Molecular epidemiology of measles virus

seminars in VIROLOGY, Vol 6, 1995: pp 379-386

Molecular epidemiology of measles virus Paul A. Rota, Jennifer S. Rota and WiUiam J. Bellini

Live-attenuated vaccines for MV were introduced in the 1960s, and vaccination programs have greatly reduced the incidence of measles in developed countries. Still, sporadic outbreaks and some sustained epidemics of measles continue to occur. For example, approximately 50,000 cases of measles were reported in the U.S. between 1988 and 1991, a 10-fold increase in the n u m b e r of cases reported for the previous 3-year period. ~ One reason for this resurgence of measles cases was the failure to maintain high vaccination rates, particularly in urban areas. 6 Initiating a two-dose vaccination schedule along with aggressive childhood vaccination programs resulted in a historic low n u m b e r of cases (312) in 1993 and only 915 cases in 1994. 7.8 The success of recent mass vaccination campaigns in the Caribbean, and South and Central America has once again suggested that global eradication of measles may be possible. While reaching this goal will require continued commitment to increase vaccination coverage levels, the laboratory will play a critical role in monitoring the success of measles control strategies by developing more sensitive diagnostic reagents and by providing genetic analysis of measles strains. This chapter will review progress made in characterizing the genetic heterogeneity among wildtype MV and describe how this genetic information has already made valuable contributions toward understanding the epidemiology of MV.

Genetic analysis of viruses associated with recent outbreaks of measles in the United States indicated that at least four genotypes were present during 1994 and 1995. None of these more recent genotypes were related to the genotype responsible for the resurgence of measles cases in the United States between 1989 and 1992. The sequence data confirmed that the majority of measles cases that occurred in the United States between 1994 and 1995 were the result of international importation of virus. The data also suggested that transmission of the genotype associated with the resurgence had been interrupted by aggressive control measures. Therefore, molecular epidemiologic studies will provide a powerful means to measure the success of measles control strategies.

Key words: genetic variation / measles virus / molecular epidemiology / genetic variation ©1995 Academic Press Ltd MEASLES WRUS (MV), a negative-stranded RNA virus (Figure 1), is a m e m b e r of the morbillivirus genus of the Paramyxoviridae. MV is most closely related to a n u m b e r of animal morbilliviruses, such as canine distemper virus and rinderpest virus. MV infects only humans and subhuman primates and there are no known reservoirs for the virus outside of the human population. MV is highly contagious and causes a disease characterized by high fever, cough, coryza, conjunctivitis and the appearance of a maculopapular rash. Most individuals recover from acute measles, but severe complications can include giant cell pneumonia, inclusion body encephalitis (MIBE) and, rarely, subacute-sclerosing panencephalitis (SSPE). Since measles infection is associated with immunosuppression, secondary bacterial infections are common. In developing countries, fatality rates for measles can be as high as 15% and measles causes approximately one million deaths among infants and children in these countries every year. a-4

Genetic analysis of vaccine str~in.q

From the Measles Virus Section, Respiratory and Enteric Viruses Branch, Division of Viral and RickettsialDiseases, National Center for Infectious Diseases, Centersfor Disease Control and Prevention, Atlanta, GA30333, USA ©1995 Academic Press Ltd 1044-5773/95/060379 + 08 $12.00/0

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The live-attenuated measles vaccine currently used in the United States, Moraten, was derived from the prototype Edmonston strain which was originally isolated in 1954. 9 Edmonston has also been the progenitor strain for many of the measles vaccines currently used worldwide. Still, other measles vaccines were developed using locally isolated strains of measles in Russia, China and Japan. 1° Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, the sequences of the hemaggiutinin (H), fusion (F), nucleoprotein (N), and matrix (M) genes from all o f the vaccine

P. A. Rota et al

the observation that wild-type viruses isolated during the 1950s and 1960s were also genetically similar to wtEd suggest that a single genotype of MV had nearly worldwide distribution at one time. ~2 Sequence comparisons of the su'uctural genes of vaccine and wild-type strains of MV have not identified nucleotide or amino acid substitution(s) that consistently differentiate between all vaccine and wildtype strains. The continuing efforts to locate potential virulence markers are now focusing on the RNA dependant RNA polymerase (L gene) and the noncoding terminal and intergenic regions of MV. l~ These efforts will be aided by the development of new

strains differed by no more than 0.6% at the nucleotide level. In fact, the sequence of the entire 15,894 nucleotide genome of the Edmonston-derived, AIK-C strain was found to have only 56 nucleotide substitutions compared with Edmonston. 1~ Several non-Edmonston-devived vaccines contained e n o u g h genetic heterogeneity to confirm their indep e n d e n t origins but, overall, the sequences of both Edmonston-derived and non Edmonston-derived vaccine strains were more closely related to the sequence of a low-passage seed of the original Edmonston strain (wtEd) than they were to most of the recent wild-type viruses described below. These data combined with

50S negative- sense genomic RNA of measles virus

N

3'

I

P/C/V

J

F

M

I

H

I

]

L I

monocistronic mRNAs r

--tP"

H

Figure 1. Schematic representation of the structure of measles virus. Upper panel shows the arrangement of the viral genome. The MV genomic RNA is 15893 nucleotides in length. Arrows indicate monocistronic messages for each gene and number of arrows represents relative abundance of each mRNA (N, nucleoprotein; P, phosphoprotein; C, C protein; V, V protein; M, matrix; F, fusion; H, hemagglutinin; L, polymerase). Three gene products are coded for in the measles P gene. The P and C proteins are coded for on the same mRNA and are generated by translation from alternative start codons in overlapping reading frames. The V protein is translated from a mRNA generated by RNA editing. 3 2 '3 3 Lower panel shows diagram of the MV virion. 380

5'

Molecular epidmniology of measles virus Table 1. Comparison of sequences obtaiqed from me,~sles cases with severe outcomes to vaccine and wild-type strains of MV. Case description Giant cell pneumonia, USA, 1989 Giant cell pneumonia, USA, 1991 MIBE, USA, 1989 MIBE, USA, 1989

Gene*

% VacJ- % WT$

H

0.3

3.0

H N N

0.4 2.5 2.9

3.0 0.3 0.4

* Gene sequenced. t Percentdifference from Moraten vaccinestrain. $ Percent difference from Chicago-1wild-tupestrain from 1989. in-vitro and in-vivo assays for attenuation as well as the eventual application of infectious clone technology. 14 The database of genetic information for measles vaccines has contributed to investigations of measles cases with severe outcomes (Table 1). Analysis of MV sequences amplified from tissue specimens was used to confirm the presence of either vaccine or wild-type viruses in these cases.

G e n e t i c a n d a n t i g e n i c diversity o f wild-type measles viruses Measles has been considered to be an antigenically and genetically stable monotypic virus and, until recently, most genetic studies had focused on the

characteristics of measles viruses associated with severe neurologic diseases such as MIBE and SSPE. However, it has become apparent that multiple lineages of wild-type viruses are present. Genetic heterogeneity has been described for the N, F, H, M, phosphoprotein (P), and polymerase (L) genes. 15-~5 The greatest degree of genetic diversity is f o u n d in the C-terminal 450 nucleotides of the coding region of the N gene, 25 though the entire coding regions of the H and N genes have approximately the same degree of nucleotide sequence diversity. Compared to the H and N genes, the F, M, and L genes of wild-type MVs are relatively conserved. 12A7'2° Genetic heterogeneity has also given rise to antigenic variation a m o n g wild-type MV. Monoclonal antibodies have detected changes in epitopes on the H and N proteins of recent wild-type viruses. 26"27 Antigenic changes in the H protein have also been detected using serum specimens from either vaccinated or recently infected humans. While antibody induced by vaccination was able to neutralize wildtype MV, the results indicated that the wild-type H protein contained new a n d / o r altered epitopes compared to the H protein from vaccine virus. 27 Since the H protein is the major target for virus neutralizing antibodies, the antigenic properties of wild-type MV will have to be monitored to assess the stability of cross-reactive epitopes. Current research efforts to precisely define the epitopes on the H and N proteins that are targets of the h u m a n i m m u n e response will

Table 2. Geographic distribution ofgenotypes of measles virus associated with recent outbreaks (1988-1995) Group 1 Edmonston* all vaccines* Argentina Russia China

Group 4 1954 1950s-1960s 1991 1991 1993

United States (NewJersey) Spain England

Group 2 United States England Guam Japan China Canada

Group 5 1988-1992 1988-1993 1994 1983-1994 1993 1988

United States (Tennessee) Spain Netherlands Germany

Group 3 United States (Colorado) 'rfiail[a°anan d

1994-1995 1993-1995 1993-1994

1994 1992 1991 1992-1993

Group 6 1994-1995 1994 1993

Gambia Gabon* Cameroons* Zambia Kenya

* Reference strains were added for clarification. 381

1991 1984 1983 1992 1994

P. A. Rota et al

help put the sequence data into a more meaningful context. Biochemical and genetic analyses of diverse wildtype viruses have contributed to understanding the basic biology of MV. For example, the H proteins of some wild-type viruses contain an additional N-linked giycosylation site at amino acid 416, 2°'2s while other wild-type H proteins are missing the glycosylation site at amino acid 238. The H proteins of many of the wildtype viruses do not agglutinate vervet monkey erythrocytes in isotonic conditions 2s though some exhibit salt-dependent agglutination (unpublished observations). The biological differences between strains

have been described more extensively in a recent review, is

Temporal a n d g e o g r a p h i c d i s t r i b u t i o n o f genetic groups of MV Currently circulating wild-type MVs can be divided into five or six distinct genetic groups based on sequencing of the H a n d / o r N genes (Table 2, Figure 2). Though the boundaries between these groups and the nomenclature used vary somewhat between individual reports, 19'21 the assignment of genetic groups

Tennessee, USA:1994 Illinois, USA:1994 Madrid, Spain: 1992 Ithoven, Netherlands:1991 F

Group 5

Thailand: 1993

New Mexico, USA:1995 Colorado, USA:1994-1995 E Palau:1993

Washington, USA:1994 Nebraska, USA:1994 (Japan:1994) Japan: 1988 Guam:1994 I U.S. 1989-1992 Vermont & New Jersey, USA:1994 Madrid,Spain:1993-94 Michigan, Colorado, USA:1995 England: 1993 Gambia: 1991 I Gabon:1984 Zambia: 1993 r - Cameroons:1983 I New York, USA: 1994 (Kenya:1994) m ~ wt-Edmonston -- vaccines Me China: 1993 = 10NT Argentina:1991 Moscow, Russia:1991

1

Group 3

Group 2

Group 4

Group 6

Group 1

Figure 2. Genetic relationships of MV isolated from recent outbreaks. Phenogram is based on the sequences of the H gene. 382

Molecular epidemiology of measles virus as described below are well supported by phylogenetic analysis. Group 1 appears to have had a very wide distribution during the pre-vaccine era and contains the prototype Edmonston strain and all of the known vaccine strains. This group also contains several wildtype viruses that were isolated in the U.S. and Europe in the 1950s and 1960s. Sequences of MV from this group were also detected in RNA extracted from formalin-fixed lung tissue of Brazilian children who had died with giant cell p n e u m o n i a during the 1970s (unpublished observations). Wild-type viruses from group 1 have been isolated in Argentina, Russia and China in 1991-1993. Each of these strains has several unique nucleotide substitutions in the H, N, or F genes that differentiate them from the vaccine strain or Edmonston so it is unlikely that they represent laboratory contaminants. G r o u p 2 contains viruses that were associated with the resurgence of measles cases in the U.S. between 1989 and 1991. Also included in this diverse group are recent isolates from Japan, China, Canada and Guam and there is evidence that viruses from group 2 circulated in J a p a n as early as 1983. 22 Groups 3 and 4 were defined very recently after analysis of viruses associated with outbreaks occurring between 1993 and 1995. Group 5 contains viruses from recent outbreaks that were related to a strain that was

isolated in the U.S. in 1977. Viruses within group 6 show a high degree of genetic heterogeneity and these strains have been isolated in Africa or traced to direct importations from African countries. Several other genetic groups that are distinct from those described above have been identified. However, viruses from these groups have not b e e n isolated in at least 10 years and most likely represent extinct or inactive lineages, a2 Aside from the African group (group 6), there appears to be no geographic restriction to the circulation of these c o n t e m p o r a r y genotypes of MV. Rather, it seems that multiple genotypes can co-circulate in any area. T h e true extent of genetic diversity a m o n g wild-type MV is still unknown. T h e r e have been very few recent isolations from many parts of the world including most of South America, Asia and Africa. In fact, preliminary sequence analysis of recently isolated strains from China indicated the existence and widespread circulation of a previously undescribed lineage of MV (unpublished observations).

Genetic stability o f c i r c u l a t i n g genotypes T h e a m o u n t of genetic heterogeneity that occurs within a genetic group during an outbreak was addressed by sequencing the H and N genes from 12 wild-type MVs isolated t h r o u g h o u t the U.S. between 1989 and mid-1992. These viruses were isolated over a three-year span from a variety of cases ranging from uncomplicated measles to fatal infections. T h e H genes all contained the same 53 nucleotide substitutions relative to the sequence o f w t E d (Figure 3) and, overall, the sequences of the H and N genes of these viruses varied by less than 0.5%. 21 This observation demonstrated that MVs associated with a single epidemic are nearly h o m o g e n e o u s and suggests that the virus undergoes little genetic change during continuous person-to-person transmission. It is also a p p a r e n t that a single, p r e d o m i n a n t viral genotype was responsible for the bulk of the 50,000 measles cases that occurred during the resurgence of measles in the U.S. (Figure 4).

- IL-2 1989 .~IL-3 1989 IL-4 1989 CA 1990

fL

TX 1992 TX-1 1989 TX-2 1989 -1 1989 1989 1990

I

Wt Edm 1954

I = 6 nucleotides

Diversity of genotypes isolated in 1994--95 in the U.S.

Figure 3. Genetic variation of MV within an epidemic. Phenogram shows the genetic relationships between the H genes of 10 wild-type MVs isolated during the resurgence of MV in the United States between 1988 and 1992. Recent isolates from Illinois (ILL California (CA), Texas (TX) and Pennsylvania (PA) were compared to a low passage seed of the Edmonston strain (wtEd).

In 1994 and 1995, several sporadic outbreaks of measles occurred in the U.S. Analysis of sequence data indicated that at least four distinct genotypes 383

P• A. Rota et al

USA: 1988-1992

Key to genotypes Group 2 [~

Group3 Group 4

Group 5 Group 6

USA: 1994-1995 •

IL

[~ P - ~ r ~

Group3,4

fq

Japan

England

-

Kenya Spain

Asia ?

ral Europe?

Figure 4. Change in distribution of genotypes of MV in the United States between 1988-1992 and 1994-1995. States from which MV isolates were obtained are shaded to indicate the viral genotype. On lower panel, solid arrows indicate confirmed or suspected (?) sources of imported viruses. Dashed arrows indicate spread of MV after a single suspected importation into Las Vegas. No virus was isolated from states without shading, but arrows indicate possible spread based on epidemiological linkage• were present (Figures 2,4). None of the viruses isolated in the U.S. were similar to the genotype (group 2) that had circulated widely during the resurgence. Therefore, the outbreaks occurring in 1994--1995 resulted from importation of virus into the U.S. In some cases, the index case could clearly be traced to an importation while in others the source of virus was n o t identified. One of the largest outbreaks, which occurred in New Jersey in early 1994, was traced to a Spanish

student newly arrived to a university campus. This was confirmed by sequence analysis of MV isolated from cases in New Jersey that were nearly identical to viruses that had been isolated in Spain in 1993 (Figure 4). Single measles cases in Vermont and New York were the result of infected children returning from England and Kenya, respectively• In both cases, sequence analysis of the viral isolates confirmed the source of the viruses (Figures 2,4)• Later in 1994, an outbreak at a school in Illinois was 384

Molecular epidemiology of measles virus believed to have been the result of transmission of virus from New Jersey. However, sequence analysis indicated that the virus from Illinois was of a different genotype (Figures 2,4). The MV from Illinois was more closely related to viruses which had circulated in Central Europe and to a virus that had been isolated from an earlier outbreak in Tennessee. In this case, sequence data clearly linked the outbreaks in Tennessee and Illinois to imported viruses even though no index case was identified in either outbreak. ~l In late 1994, measles outbreaks occurred in several states, including Colorado, and standard epidemiology traced the index cases in all of the states to a casino in Las Vegas, Nevada. T h o u g h no source was identified for this outbreak, sequence analysis showed that isolates from Colorado were most closely related to a virus that had been isolated from a Japanese student arriving in Nebraska earlier in the year. The likelihood that these viruses were brought to the U.S. from Asia was strengthened by the observation that a similar virus was isolated in Thailand in early 1993 (Figure 2). An unknown source outbreak in New Mexico in February 1995 was also linked to the Las Vegas casino based on sequence data (Figures 2,4). In May 1995, another measles outbreak occurred in Colorado and, again, no source was identified. The genotype of MV associated with this outbreak was similar to that of the New Jersey isolate from 1994. This same genotype was f o u n d in Michigan where five cases were traced to a German visitor (Figure 4). This genotype (group 4) appears to be circulating widely in many areas in Europe, including Germany, Spain and England. Therefore, molecular epidemiology was used to confirm more classic epidemiologic links or, in some cases, to suggest epidemiologic links where none were apparent. While sequence data cannot always identify the geographic origin of the virus with certainty, this data can clearly show whether or not individual outbreaks are linked and identify predominant, 'indigenous' genotypes.

Still, the diagnosis of measles is usually based on clinical presentation a n d / o r serologic confirmation. In most cases, specimens for virus isolation or for PCR amplification of MV RNA are not obtained. There needs to be a concerted effort to obtain specimens from as many sources as possible. RT-PCR will be used for rapid genotyping and to improve the sensitivity of virus detection in clinical specimens. 3°'aa The genetic analysis of wild-type viruses from 1994 and 1995 showed that the genotype that had circulated widely during the resurgence of measles cases in the U.S. between 1989 and 1992 was no longer present. This suggests that the improved measles control programs that were initiated in the early 1990s may have interrupted transmission of what had been the indigenous lineage of virus. In fact, there was a sixweek period in 1993 during which no indigeneous measles cases were reported. 7 If the interruption of indigenous measles transmission can be accomplished by maintaining high immunization levels, there is hope for eventual eradication of measles. The U.S. has established 1996 as a target date for the elimination of indigenous transmission of measles and continued molecular surveillance of wild-type strains will be necessary to monitor progress towards this goal.

Acknowledgements The authors thank Wen-bo Xu, Sirima Pattamdilok, Elsa Baumeister, Stanislav Markushin and Matilda Sequeira for contributing unpublished data. Financial support was provided by the World Health Organization and the National Vaccine Program.

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