- Email: [email protected]
Monitoring of measles elimination using molecular epidemiology
Vaccine 19 (2001) 2245– 2249 www.elsevier.com/locate/vaccine
Monitoring of measles elimination using molecular epidemiology Mick N. Mulders *, Anh T. Truong, Claude P. Muller Department of Immunology, World Health Organization Collaborating Center for Measles, Laboratoire National de Sante´, P.O. Box 1102, L-1011 Luxembourg, Luxembourg
Abstract The different measles virus genotypes are confined to more or less distinct geographic regions. Molecular characterization of virus isolates has been successfully used to determine epidemiological links between cases and the geographic origin of imported viruses. In Europe, indigenous measles has been eliminated in some countries, but in others the disease is still endemic. Intra-outbreak variability can be used to differentiate between sporadic endemic cases and a ‘pseudo-outbreak’ of unrelated imported cases. The interruption of virus circulation by mass vaccination campaigns could be demonstrated by comparing the variability of pre-campaign viruses with post-campaign isolates. Simplified tools are being developed that could bring genotyping within reach of laboratories that do not have the possibility of sequencing. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Measles; Genotype; Epidemiology
1. Introduction Many regions have adopted a strategy to eliminate measles: the Americas before the end of last year, Europe by 2007, and the Eastern Mediterranean by 2010. Despite this global commitment, measles remains endemic both in many developing and some industrialized countries. In 1998, global measles vaccination coverage even declined to 72% from 79% the year before [1], with Africa and South-East Asia having the lowest coverage rates. As a result of the high infectivity of measles, 90–95% of the population need to be immune to eliminate indigenous measles. It has become clear that in most countries sufficiently high vaccine coverage cannot be reached by routine vaccination alone. Therefore other vaccination strategies such as mass campaigns have been implemented in particular in the developing countries with the main goal of interrupting MV transmission and circulation. Measles virus is monotypic and belongs to the genus morbillivirus (family Paramyxoviridae). The virus genome is a linear single stranded RNA molecule of negative polarity, with a length of about 15 900 ribonucleotides, and encodes its own RNA-dependent RNA polymerase. Due to the lack of proofreading, mutations * Corresponding author. Tel: +352-490626; fax: +352-490686. E-mail address: [email protected] (M.N. Mulders).
accumulate, that can be exploited to determine the relative relatedness between viruses, and their transmission pathways within countries or between continents. Here we discuss molecular epidemiology as a tool to monitor measles elimination efforts.
2. Measles virus genotypes and their geographic distribution Genetic classification of MV is based on either the C-terminal hypervariable region (456 nt) of the nucleoprotein (N) gene or on the full length hemagglutinin (H) gene. As a result of the commitment of a number of laboratories, an increasingly comprehensive picture of the geographic distribution of the different MV genotypes and clades emerges. The standardized nomenclature of the WHO for describing the genetic characteristics of wild-type (wt) measles virus isolates [2] distinguishes eight clades, designated A – H, and within these clades there are at least 17 genotypes (A, B1 –B3, C1, C2, D1 –D6, E, F, G1, G2, H; Fig. 1). To each genotype a reference strain has been assigned. Some genotypes circulate in geographically more or less confined regions, while other genotypes appear to be extinct. Clade A consists of vaccine and vaccine-related wt viruses [3], and it is thought to have been a major
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 4 5 3 - 9
2246
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249
genotype before routine vaccination was implemented in the late 1960s. However, before the introduction of B95a cell line, which is 10 000-fold more efficient for MV isolation [4], most MV were isolated on Vero cells,
which may have introduced a selection bias. In recent years viruses belonging to genotype A, have been isolated in the US [3], the UK [5], in South Africa [6], China [7], and Russia [8], indicating the continued
Fig. 1. Based on the sequence alignment of the 456 nt long C-terminal hypervariable region of the N-gene, a phylogenetic tree was contructed using ClustalX [28]. Genotype reference strains are displayed in bold face. Genbank accession numbers are given in brackets. WHO country codes are used. * Some sequences were shorter than the length of the alignment. WHO genotype reference sequences are printed in bold.
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249
circulation for many decades. Clade B comprises genotypes B1, B2 and B3 suggested by us [9,10]. Isolates belonging to these genotypes can be found mainly in equatorial Africa [9,10]. Clade C consists of genotypes C1 and C2, and is isolated mainly in Europe, but also in Japan [11,12]. We experienced in Luxembourg in 1996 a large-scale outbreak involving 110 cases, which was caused by a genotype C2 virus [12]. Clade D viruses were found on most continents [6,8,11,13]. This clade is subdivided into genotypes D1 to D6, with a seventh one found in the UK, and more recently by us in Nepal [14]. Genotype D1 is thought to be inactive, while genotype D2 has been found only in southern Africa [6]. Clades E and F are thought to be extinct. Clade G was also considered to be inactive, but recently, we and others reported the isolation of viruses belonging to a new genotype G2 within this clade [14 – 16]. These viruses were found in Indonesia and Malaysia [14,16]. Clade H viruses dominate mainland China. Both G2 and H viruses have also been exported to The Netherlands [15]. Definitions for genotype and/or clade demarcation are based on the genetic divergence between different clusters and have not been standardized, like for poliovirus and other enteroviruses [17,18]. The maximum variation observed in the distance matrix, on which the dendrogram of Fig. 1 is based, was 13.6% in the C-terminal region of the N gene. H gene sequence variation is 7% at maximum. The genetic divergence within the variable N region is between 1.7% for clade A and 9.6% for clades C and D viruses (clade B: 4.8%; clade G: 5.7%, clade H: 5.9%). The sequence variation within clades C and D is high, mainly because genotypes C1 and C2 are quite distinct from each other, as is D6 from the other D genotypes. The maximum variation within one genotype fluctuates between 1.7% for A and 6.0% for C1, with B3: 3.7%; C2: 3.3%; D1: 2.2%; D3: 2.1%; D4: 5.4%; D5: 2.9%; D6: 1.8%; d7: 3.5% (data based on distance matrix from dendrogram shown in Fig. 1). For future genotype classification of new MV strains, we suggest to take into account not only the clustering pattern obtained in the phylogenetic analysis, but also a genotype demarcation of approx. 3.5% and a clade demarcation of approx. 7%. Also, new genotypes should perhaps be tentatively assigned a lower case letter (e.g. d7) until it is accepted by the WHO nomenclature committee.
3. Molecular epidemiology to monitor vaccination campaigns Comparative sequence analysis, i.e. molecular epidemiology, can provide the tools to monitor virus circulation and progress of elimination efforts. In the US, where measles has become a rare disease due to
2247
intense vaccination efforts, all MV isolates are sequenced in order to determine the epidemiological link between cases and the geographic origin of the virus isolate. Of the 100 cases that were reported in the US in 1999, 66 could be identified as importations, while 24 were from four localized outbreaks [19]. In some parts of Europe measles elimination has been achieved, but in others the disease is still endemic. Since case notification is usually insufficient, it is difficult to distinguish between cases caused by the epidemic or endemic virus and a ‘pseudo-outbreak’ of sporadic unrelated cases due to virus reintroduction. The 1996-outbreak in Luxembourg was caused by C2 viruses with a sequence variability of 0.2% in the C-terminus of the N-gene [12]. However, sporadic cases continued to occur during the following year, suggesting that the virus persistently circulated. Although some of the viruses collected later were of the same genotype, the sequence divergence with the outbreak virus was 3–4-fold higher than the maximum divergence found among outbreak viruses. We concluded that the viruses were unrelated to the outbreak. It appeared that circulation of the outbreak virus had ceased and that subsequent sporadic measles cases were due to reintroduction of virus. Despite the local outbreak among a group of unvaccinated children, the immunity of the general population was sufficient to interrupt virus transmission. Similarly, the interruption of virus circulation by mass vaccination campaigns could be demonstrated by comparing the variability of pre-campaign viruses with post-campaign isolates. Molecular epidemiological analysis would provide the means to determine whether cases are related and the virus continues to be transmitted within the region covered by the campaign, or whether cases are unrelated and due to reintroduction of virus. Since mass vaccination campaigns are implemented mostly in developing countries it is a challenge to bring genomic characterization within reach of laboratories that are equipped for PCR, but not necessarily for sequencing. Viral RNA has been detected with high efficiency in blood (serum: 75% [20,21]; plasma: 100%, within 3 days of onset of rash; and in peripheral blood mononuclear cells: 96% [22]). However, both virus and viral RNA can also be obtained by non-invasive procedures from throat swabs (81 –100%) [20,21], nasopharyngeal secretions (96%) [22], and urine (83%) [23]. To characterize the viral genome, analysis of restriction fragment length polymorphism (RFLP) [24] and of the differential mobility of heteroduplexes [25] have been proposed. RFLP was successfully used to differentiate between wt and vaccine-derived [24]. An example of a heteroduplex mobility assay (HMA) is shown in Fig. 2. Lanes 3–7 show the HMA patterns of isolates from Janakpur and Pokhara, Nepal. These viruses belong to
2248
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249
References
Fig. 2. HMA analysis of PCR-amplified carboxyl-terminal N gene fragments from nine Asian wt MV isolates. Lane 1 shows the homoduplex control (Edmonston-wt.USA/1954). In lanes 2– 10, the Edmonston derived PCR fragment was annealed to PCR fragments from: MVi/Taipei.Taiwan/94 (lane 2), MVi/Janakpur.NEP/2.99/1 (lane 3), MVi/Janakpur.NEP/2.99/2 (lane 4), MVi/Hetauda.NEP/2.99 (lane 5), MVi/Kathmandu.NEP/5.99 (lane 6), MVi/Pokhara.NEP/ 5.99 (lane 7), MVi/Amsterdam.NET/3.98 (lane 8), MVi/Amsterdam.NET/4.97 (lane 9), and MVi/Amsterdam.NET/27.97 (lane 10). Heteroduplex fragments were resolved on a polyacrylamide gel (MDE, BioWhittaker Molecular Applications, Rockland ME, USA) at 150 V for 8 h and stained with SYBR-green (BMA).
the newly proposed d7 genotype [14]. The strain from Pokhara had a slightly different HMA pattern (lane 7) and together with MVi/Amsterdam.NET/3.98 (lane 8) belonged to genotype D4 [14]. The reference viruses of clades G (lane 9) and H (lane 10) as well as a Taiwanese strain representing genotype D3 (lane 2) revealed clearly different heteroduplex mobility patterns when compared to the more homogenous ‘d7’ (or ‘D7’) MV isolates. These results emphasize the value of the HMA for rapid and simple genetic pre-screening of MV isolates. Previously, dried plasma spots (DPSs) on filter paper membranes have been used successfully to detect RNA of HIV-1 [26], of hepatitis C [27], and other viruses. DPSs eliminate the need for cumbersome and expensive protocols for sample collection, storage and shipment. Genomic amplification of the genetic material from DPS, combined with techniques such as probe hybridization, HMA, and RFLP, could bring genotyping within the reach of laboratories that do not have the possibility of sequencing. With a simplified technology monitoring of measles elimination efforts including mass campaigns by molecular tools could be performed in most countries.
Acknowledgements We would like to thank Wim Ammerlaan, Frank Hanses and Stephanie Kreis for their contribution to these studies. This work has been supported by the Ministe`re de la Coope´ration, Ministe`re de la Sante´, Recherche Biome´dicale a.s.b.l. and the Centre de Recherche Public — Sante´.
[1] World Health Organization. Measles — progress towards global control and regional elimination, 1998– 1999. Wkly Epidemiol Rec 1999;74:429– 34. [2] World Health Organization. Expanded Programme on Immunization (EPI). Standardization of the nomenclature for describing the genetic characteristics of wild-type measles viruses. Wkly Epidemiol Rec 1998;73:265– 9. [3] Rota JS, Wang ZD, Rota PA, Bellini WJ. Comparison of sequences of the H, F, and N coding genes of measles virus vaccine strains. Virus Res 1994;31:317– 30. [4] Kobune F, Sakata H, Sugiura A. Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus. J Virol 1990;64:700– 5. [5] Outlaw MC, Pringle CR. Sequence variation within an outbreak of measles virus in the Coventry area during spring/summer 1993. Virus Res 1995;39:3– 11. [6] Kreis S, Vardas E, Whistler T. Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype. J Gen Virol 1997;78:1581– 7. [7] Xu W, Tamin A, Rota JS, Zhang L, Bellini WJ, Rota PA. New genetic group of measles virus isolated in the People’s Republic of China. Virus Res 1998;54:147– 56. [8] Santibanez S, Heider A, Gerike E, Agafonov A, Schreier E. Genotyping of measles virus isolates from central Europe and Russia. J Med Virol 1999;58:313– 20. [9] Truong AT, Kreis S, Ammerlaan W, et al. Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates. Virus Res 1999;62:89– 95. [10] Hanses F, Truong AT, Ammerlaan W, et al. Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa. J Gen Virol 1999;80:871– 7. [11] Rima BK, Earle JA, Baczko K, et al. Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture. J Gen Virol 1997;78:97– 106. [12] Hanses F, van Binnendijk R, Ammerlaan W, et al. Genetic variability of measles viruses circulating in the Benelux. Arch Virol 2000;145:541– 51. [13] Katayama Y, Shibahara K, Kohama T, Homma M, Hotta H. Molecular epidemiology and changing distribution of genotypes of measles virus field strains in Japan. J Clin Microbiol 1997;35:2651– 3. [14] Truong AT, Ammerlaan W, de Swart RL, et al. Molecular epidemiological analysis of measles virus strains from five Asian countries. 2001, in preparation. [15] de Swart RL, Wertheim-van Dillen PM, van Binnendijk RS, Muller CP, Frenkel J, Osterhaus AD. Measles in a Dutch hospital introduced by an immuno-compromised infant from Indonesia infected with a new virus genotype. Lancet 2000;355:201– 2 letter. [16] Rota PA, Liffick S, Rosenthal S, Heriyanto B, Chua KB. Measles genotype G2 in Indonesia and Malaysia. Lancet 2000;355:1557– 8 letter. [17] Mulders MN, Salminen M, Kalkkinen N, Hovi T. Molecular epidemiology of coxsackievirus B4 and disclosure of the correct VP1/2A(pro) cleavage site: evidence for high genomic diversity and long-term endemicity of distinct genotypes. J Gen Virol 2000;81:803– 12. [18] Mulders MN, Lipskaya GY, van der Avoort HG, Koopmans MP, Kew OM, van Loon AM. Molecular epidemiology of wild poliovirus type 1 in Europe, the Middle East, and the Indian subcontinent. J Infect Dis 1995;171:1399– 405. [19] Centers for Disease Control and Prevention. Measles — United States, 1999. MMWR Morb Mortal Wkly Rep 2000;49:557–60.
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249 [20] Jin L, Richards A, Brown DW. Development of a dual targetPCR for detection and characterization of measles virus in clinical specimens. Mol Cell Probes 1996;10:191–200. [21] Matsuzono Y, Narita M, Ishiguro N, Togashi T. Detection of measles virus from clinical samples using the polymerase chain reaction. Arch Pediatr Adolesc Med 1994;148:289–93. [22] Nakayama T, Mori T, Yamaguchi S, et al. Detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability. Virus Res 1995;35:1– 16. [23] Rota PA, Khan AS, Durigon E, Yuran T, Villamarzo YS, Bellini WJ. Detection of measles virus RNA in urine specimens from vaccine recipients. J Clin Microbiol 1995;33:2485–8. [24] Kawashima H, Mori T, Takekuma K, Hoshika A, Hata M, Nakayama T. Polymerase chain reaction detection of the hemag-
.
[25] [26]
[27]
[28]
2249
glutinin gene from an attenuated measles vaccine strain in the peripheral mononuclear cells of children with autoimmune hepatitis. Arch Virol 1996;141:877– 84. Kreis S, Whistler T. Rapid identification of measles virus strains by the heteroduplex mobility assay. Virus Res 1997;47:197–203. Cassol S, Gill MJ, Pilon R, et al. Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper. J Clin Microbiol 1997;35:2795–801. Abe K, Konomi N. Hepatitis C virus RNA in dried serum spotted onto filter paper is stable at room temperature. J Clin Microbiol 1998;36:3070– 2. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL:X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25:4876– 82.
Monitoring of measles elimination using molecular epidemiology Mick N. Mulders *, Anh T. Truong, Claude P. Muller Department of Immunology, World Health Organization Collaborating Center for Measles, Laboratoire National de Sante´, P.O. Box 1102, L-1011 Luxembourg, Luxembourg
Abstract The different measles virus genotypes are confined to more or less distinct geographic regions. Molecular characterization of virus isolates has been successfully used to determine epidemiological links between cases and the geographic origin of imported viruses. In Europe, indigenous measles has been eliminated in some countries, but in others the disease is still endemic. Intra-outbreak variability can be used to differentiate between sporadic endemic cases and a ‘pseudo-outbreak’ of unrelated imported cases. The interruption of virus circulation by mass vaccination campaigns could be demonstrated by comparing the variability of pre-campaign viruses with post-campaign isolates. Simplified tools are being developed that could bring genotyping within reach of laboratories that do not have the possibility of sequencing. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Measles; Genotype; Epidemiology
1. Introduction Many regions have adopted a strategy to eliminate measles: the Americas before the end of last year, Europe by 2007, and the Eastern Mediterranean by 2010. Despite this global commitment, measles remains endemic both in many developing and some industrialized countries. In 1998, global measles vaccination coverage even declined to 72% from 79% the year before [1], with Africa and South-East Asia having the lowest coverage rates. As a result of the high infectivity of measles, 90–95% of the population need to be immune to eliminate indigenous measles. It has become clear that in most countries sufficiently high vaccine coverage cannot be reached by routine vaccination alone. Therefore other vaccination strategies such as mass campaigns have been implemented in particular in the developing countries with the main goal of interrupting MV transmission and circulation. Measles virus is monotypic and belongs to the genus morbillivirus (family Paramyxoviridae). The virus genome is a linear single stranded RNA molecule of negative polarity, with a length of about 15 900 ribonucleotides, and encodes its own RNA-dependent RNA polymerase. Due to the lack of proofreading, mutations * Corresponding author. Tel: +352-490626; fax: +352-490686. E-mail address: [email protected] (M.N. Mulders).
accumulate, that can be exploited to determine the relative relatedness between viruses, and their transmission pathways within countries or between continents. Here we discuss molecular epidemiology as a tool to monitor measles elimination efforts.
2. Measles virus genotypes and their geographic distribution Genetic classification of MV is based on either the C-terminal hypervariable region (456 nt) of the nucleoprotein (N) gene or on the full length hemagglutinin (H) gene. As a result of the commitment of a number of laboratories, an increasingly comprehensive picture of the geographic distribution of the different MV genotypes and clades emerges. The standardized nomenclature of the WHO for describing the genetic characteristics of wild-type (wt) measles virus isolates [2] distinguishes eight clades, designated A – H, and within these clades there are at least 17 genotypes (A, B1 –B3, C1, C2, D1 –D6, E, F, G1, G2, H; Fig. 1). To each genotype a reference strain has been assigned. Some genotypes circulate in geographically more or less confined regions, while other genotypes appear to be extinct. Clade A consists of vaccine and vaccine-related wt viruses [3], and it is thought to have been a major
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 4 5 3 - 9
2246
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249
genotype before routine vaccination was implemented in the late 1960s. However, before the introduction of B95a cell line, which is 10 000-fold more efficient for MV isolation [4], most MV were isolated on Vero cells,
which may have introduced a selection bias. In recent years viruses belonging to genotype A, have been isolated in the US [3], the UK [5], in South Africa [6], China [7], and Russia [8], indicating the continued
Fig. 1. Based on the sequence alignment of the 456 nt long C-terminal hypervariable region of the N-gene, a phylogenetic tree was contructed using ClustalX [28]. Genotype reference strains are displayed in bold face. Genbank accession numbers are given in brackets. WHO country codes are used. * Some sequences were shorter than the length of the alignment. WHO genotype reference sequences are printed in bold.
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249
circulation for many decades. Clade B comprises genotypes B1, B2 and B3 suggested by us [9,10]. Isolates belonging to these genotypes can be found mainly in equatorial Africa [9,10]. Clade C consists of genotypes C1 and C2, and is isolated mainly in Europe, but also in Japan [11,12]. We experienced in Luxembourg in 1996 a large-scale outbreak involving 110 cases, which was caused by a genotype C2 virus [12]. Clade D viruses were found on most continents [6,8,11,13]. This clade is subdivided into genotypes D1 to D6, with a seventh one found in the UK, and more recently by us in Nepal [14]. Genotype D1 is thought to be inactive, while genotype D2 has been found only in southern Africa [6]. Clades E and F are thought to be extinct. Clade G was also considered to be inactive, but recently, we and others reported the isolation of viruses belonging to a new genotype G2 within this clade [14 – 16]. These viruses were found in Indonesia and Malaysia [14,16]. Clade H viruses dominate mainland China. Both G2 and H viruses have also been exported to The Netherlands [15]. Definitions for genotype and/or clade demarcation are based on the genetic divergence between different clusters and have not been standardized, like for poliovirus and other enteroviruses [17,18]. The maximum variation observed in the distance matrix, on which the dendrogram of Fig. 1 is based, was 13.6% in the C-terminal region of the N gene. H gene sequence variation is 7% at maximum. The genetic divergence within the variable N region is between 1.7% for clade A and 9.6% for clades C and D viruses (clade B: 4.8%; clade G: 5.7%, clade H: 5.9%). The sequence variation within clades C and D is high, mainly because genotypes C1 and C2 are quite distinct from each other, as is D6 from the other D genotypes. The maximum variation within one genotype fluctuates between 1.7% for A and 6.0% for C1, with B3: 3.7%; C2: 3.3%; D1: 2.2%; D3: 2.1%; D4: 5.4%; D5: 2.9%; D6: 1.8%; d7: 3.5% (data based on distance matrix from dendrogram shown in Fig. 1). For future genotype classification of new MV strains, we suggest to take into account not only the clustering pattern obtained in the phylogenetic analysis, but also a genotype demarcation of approx. 3.5% and a clade demarcation of approx. 7%. Also, new genotypes should perhaps be tentatively assigned a lower case letter (e.g. d7) until it is accepted by the WHO nomenclature committee.
3. Molecular epidemiology to monitor vaccination campaigns Comparative sequence analysis, i.e. molecular epidemiology, can provide the tools to monitor virus circulation and progress of elimination efforts. In the US, where measles has become a rare disease due to
2247
intense vaccination efforts, all MV isolates are sequenced in order to determine the epidemiological link between cases and the geographic origin of the virus isolate. Of the 100 cases that were reported in the US in 1999, 66 could be identified as importations, while 24 were from four localized outbreaks [19]. In some parts of Europe measles elimination has been achieved, but in others the disease is still endemic. Since case notification is usually insufficient, it is difficult to distinguish between cases caused by the epidemic or endemic virus and a ‘pseudo-outbreak’ of sporadic unrelated cases due to virus reintroduction. The 1996-outbreak in Luxembourg was caused by C2 viruses with a sequence variability of 0.2% in the C-terminus of the N-gene [12]. However, sporadic cases continued to occur during the following year, suggesting that the virus persistently circulated. Although some of the viruses collected later were of the same genotype, the sequence divergence with the outbreak virus was 3–4-fold higher than the maximum divergence found among outbreak viruses. We concluded that the viruses were unrelated to the outbreak. It appeared that circulation of the outbreak virus had ceased and that subsequent sporadic measles cases were due to reintroduction of virus. Despite the local outbreak among a group of unvaccinated children, the immunity of the general population was sufficient to interrupt virus transmission. Similarly, the interruption of virus circulation by mass vaccination campaigns could be demonstrated by comparing the variability of pre-campaign viruses with post-campaign isolates. Molecular epidemiological analysis would provide the means to determine whether cases are related and the virus continues to be transmitted within the region covered by the campaign, or whether cases are unrelated and due to reintroduction of virus. Since mass vaccination campaigns are implemented mostly in developing countries it is a challenge to bring genomic characterization within reach of laboratories that are equipped for PCR, but not necessarily for sequencing. Viral RNA has been detected with high efficiency in blood (serum: 75% [20,21]; plasma: 100%, within 3 days of onset of rash; and in peripheral blood mononuclear cells: 96% [22]). However, both virus and viral RNA can also be obtained by non-invasive procedures from throat swabs (81 –100%) [20,21], nasopharyngeal secretions (96%) [22], and urine (83%) [23]. To characterize the viral genome, analysis of restriction fragment length polymorphism (RFLP) [24] and of the differential mobility of heteroduplexes [25] have been proposed. RFLP was successfully used to differentiate between wt and vaccine-derived [24]. An example of a heteroduplex mobility assay (HMA) is shown in Fig. 2. Lanes 3–7 show the HMA patterns of isolates from Janakpur and Pokhara, Nepal. These viruses belong to
2248
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249
References
Fig. 2. HMA analysis of PCR-amplified carboxyl-terminal N gene fragments from nine Asian wt MV isolates. Lane 1 shows the homoduplex control (Edmonston-wt.USA/1954). In lanes 2– 10, the Edmonston derived PCR fragment was annealed to PCR fragments from: MVi/Taipei.Taiwan/94 (lane 2), MVi/Janakpur.NEP/2.99/1 (lane 3), MVi/Janakpur.NEP/2.99/2 (lane 4), MVi/Hetauda.NEP/2.99 (lane 5), MVi/Kathmandu.NEP/5.99 (lane 6), MVi/Pokhara.NEP/ 5.99 (lane 7), MVi/Amsterdam.NET/3.98 (lane 8), MVi/Amsterdam.NET/4.97 (lane 9), and MVi/Amsterdam.NET/27.97 (lane 10). Heteroduplex fragments were resolved on a polyacrylamide gel (MDE, BioWhittaker Molecular Applications, Rockland ME, USA) at 150 V for 8 h and stained with SYBR-green (BMA).
the newly proposed d7 genotype [14]. The strain from Pokhara had a slightly different HMA pattern (lane 7) and together with MVi/Amsterdam.NET/3.98 (lane 8) belonged to genotype D4 [14]. The reference viruses of clades G (lane 9) and H (lane 10) as well as a Taiwanese strain representing genotype D3 (lane 2) revealed clearly different heteroduplex mobility patterns when compared to the more homogenous ‘d7’ (or ‘D7’) MV isolates. These results emphasize the value of the HMA for rapid and simple genetic pre-screening of MV isolates. Previously, dried plasma spots (DPSs) on filter paper membranes have been used successfully to detect RNA of HIV-1 [26], of hepatitis C [27], and other viruses. DPSs eliminate the need for cumbersome and expensive protocols for sample collection, storage and shipment. Genomic amplification of the genetic material from DPS, combined with techniques such as probe hybridization, HMA, and RFLP, could bring genotyping within the reach of laboratories that do not have the possibility of sequencing. With a simplified technology monitoring of measles elimination efforts including mass campaigns by molecular tools could be performed in most countries.
Acknowledgements We would like to thank Wim Ammerlaan, Frank Hanses and Stephanie Kreis for their contribution to these studies. This work has been supported by the Ministe`re de la Coope´ration, Ministe`re de la Sante´, Recherche Biome´dicale a.s.b.l. and the Centre de Recherche Public — Sante´.
[1] World Health Organization. Measles — progress towards global control and regional elimination, 1998– 1999. Wkly Epidemiol Rec 1999;74:429– 34. [2] World Health Organization. Expanded Programme on Immunization (EPI). Standardization of the nomenclature for describing the genetic characteristics of wild-type measles viruses. Wkly Epidemiol Rec 1998;73:265– 9. [3] Rota JS, Wang ZD, Rota PA, Bellini WJ. Comparison of sequences of the H, F, and N coding genes of measles virus vaccine strains. Virus Res 1994;31:317– 30. [4] Kobune F, Sakata H, Sugiura A. Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus. J Virol 1990;64:700– 5. [5] Outlaw MC, Pringle CR. Sequence variation within an outbreak of measles virus in the Coventry area during spring/summer 1993. Virus Res 1995;39:3– 11. [6] Kreis S, Vardas E, Whistler T. Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype. J Gen Virol 1997;78:1581– 7. [7] Xu W, Tamin A, Rota JS, Zhang L, Bellini WJ, Rota PA. New genetic group of measles virus isolated in the People’s Republic of China. Virus Res 1998;54:147– 56. [8] Santibanez S, Heider A, Gerike E, Agafonov A, Schreier E. Genotyping of measles virus isolates from central Europe and Russia. J Med Virol 1999;58:313– 20. [9] Truong AT, Kreis S, Ammerlaan W, et al. Genotypic and antigenic characterization of hemagglutinin proteins of African measles virus isolates. Virus Res 1999;62:89– 95. [10] Hanses F, Truong AT, Ammerlaan W, et al. Molecular epidemiology of Nigerian and Ghanaian measles virus isolates reveals a genotype circulating widely in western and central Africa. J Gen Virol 1999;80:871– 7. [11] Rima BK, Earle JA, Baczko K, et al. Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture. J Gen Virol 1997;78:97– 106. [12] Hanses F, van Binnendijk R, Ammerlaan W, et al. Genetic variability of measles viruses circulating in the Benelux. Arch Virol 2000;145:541– 51. [13] Katayama Y, Shibahara K, Kohama T, Homma M, Hotta H. Molecular epidemiology and changing distribution of genotypes of measles virus field strains in Japan. J Clin Microbiol 1997;35:2651– 3. [14] Truong AT, Ammerlaan W, de Swart RL, et al. Molecular epidemiological analysis of measles virus strains from five Asian countries. 2001, in preparation. [15] de Swart RL, Wertheim-van Dillen PM, van Binnendijk RS, Muller CP, Frenkel J, Osterhaus AD. Measles in a Dutch hospital introduced by an immuno-compromised infant from Indonesia infected with a new virus genotype. Lancet 2000;355:201– 2 letter. [16] Rota PA, Liffick S, Rosenthal S, Heriyanto B, Chua KB. Measles genotype G2 in Indonesia and Malaysia. Lancet 2000;355:1557– 8 letter. [17] Mulders MN, Salminen M, Kalkkinen N, Hovi T. Molecular epidemiology of coxsackievirus B4 and disclosure of the correct VP1/2A(pro) cleavage site: evidence for high genomic diversity and long-term endemicity of distinct genotypes. J Gen Virol 2000;81:803– 12. [18] Mulders MN, Lipskaya GY, van der Avoort HG, Koopmans MP, Kew OM, van Loon AM. Molecular epidemiology of wild poliovirus type 1 in Europe, the Middle East, and the Indian subcontinent. J Infect Dis 1995;171:1399– 405. [19] Centers for Disease Control and Prevention. Measles — United States, 1999. MMWR Morb Mortal Wkly Rep 2000;49:557–60.
M.N. Mulders et al. / Vaccine 19 (2001) 2245–2249 [20] Jin L, Richards A, Brown DW. Development of a dual targetPCR for detection and characterization of measles virus in clinical specimens. Mol Cell Probes 1996;10:191–200. [21] Matsuzono Y, Narita M, Ishiguro N, Togashi T. Detection of measles virus from clinical samples using the polymerase chain reaction. Arch Pediatr Adolesc Med 1994;148:289–93. [22] Nakayama T, Mori T, Yamaguchi S, et al. Detection of measles virus genome directly from clinical samples by reverse transcriptase-polymerase chain reaction and genetic variability. Virus Res 1995;35:1– 16. [23] Rota PA, Khan AS, Durigon E, Yuran T, Villamarzo YS, Bellini WJ. Detection of measles virus RNA in urine specimens from vaccine recipients. J Clin Microbiol 1995;33:2485–8. [24] Kawashima H, Mori T, Takekuma K, Hoshika A, Hata M, Nakayama T. Polymerase chain reaction detection of the hemag-
.
[25] [26]
[27]
[28]
2249
glutinin gene from an attenuated measles vaccine strain in the peripheral mononuclear cells of children with autoimmune hepatitis. Arch Virol 1996;141:877– 84. Kreis S, Whistler T. Rapid identification of measles virus strains by the heteroduplex mobility assay. Virus Res 1997;47:197–203. Cassol S, Gill MJ, Pilon R, et al. Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper. J Clin Microbiol 1997;35:2795–801. Abe K, Konomi N. Hepatitis C virus RNA in dried serum spotted onto filter paper is stable at room temperature. J Clin Microbiol 1998;36:3070– 2. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL:X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25:4876– 82.