the Taoyuan area in 1994. The only H1 sequence detected in 1997, MVs/Taipei.TWN/36.97, was very closely related (99% identity) to Chinese viruses detected in 1995, MVi/Hunan.PRC/15.95/9 (DQ356792) and MVi/Hunan.PRC/18.95/18 (DQ356791). One genotype H1 sequence was detected in 2000, but information regarding the source of infection was not available. During 2001, six cases were confirmed as genotype H1. Three of these cases had traveled to China 7-21 days before onset of rash and the sequences from the six cases were distributed over three different lineages (Figure 3).

There were 26 measles cases confirmed by serology in 2002, and 10 cases were from a school outbreak that occurred from September to October. There were 17 cases with sequence information and 15 of the H1 sequences belonged to 3 different lineages. Of those, 3 were imported from China (MVs/Taipei.TWN/26.02, MVs/Taipei.TWN/27.02, and MVs/Pingtung.TWN/33.02). Nine of the H1 isolates associated with the 2002 outbreak (six of these were either household or school clustering) had identical sequences and formed a separate lineage (Table 1, Figure 3) which included identical sequences from viruses isolated from Japan and China, MVi/Tokyo.JPN/23.01 (AB095428), MVi/Fuji.JPN/21.02 (AB095430), MVi/Kawasaki.JPN/36.01 (AB095429), MVi/Shanxi.PRC/13.02/1 (DQ356858), and MVi/Shanghai.PRC/30.02/1 (DQ356839), suggesting that these viruses were introduced by importation.

The genotype H1 sequence detected in 2003 was from an eleven months old infant returning from a visit to China. The two cases with genotype H1 in 2005 were in the same family and there was no history of travel. The six genotype H1 sequences detected in 2006-2007 were all imported from China (Table 1). Among the eight genotype H1 sequences detected in 2008 (Table 1), four had a travel history to China. The case associated with MVs/Kaohsiung.TWN/45.08 was responsible for nosocomial transmission in two hospitals and the index case represented by MVs/Kaohsiung/52.08 was identified later. In this outbreak, eight cases were confirmed and only four had adequate samples for PCR testing. One of the genotype H1 sequences obtained in 2008, MVs/Taoyuan.TWN/29.08, was detected by the laboratory surveillance system but no source was identified. A search of GenBank found an identical sequence from case reported in Hong Kong, MVs/HongKong.CHN/17.08 (EU870594). Therefore, there were multiple introductions of genotype H1 viruses into Taiwan from China and the epidemiologic information documented importation for 17 of 50 cases. The sequences from the recent genotype H1 viruses that were imported into Taiwan showed 100% sequence identity with genotype H1 viruses that were circulating in China or were associated with international importation of virus. For example, MVs/Tainan.TWN/33.07 (EU914238) had 100% identity with MVi/Sichuan.PRC/28.04/1 (EU557230) and MVi/Chongqing.PRC/20.04/1 (EU557209). MVs/Tainan.TWN/31.07/1 (EU914237) shared identical sequences with MVs/Aberdeen.GRB/10.06 (EF079131), MVi/Shanghai.PRC/22.06/11 (DQ902857) and MVs/Oregon.USA/8.06 (DQ888762).

Among the 50 H1 sequences characterized, forty-five cases were clustered with the reference strain for genotype H1 (Figure 3) and the other five cases, displayed in a separate lineage, were closely related to two genotype H1 viruses from China, MVi/Hunan.PRC/18.95/18 (DQ356791) and MVi/Hunan.PRC/15.95/9 (DQ356792).

Only three cases were confirmed to be associated with genotype H2, one was in 1994 from the Taoyuan outbreak and the other two were from an outbreak in a hospital in 1998. Although no information regarding the source of these cases was available, these are more closely related to the isolates from Vietnam [26].

Specimens including throat swabs, urine, whole blood, or serum were collected from suspected cases and tested in the laboratory at the Taiwan CDC. During the period of 1992-2008, various specimens from 896 suspected patients were collected for laboratory tests. Specimens were processed with RT-PCR amplification from a total of 174 patients, including 42 with only one serum collected at the acute phase and tested negative for measles IgM and IgG, the other 132 cases were serologically confirmed with positive measles IgM. Finally, 74 confirmed measles cases, including 16 virus isolates, were sequenced and used for genetic characterization. Here, a confirmed case is defined serologically either by a positive IgM, or a significant rise (4× or higher) of IgG titer between acute and convalescence phase, or a positive RT-PCR result from various clinical samples (serum, throat swab or urine). Any confirmed case that has travel history abroad 7-23 days before onset of rash is classified as an imported case [35].

Serum specimens were tested for measles IgM and IgG antibodies using Enzygnost Anti-Marsen-Virus/IgM, Enzygnost Anti-Marsen-Virus/IgG (Dade Behring, Marburg, Germany) following the manufacturer's instructions.

Clinical specimens including throat swab, urine sediments and lymphocytes were inoculated onto B95a cells, a marmoset B lymphoblastoid cell line transformed by Epstein-Barr virus [36], and observed for the presence of cytopathic effect (CPE). Inoculated cells were blind passaged up to two times before discarding those with no evident CPE.

RNA was extracted from infected cells or directly from clinical specimens by using the Viral RNA mini kit (Qiagen Inc., Chatsworth, CA) following the manufacturer's instructions. To obtain the sequence of the 450 nt region of the measles N gene that is required for genotyping, RT-PCR was performed by using a one-step RT-PCR kit (Qiagen) with reverse primer MV64 (nt 1719-1739, 5'-TATAACAATGATGGAGGGTAG-3') and forward primer MV59 (nt 866-889., 5'-GATATGTGACATTGATACATATAT-3'). Primer concentration was 0.4 μM each. The PCR cycling conditions were reverse transcription at 50°C for 30 min, initial PCR activation step by 95°C for 15 min, followed by 35 cycles of 30 s at 94°C, 30 s at 51°C and 1 min at 72°C, with a final extension for 5 min at 72°C. A nested PCR was then performed on the resulting PCR product of 873 bps using HotStartTaq Master Mix Kit (Qiagen) with primers MV60 and MV63 (0.2 μM each) as described elsewhere [37], and the cycling conditions were 95°C for 15 min followed by 30 cycles of 30 s at 94°C, 30 s at 63°C and 1 min at 72°C, with a final extension for 5 min at 72°C. Sequencing reactions were performed by using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Forster City, CA) with the same primers as those used for the nested PCR. Nucleotide sequences were analyzed with MegAlign version 3.1.7. Phylogenetic trees were drawn with MEGA version 4.1 by neighbor-joining using 1000 bootstrap replicates. The wild-type MeV isolates and genotype sequences from Taiwan were named as recommended by WHO [38]. The WHO-designated reference sequences [39] for each genotype were obtained from GenBank.