Virus Genes DOI 10.1007/s11262-015-1173-1

Long-term transmission of measles virus in Central and continental Western Europe S. Santibanez • J. M. Hu¨bschen • C. P. Muller • F. Freymuth • M. M. Mosquera • M. Ben Mamou • M. N. Mulders • K. E. Brown • R. Myers • A. Mankertz

Received: 20 August 2014 / Accepted: 16 January 2015 Ó Springer Science+Business Media New York 2015

Abstract The World Health Organization (WHO) has adopted an elimination goal for measles and rubella, which is supposed to be met in the WHO European Region (EUR) by 2015. For verification of elimination, it is required that the genotyping data of detected measles viruses provide evidence for the interruption of endemic transmission. In order to record and assess the extent of endemic measles virus (MV) circulation in a part of the EUR, we analyzed transmission chains of the epidemiologically most relevant MV variants identified in Central and continental Western Europe (CCWE) from 2006 to 2013. Based on MV sequence data deposited in the WHO global database for molecular surveillance of measles (MeaNS), the circulation period was calculated for each MV variant at the country-level and for the entire region of CCWE. The MV variants ‘‘D5-Okinawa,’’ ‘‘D4-Hamburg,’’ ‘‘D4-Manchester,’’ and ‘‘D8-Frankfurt-Main’’ spread widely in CCWE; they caused large and longS. Santibanez (&)
A. Mankertz WHO European Regional Reference Laboratory for Measles and Rubella, Robert Koch Institute, Seestr. 10, D-13353 Berlin, Germany e-mail: [email protected] J. M. Hu¨bschen
C. P. Muller WHO European Regional Reference Laboratory for Measles and Rubella, Institute of Immunology, Luxembourg Institute of Health/Laboratoire National de Sante´, 20A Rue Auguste Lumie`re, L-1950 Luxembourg, Luxembourg F. Freymuth National Reference Centre for Measles and Respiratory Paramyxoviridae, CHU Caen, Caen, France

lasting outbreaks with secondary spread that resulted in additional outbreaks. Nation-wide outbreaks (epidemics) with thousands of measles cases occurred in four countries (Switzerland, France, Bulgaria, and Romania) and were characterized by continuous detection of the same MV variant for more than 12 months suggesting endemic transmission. In the entire region of CCWE, the circulation period of the four predominant MV variants ranged from 18 to 44 months. The long-lasting MV transmission which affected predominantly unvaccinated individuals in different hard-to-reach groups and in the general population is not consistent with the measles elimination goal. Additional efforts are necessary to meet the elimination target in the EUR. Keywords Measles virus
Molecular surveillance
Measles elimination

M. M. Mosquera CIBER en Epidemiologia y Salud Publica, CIBERESP, Madrid, Spain M. B. Mamou WHO Regional Office for Europe, UN City, Marmorvej 51, DK-2100 Copenhagen Ø, Denmark M. N. Mulders World Health Organization Department of Immunization, Vaccines and Biologicals, Geneva, Switzerland K. E. Brown
R. Myers Virus Reference Department, Microbiology Services, Public Health England, London, UK

M. M. Mosquera Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain

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Introduction Measles virus (MV), a member of the genus Morbillivirus in the family Paramyxoviridae, is an aerosol-borne and highly contagious human pathogen. MV infection can lead to serious or even fatal complications like pneumonia, subacute sclerotic panencephalitis (SSPE), or measles inclusion body encephalitis (MIBE) and has caused millions of deaths. MV-induced immunosuppression results in increased susceptibility to opportunistic infections and is the major cause of infant death associated with acute measles [1]. The globally used live-attenuated measles vaccine confers a long-term immunity [2]. From 2000 to 2012, world-wide increase in coverage of routine immunization with measles-containing vaccines (MCV), plus supplementary immunization activities led to a 77 % decrease in reported annual measles incidence globally and a 78 % decline in estimated annual deaths from 562,400 to 122,000 [3]. The WHO European Region (EUR) has adopted an elimination goal for endemic measles by 2015. Endemic transmission is defined as continuous transmission of indigenous or imported MV that persists for C12 months in a defined geographical area [4]. A vaccination coverage of C95 % with two doses of a measles-containing vaccine (MCV) must be achieved and maintained in order to terminate endemic transmission and reach a low incidence of \1 case per 1 million population [5]. Genetic characteristics of detected wild-type MV combined with case-based epidemiological data are used to describe the transmission pathways of circulating MV variants. This information is required to identify endemic variants, and finally, to document interruption of transmission of endemic measles in the EUR [4]. The MV genome is a non-segmented, negative sense RNA that contains six genes which code for six structural proteins (N, P, M, F, H, and L) plus two non-structural proteins (C and V) [6]. The genes are separated by conserved intergenic trinucleotides [7]. The coding regions are preceded and followed by untranslated regions (UTRs) which include conserved transcription start and stop sequences, leading to the transcription of monocistronic mRNAs [8]. The length of the MV genome of 15,894 nucleotides (nt) is highly conserved; the first report demonstrating variation in genome size of wild-type MV has been published recently [8]. Although MV is considered a monotypic virus, genetic and antigenic variation has been detected in wild-type viruses with highest variability in the N, P, and H genes [9]. According to the WHO nomenclature using the nucleotide sequences of the genes N and H, wild-type MV are divided into eight clades (A–H) and further subdivided into 24 genotypes. Each genotype is represented by one or two WHO reference strains. The 450

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nt encoding the 150 C-terminal amino acids of the N protein (N-450 region) are a highly variable part of the MV genome and are accordingly used for genotype assignment [10]. A global database for molecular surveillance of measles (measles nucleotide surveillance, MeaNS) was started in 2008 as a joint project of the Public Health England (formerly Health Protection Agency) and the WHO [11]. The database collects sequence data of the N-450 region and the complete coding regions of the N and H genes. Case-based epidemiological information is necessary to create a name for a submitted sequence according to the convention of the WHO [10]. For the purpose of tracking transmission pathways, direct comparison of sequence data of detected MV is needed. Experiences from recent large outbreaks have shown that sequence identity within the N-450 region provides a useful mechanism for tracing the spread of certain measles virus variants [12]. Designating the N-450 sequence of an epidemiological relevant MV variant as ‘‘sequence variant’’ with a corresponding ‘‘Named Strain’’ listed in the MeaNS database further simplifies monitoring of transmission chains in a selected geographic area. Since the start of molecular surveillance of measles in the 1990s, substantial progress has been made globally and in the EUR. Monitoring of MV circulation in the past two decades shifted from analysis of randomly collected samples and data in a few countries to a systematic, area-wide, and continuous surveillance including reporting to the WHO on a regular basis. Molecular surveillance of measles is supported and coordinated by the WHO Global Measles and Rubella Laboratory Network (M/R LabNet) comprising 723 laboratories in 187 member states. The standardized nomenclature is used to describe the genetic diversity of wild-type MV and is regularly updated by the WHO. LabNet members are encouraged to submit MV sequence data plus the relevant case-based epidemiological information as soon as available to MeaNS. The data are thus accessible to all contributing users in an almost real-time modus and users are able to recognize and analyze MV transmission chains at an early stage in a global context. Since the beginning of molecular surveillance, the pattern of predominant MV genotypes has changed repeatedly in the WHO EUR. The indigenous European genotypes C2 and D6 were first detected in 1990 and were widespread throughout the region until approximately 2005. Genotype D6 circulated in the entire EUR [13], whereas detection of genotype C2 was restricted to the western and central parts [14]. From 2004 onward, spread of imported MV genotypes originating from areas outside the EUR such as B2, B3, D4, D5, D8, D9, G3, and H1 has been observed in all parts of the region. Several imported MV variants spread widely and initiated large outbreaks including several nation-wide outbreaks (epidemics) [15]. These outbreaks

Virus Genes

have been described mostly from a national perspective. The reports show the geographical and temporal distribution of cases, the genetic MV variant(s) identified with the source of importation, and the transmission pathway within the country. However, circulation of the predominant MV variants in the EUR was not restricted to a certain country. MV variants responsible for a large outbreak in one country were frequently exported to other countries of the EUR, thereby initiating secondary spread. The resulting chains of MV transmission have not yet been systematically analyzed in a supranational context or for the whole WHO EUR. Based on molecular data collected in the MeaNS database and published epidemiological information, the present study analyzes transmission chains of four epidemiologically relevant MV variants belonging to genotypes D4, D5, and D8 in Central and continental Western Europe (CCWE, Israel, and Turkey are included) from 2006 to 2013. Our objective was to record and assess the extent of endemic MV circulation within this part of the EUR.

Analysis of MV transmission chains MV variant ‘‘D5-Okinawa’’—the epidemic in Switzerland An increasing measles activity was recorded in the Canton of Lucerne from November 2006 onward. The sequence of the circulating MV (MVs/Lucerne.CHE/46.06) showed identity to the N-450 sequence of a genotype D5 virus named ‘‘D5-Okinawa’’ (Named Strain of the WHO: MVs/ Okinawa.JPN/37.06[D5]) indicating a putative import from Asia (Fig. 1). Prior to the epidemic in Switzerland, MV genotype D5 had been mainly detected in Cambodia and Thailand [16]. ‘‘D5-Okinawa’’ had been associated with one outbreak which started in Okinawa/Japan shortly before ‘‘D5-Okinawa’’ appeared in Switzerland [17]. The spread of ‘‘D5-Okinawa’’ to other Swiss cantons resulted in an epidemic consisting of three waves with peaks of reported case numbers in August 2007, March 2008, and March 2009 [18]. ‘‘D5-Okinawa’’ was continuously and nearly exclusively detected in Switzerland from November 2006 until June 2008 and was distributed country-wide (MVs/Zug.CHE/29.07[D5], MVs/Zurich.CHE/37.07[D5], MVs/Chur.CHE/05.08[D5], MVs/Biel.CHE/14.08[D5]). It was solely responsible for the first and the second wave of the epidemic, whereas three distinct genotypes (B3, ‘‘D4Enfield,’’ and ‘‘D5-Okinawa’’) contributed to the third wave. From January to March 2009, ‘‘D5-Okinawa’’ was detected mainly in the western part (French-speaking cantons) of Switzerland (MVs/Fribourg.CHE/05.09[D5], MVs/Lausanne.CHE/05.09[D5]). The MV genotype

pattern suggests that most of the 4,415 cases notified by the Swiss Federal Office of Public Health during the course of the epidemic (November 2006–September 2009) were associated with ‘‘D5-Okinawa.’’ The spread of this virus variant resulted in secondary outbreaks in Germany, Austria, and France, and additional cases in other countries [19] (Fig. 2). The first recognized secondary outbreak with 90 notified cases lasted from January to May 2007 in Lower Bavaria/Germany (MVs/Passau.DEU/11.07[D5]) [20]. Subsequent spread from Lower Bavaria to Upper Austria (MVs/Braunau.AUT/17.07[D5]) as well as to Lower Saxony/Germany (MVs/Hannover.DEU/21.07[D5]) initiated only limited local transmission. At the beginning of 2008, additional outbreaks were linked to cases in Switzerland and occurred in southern Baden-Wuerttemberg/Germany (MVs/Freiburg.DEU/05.08[D5], MVs/ Loerrach.DEU/07.08[D5]). Subsequent spread to several other locations in Baden-Wuerttemberg (MVs/Stuttgart.DEU/09.08[D5], MVs/Heilbronn.DEU/13.08[D5]) was observed until May 2008 [21]. A few cases in North Rhine-Westphalia (MVs/Mettmann.DEU/14.08[D5], MVs/ Bielefeld.DEU/16.08[D5]) were probably linked to cases in Baden-Wuerttemberg. In March 2008, an outbreak associated with ‘‘D5-Okinawa’’ started among Austrian and German students attending an anthroposophic school in the province of Salzburg/Austria. The presumed index case was a visitor from an anthroposophic school in Switzerland at the beginning of March. ‘‘D5-Okinawa’’ was continuously detected in the province of Salzburg/Austria (MVs/ Salzburg.AUT/12.08[D5]) and the neighboring southeastern Upper Bavaria/Germany (MVs/Bad Reichenhall.DEU/13.08[D5], MVs/Traunstein.DEU/15.08[D5]) until May 2008. Further spread of this virus variant to four other provinces in Austria (MVs/Vienna.AUT/16.08[D5], MVs/Zams.AUT/18.08[D5], MVs/Hall.AUT/20.08[D5], MVs/Linz.AUT/27.08[D5]) and other parts of Bavaria resulted in secondary outbreaks observed until July 2008; the largest secondary outbreak with 83 notified cases occurred in Muehldorf am Inn, Upper Bavaria (MVs/Muehldorf am Inn.DEU/17.08[D5]). In Austria, 394 cases were supposedly linked to the index case in Salzburg; most of them (N = 364) were reported from the provinces of Salzburg and Upper Austria [22]. In France, ‘‘D5-Okinawa’’ occurred initially only sporadically in the Iˆle-deFrance region in June 2007. Continuous and nation-wide circulation of this virus variant started probably in the Champagne-Ardenne region in February 2008 (MVs/Reims.FRA/07.08[D5]) and lasted until March 2009 [23]. The geographic distribution of cases with identification of ‘‘D5Okinawa’’ indicates spread to 14 of the 27 French regions. The highest proportion among the nation-wide detections accounted to the Rhoˆne-Alpes region where ‘‘D5-Okinawa’’ had appeared particularly frequently around the end

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Virus Genes MVs/Manchester.GBR/10.09 MVs/Lisieux.FRA/27.07 100

MVs/Maramures.ROU/04.11

Genotype D4

MVs/Hamburg.DEU/03.09

99 73

MVs/Enfield.GBR/14.07

MVi/Montreal.CAN/0.89 D4 U01976 MVi/Victoria.AUS/12.99 D9 AF481485 MVi/Illinois.USA/0.89/1 D3 U01977 MVi/Palau/0.93 D5 L46758 MVi/Bangkok.THA/0.93/1 D5 AF079555

72

Genotype D5

MVs/Okinawa.JPN/37.06

75 99

MVs/Lucerne.CHE/46.06 99

MVi/Manchester.GBR/30.94 D8 AF280803

Genotype D8

MVs/Frankfurt-Main.DEU/17.11 MVi/Menglian.Yunnan.CHN/47.09 D11 GU4405 MVi/Victoria.AUS/16.85 D7 AF243450 MVi/Illinois.USA/50.99 D7 AY037020

80

MVi/Bristol.GBR/0.74 D1 D01005 MVi/New Jersey.USA/0.94/1 D6 L46750 MVi/Johannesburg.ZAF/0.88/1 D2 U64582 MVi/Kampala.UGA/51.01/1 D10 AY923185 96

MVi/Amsterdam.NLD/49.97 G2 AF171232

75

MVi/Gresik.IDN/17.02 G3 AY184217

88

MVi/Berkeley.USA/0.83 G1 U01974 MVi/Hunan.CHN/0.93/7 H1 AF045212 MVi/Beijing.CHN/0.94/1 H2 AF045217

96

MVs/Madrid.ESP/0.94(SSPE) F X84865

76

100

MVi/Maryland.USA/0.77 C2 M89921 MVi/Erlangen.DEU/0.90 C2 X84872

71

MVi/Tokyo.JPN/0.84 C1 AY043459 MVi/Goettingen.DEU/0.71 E X84879 MVi/Maryland.USA/0.54 A U01987 MVi/Libreville.GAB/0.84 B2 U01994 MVi/Yaounde.CMR/12.83 B1 U01998

74 94

MVi/New York.USA/0.94 B3 L46753 MVi/Ibadan.NGA/0.97/1 B3 AJ232203

0.01

Fig. 1 Phylogenetic relationship between the predominant MV variants in CCWE, 2006–2013, and the WHO MV reference strains for each genotype. The MV variants analyzed in this study are marked with different colors; the same color code is used in Fig. 3. Phylogenetic analysis is based on a 450-nt sequence encoding the

C-terminus of the MV N-protein. The tree was constructed using the Neighbor-Joining method and the Kimura 2-parameter model of MEGA version 4 [37]. Only bootstrap values (1000 replicates) of at least 70 are shown. The scale bar indicates deviation of 1 nt per 100-nt sequence

of the year 2008. Resurgence of this virus variant in the French-speaking cantons of Switzerland in January 2009 might therefore be explained by re-importation from the neighboring Rhoˆne-Alpes region [18].

The transmission pattern of ‘‘D5-Okinawa’’ shows for Switzerland a continuous transmission for a period of 20 months indicating endemic transmission (C12 months), and for France an almost continuous transmission for a

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Virus Genes Fig. 2 Transmission pathway of MV variant ‘‘D5-Okinawa’’ in CCWE, 2007–2009. Transmissions with a known epidemiological link are marked by an arrow; detections without information on MV importation are indicated by asterisks

NOR

2007 2008 2009

DNK

DEU

FRA CHE

14-month period. In summary, our data show circulation of this virus variant over a 29-month period (November 2006– March 2009) in CCWE (Fig. 3). MV variant ‘‘D4-Hamburg’’—the epidemic in Bulgaria A measles outbreak started in Hamburg/Germany in December 2008, reached its peak in February and March 2009 and lasted until June 2009 [24]. The presumed index case came from London in November 2008 and showed measles symptoms in early December. The sequence of the MV identified for this outbreak showed a new variant of genotype D4 named ‘‘D4-Hamburg’’ (Named Strain of the WHO: MVs/Hamburg.DEU/03.09[D4]). ‘‘D4-Hamburg’’ is closely related to ‘‘D4-Enfield’’ (Named Strain of the WHO: MVs/Enfield.GBR/14.07/[D4]) which became endemic in the United Kingdom in 2008 (ECDC, 2008) (Fig. 1). The regional spread of ‘‘D4-Hamburg’’ in Germany resulted in expansion of the outbreak to Lower Saxony, a federal state south of Hamburg (MVs/Harburg.DEU/06.09[D4], MVs/ Wildeshausen.DEU/21.09[D4]). During the course of this

AUT

outbreak, 216 cases were notified in Hamburg and 53 cases in Lower Saxony [25]. In Bulgaria, measles virus started to circulate in 2009, after an 8-year era of absence of indigenous transmission [11]. The outbreak started in the northeast of the country where MV ‘‘D4-Hamburg’’ was detected from April 2009 onward (MVs/Shumen.BGR/15.09/1[D4], MVs/Silistra.BGR/21.09/1[D4]). The index case in the Bulgaria outbreak was a member of a Roma community from Hamburg and visited Razgrad district in northeastern Bulgaria. The rapidly developing epidemic proceeded from northeast to southwest Bulgaria (MVs/Blagoevgrad.BGR/02.10[D4], MVs/Plovdiv.BGR/03.10/1[D4]); the peak of the recorded case numbers was reached in March 2010. From April 2009 until the second half of 2010, a total of 24,253 cases were reported; 24 cases were fatal [26]. ‘‘D4-Hamburg’’ has been detected in Bulgaria ultimately in March 2011 (MVs/VelikoTarnovo.BGR/10.11/1[D4]). Approximately, 90 % of the affected patients belonged to the ethnic minority of Roma. During the epidemic, spread of ‘‘D4-Hamburg’’ to other Balkan countries and over long distances had initiated secondary outbreaks in several parts of Europe [27]. In 2009,

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Virus Genes

three outbreaks with a total of 54 cases occurred in Roma communities in Poland (MVs/Lodz.POL/27.09[D4], MVs/ Pulawy.POL/28.09[D4]). Furthermore, spread from Bulgaria to Ireland (MVs/Kerry.IRL/40.09[D4]) and from there into Northern Ireland/United Kingdom (MVs/Belfast.GBR/ 50.09[D4]) resulted in small outbreaks in both countries. In 2010, ‘‘D4-Hamburg’’ had initiated secondary outbreaks in the Balkan region affecting Greece with 91 laboratory-confirmed cases linked to Bulgarian Roma people (MVs/ Amaliada.GRC.12.10[D4], MVs/Pyrgos.GRC/19.10[D4]), the former Yugoslav Republic of Macedonia with 908 reported cases (August 2010–August 2011; MVs/Kumanovo.MKD/35.10/1[D4], MVs/Skopje.MKD/44.10/1[D4]) [28], and Serbia with 363 reported cases (December 2010 August 2011; MVs/Leskovac.SRB/08.11/1[D4]) (J. Nedeljkovic´, personal communication, November 2014). Moreover, ‘‘D4-Hamburg’’ occurred sporadically in Austria (MVs/Graz.AUT/12.10[D4], MVs/Vienna.AUT/13.10 [D4]), Romania (MVs/Timis.ROU/18.10/1[D4]), and Turkey (MVs/Istanbul.TUR/20.10[D4]). In the same year, multiple re-importations of this MV variant from Bulgaria into Germany had caused an outbreak in Munich/Bavaria (MVs/Muenchen.DEU/22.10[D4]) and small outbreaks in Mannheim/Baden-Wuerttemberg (MVs/Mannheim.DEU/ 07.10[D4]) and Essen/North Rhine-Westphalia. Several other sporadic cases resulted also from re-importation from Bulgaria (MVs/Eichstaett.DEU/18.10[D4], MVs/Ludwigshafen.DEU/21.10[D4]). The outbreak in Munich with 48 cases reported from June to August 2010 has shown an initial virus transmission among Bulgarian Roma residing in a migrant camp (28 cases) with subsequent spread into the general population. At the end of 2010, small outbreaks associated with ‘‘D4-Hamburg’’ occurred in Germany in homes for migrants in Neumuenster/Schleswig–Holstein (MVs/Neumuenster.DEU/47.10/1[D4]) and Karlsruhe/ Baden-Wuerttemberg (MV/Karlsruhe.DEU/48.10[D4]). In the first half of 2011, ‘‘D4-Hamburg’’ was detected in Lausanne/Switzerland in a sporadic case that became infected in Serbia (MVs/Lausanne.CHE/02.11[D4]), and during an outbreak in anthroposophic schools in Gent/Belgium (MVs/ Ghent.BEL/09.11/1[D4]) with 56 affected cases [29]. In Bulgaria, molecular surveillance was not continuously maintained during the epidemic. Identification of MV in samples that were collected five times within a 24-month period has shown the presence of MV variant ‘‘D4-Hamburg.’’ Continuous circulation of this MV variant during the entire epidemic can be therefore assumed. Our data show circulation of ‘‘D4-Hamburg’’ in CCWE for a 29-month period (January 2009–May 2011) (Fig. 3). Although ‘‘D4-Hamburg’’ was not detected during a fivemonth gap in the second half of 2009, undetected circulation in the region can be assumed.

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MV variant ‘‘D4-Manchester’’—epidemics in France and Romania France reported an increasing measles activity in mid-2008. The case numbers were evolving in three epidemic waves each higher than the one before with a total of 21,669 cases (10 fatalities) reported from October 2008 until September 2011. After a first low wave of the epidemic associated with MV variants ‘‘D5-Okinawa’’ and ‘‘D4-Manchester,’’ peaks of reported case numbers were observed for the second wave (3,429 cases) in April 2010, and for the third wave (16,466 cases) in March 2011 [30]. The MV variant, ‘‘D4Manchester’’ (Named Strain of the WHO: MVs/Manchester.GBR/10.09[D4]) is closely related to variant ‘‘D4Enfield’’ and was first detected in July 2007 in France (MVs/Lisieux.FRA/27.07[D4]) (Fig. 1). ‘‘D4-Manchester’’ became the predominant variant during the epidemic until August 2012 [30]. Moreover, ‘‘D4-Manchester’’ frequently appeared in the southwest of Germany starting in May 2010. Long-distance spread of this MV variant by 13 persons who had attended meetings in Taize´/France resulted in an outbreak with 37 cases reported from the German federal states of Baden-Wuerttemberg (MVs/Villingen-Schwenningen.DEU/37.10[D4]), North-Rhine-Westphalia, and Bavaria in September and October 2010 (Pfaff et al., 2010). In 2011, a significant increase in measles activity associated with circulation of ‘‘D4-Manchester’’ was observed in several countries bordering to France. In Belgium, ‘‘D4Manchester’’ had contributed to the resurgence of measles activity recorded in 2011 (MVs/Brussels.BEL/08.11[D4]) [29]; in Switzerland, it had caused a large outbreak with 219 notified cases in the canton of Geneva that lasted from January until August 2011 (MVs/Geneva.CHE.04.11/ 1[D4]) [31]; in Germany, it was mainly responsible for the [threefold increase in the number of measles cases recorded in Baden-Wuerttemberg in 2011 versus 2010 (526 notifications in 2011 vs. 151 in 2010) [32]. In Romania, the first measles case with detection of variant ‘‘D4-Manchester’’ had been directly imported from France in February 2010 and was identified as index case of an outbreak in Tulcea located in the southeast of the country (MVs/Tulcea.ROU/08.10[D4]). At the same time, an epidemic started in Romania that lasted more than 3 years. It was associated with ‘‘D4-Manchester’’ and its descendants of the first and second generation (MVs/Maramures.ROU/03.11[D4], Named Strain of the WHO; MVs/Ilfov.ROU/06.11[D4]). About 7,300 notified cases in Romania were attributed to this MV lineage until the end of 2012 [33]. The last detection of ‘‘D4-Manchester’’ was in August 2013 in Romania (MVs/Calarasi.ROU/32.13[D4]). Unlike the previously described MV variants ‘‘D5Okinawa’’ and ‘‘D4-Hamburg’’ that were endemic in only

Virus Genes

one country of CCWE, variant ‘‘D4-Manchester’’ established endemic transmission in two distant countries, France and Romania. For France, the data show appearance of ‘‘D4-Manchester’’ in the years 2007–2012 with continuous transmission for 20 months from January 2011 until August 2012. In Romania, ‘‘D4-Manchester’’ was detected frequently throughout a 32-month period, suggesting continuous transmission during the entire course of epidemic. In summary, ‘‘D4-Manchester’’ was active in CCWE over a period of at least 44-months (January 2010–August 2013) (Fig. 3). MV variant ‘‘D8-Frankfurt-Main’’—multiple outbreaks in Germany Dissemination of measles via an international mass gathering held in Berlin/Germany in February 2013 resulted in spread of MV within Germany (MVs/Hamburg.DEU/ 11.13[D8]) and transmission to Sweden (MVs/Malmo.SWE/ 9.13[D8]). Measles cases linked to the mass gathering showed the variant ‘‘D8-Frankfurt-Main’’ (genotype D8, Named Strain of the WHO: MVs/Frankfurt-Main.DEU/ 17.11[D8]) (Fig. 1). MV genotype D8 is endemic in India [11]; the variant ‘‘D8-Frankfurt-Main’’ was first detected in 2011 in Germany. In 2013, ‘‘D8-Frankfurt-Main’’ had initially caused an outbreak in Berlin and the neighboring federal state of Brandenburg with a total of 537 notified cases (RKI, 2013) (MVs/Berlin.DEU/10.13[D8], MVs/Potsdam.DEU/17.13[D8]). Secondary cases and outbreaks occurred in several other German federal states. In Bavaria, a large outbreak with 787 notified cases developed from April 2013 onward. All genotyped cases indicated association with MV variant ‘‘D8-Frankfurt-Main’’ (MVs/Muenchen.DEU/ 16.13[D8]) [34]. Further, small outbreaks occurred in Saxony-Anhalt, Saxony, and Thuringia. The transmission chain ‘‘D8-Frankfurt-Main’’ lasted in Germany over a 9-month period until November 2013 suggesting that it was not yet to be classified as ‘‘endemic’’ since it circulated for less than 12 months. Prior to detection in Germany, ‘‘D8-Frankfurt-Main’’ was observed in Turkey with a continuous transmission for a 9-month period from September 2012 until May 2013 (MVs/Istanbul.TUR/ 36.12[D8], MVs/Sakarya.TUR/19.13[D8]) and in Romania where it co-circulated with ‘‘D4-Manchester’’ (MVs/Gorj.ROU/26.12[D8], MVs/Suceava.ROU/41.12[D8] [33]. Judged from the national perspectives, transmission of MV variant ‘‘D8-Frankfurt-Main’’ was not endemic in any of these three countries. However, region-wide analysis in the context of CCWE indicates almost continuous persistence of ‘‘D8-Frankfurt-Main’’ over an 18-month period (June 2012– November 2013) (Fig. 3). Thus, our results demonstrate endemic transmission of a fourth MV variant not in a country but in CCWE.

Limitations and restrictions of the study This study is based on the analysis of the temporal distribution of selected MV variants in countries of CCWE. This approach is limited since deviating procedures for collection and selection of samples for MV genotyping and submission of sequence data to the MeaNS database are implemented in the individual countries. Supposedly, molecular data are under-reported due to discontinued genotyping during large outbreaks. This would result in an underestimation of the temporal extension of a given transmission chain. Furthermore, several outbreak reports that are included in the present study have been published when the outbreak was still ongoing and follow-up reports are missing, leading to an underestimation of cases attributed to a certain MV variant or genotype. It should also be noted that the term outbreak is used differently; some authors attributed exclusively cases belonging to the same chain of virus transmission to a certain outbreak, whereas other authors counted measles cases without distinguishing between different transmission chains. For these reasons, it is not possible to attribute an accurate number of measles cases to a certain transmission chain. Since the present study is restricted to analysis of geographical and temporal distribution of MV variants and epidemiological data were incomplete, it was not always possible to distinguish between indigenous transmission and importation.

Discussion Absence of endemic transmission of MV is an essential criterion in order to document that the elimination goal for measles has been achieved in a defined geographical area. With the WHO EUR approaching this goal, it is of particular importance to analyze MV transmission chains in a national and in a supranational (i.e., regional) context. This study describes the transmission chains of the most relevant MV variants identified in CCWE for the period 2006–2013. This part of the EUR consists of 38 countries with widely differing population sizes and a total population of approximately 559 million inhabitants. Published information on molecular surveillance and sequence data deposited in the MeaNS database were compiled and evaluated in order to gain a comprehensive overview of long-term transmission of MV in CCWE. CCWE has recently experienced several large outbreaks including four epidemics (nation-wide outbreaks) that were associated with MV genotypes D4, D5, and D8 imported from measles-endemic areas outside of the EUR [11]. The epidemics were observed in Switzerland (2006–2009), France (2008–2011), Bulgaria (2009–2011), and Romania (2010–2013) and were attributed to the MV variants ‘‘D5-

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Virus Genes Country

Detection by year and month

MV variant D5-Okinawa 2006 11 12

2007 1

2

3

4

5

6

7

8

9 10 11 12

4

5

6

7

8

9 10 11 12

1

2

1

2

3

4

2008 1

2

3

4

5

6

7

8

9 10 11 12

3

4

5

6

7

8

9 10 11 12

1

2

2

3

4

2009 1

2

3

Austria Belgium Denmark* France* Germany Netherlands* Norway* Romania Spain Switzerland CCWE MV variant D4-Hamburg 2009 1

2

3

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Virus Genes b Fig. 3 Geographical and temporal distribution of the predominant

MV variants ‘‘D5-Okinawa,’’ ‘‘D4-Hamburg,’’ ‘‘D4-Manchester,’’ and ‘‘D8-Frankfurt-Main’’ in CCWE. The period from the first detection to the latest detection in 2013 is shown for each MV variant. The MeaNS database served as data source.*Additional data were provided by the National Reference Laboratory. **The country name ‘‘Macedonia’’ stands for the Former Yugoslav Republic of Macedonia

Okinawa,’’ (Switzerland) ‘‘D4-Hamburg,’’ (Bulgaria) and ‘‘D4-Manchester’’ (France and Romania). Furthermore, the MV variant ‘‘D8-Frankfurt-Main’’ had caused large outbreaks in Germany in 2013. In order to get an overview on MV circulation in the entire region of CCWE, we analyzed data up-loaded to MeaNS and could thereby calculate the total circulation period for each of the four MV variants. Variant ‘‘D5Okinawa’’ initiated a continuous circulation in CCWE immediately upon its introduction into Switzerland; the transmission chain persisted over 29 months from November 2006 to March 2009. In contrast, the other variants were already circulating in Europe prior to initiating large outbreaks. The variants ‘‘D4-Hamburg’’ and ‘‘D4-Manchester’’ had probably evolved from their common ancestor ‘‘D4-Enfield’’ that was circulating in Europe since 2007. ‘‘D4’’-Hamburg was introduced from the United Kingdom into Germany in December 2008 and circulated in CCWE for 29 months until May 2011. For ‘‘D4-Manchester’’ that had appeared in CCWE already in July 2007; continuous circulation has been recorded for a 44-month period from January 2010 to August 2013. The variant ‘‘D8-Frankfurt-Main’’ was first detected in CCWE in 2011; continuous circulation can be assumed for 18 months from June 2012 to November 2013. Persistence of each of the four epidemiologically most relevant MV variants over a much longer period than 12 months in CCWE indicates endemic transmission. Circulation of MV variant ‘‘D5-Okinawa’’ was restricted mainly to Switzerland and its neighboring countries. In contrast, the variants ‘‘D4-Hamburg’’ and ‘‘D4-Manchester’’ spread over long-distance routes from Western Europe to the Balkan region and back again and resulted in outbreaks in distant regions. The difference in geographic expansion can be explained by virus spread in different groups of susceptibles: ‘‘D5-Okinawa’’ was transmitted in the German-speaking countries among members of anthroposophic communities [18], whereas ‘‘D4-Hamburg’’ was distributed across Europe by travelers of the ethnic minority of Roma [27]. Transmission of ‘‘D4-Manchester’’ in Romania was also initiated by Roma communities [33]. This demonstrates MV spread in CCWE by hard-to-reach and vaccine-skeptic populations. We identified four countries in CCWE (Switzerland, France, Bulgaria, and Romania) with endemic transmission

(C12 months) of a certain MV variant. Continuous MV transmission in these countries ranged from 20 to 32 months. Additional countries with shorter MV transmission chains of C6 and\12 months (Germany, Belgium, Greece, Italy, Serbia, Spain, and Turkey) have contributed to maintain and prolong long-term circulation in this area. However, it must be noted that most countries of CCWE did not show long-term MV circulation. Our study has shown long-lasting MV transmission in the WHO EUR which affected unvaccinated individuals in different hardto-reach groups passing it on to the general population. This situation is not consistent with the measles elimination target which has already been achieved in America [35] and Australia [36]. Additional efforts are necessary to close the identified immunity gaps in the population of CCWE. Since elimination of MV must be documented for the entire EUR, this study should be extended in order to gain a comprehensive overview on MV circulation all over Europe. Acknowledgments Sabine Santibanez, Annette Mankertz, and Judith Hu¨bschen wish to thank Ms. Petra Kurzendo¨rfer, Anne Wolbert, and Emilie Charpentier for excellent technical support. We thank Stephan Aberle, Lasse Rasmussen, Julia Dina, Kirsti Vainio, George Necula, Mihaela Lazar, Juan E. Echevarrı´a, Ana Castellanos, Samuel Cordey, Pascal Cherpillod, Jean-Luc Richard, Veronik Hutse, Viki Indenbaum, Fabio Magurano, Robert van Binnendijk, Paula Palminha, Mia Kontio, Elina Horefti, Vaso Poga, Tatjana Kolupajeva, Agata ˚ sa Wiman, Lena Wehlin, Gulay KorMako´wka, Katarina Prosenc, A ukluoglu, Stefka Ivanova, and George Mitis for the permission to cite sequence data submitted to MeaNS, David Featherstone, and all colleagues of the WHO Global Measles and Rubella Laboratory Network for continuous inspiration and cooperation.

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