Surveillance of human rotaviruses in 2007–2011, Hungary: Exploring the genetic relatedness between vaccine and field strains

Journal of Clinical Virology 55 (2012) 140–146

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Surveillance of human rotaviruses in 2007–2011, Hungary: Exploring the genetic relatedness between vaccine and field strains Brigitta László a,b,∗ , József Kónya a , Eszter Dandár b , Judit Deák c , Ágnes Farkas d , Jim Gray e , Gábor Grósz f , Miren Iturriza-Gomara g , Ferenc Jakab h , Ágnes Juhász i , Péter Kisfali j , Julianna Kovács k , György Lengyel f , Vito Martella l , Béla Melegh j , Júlia Mészáros i , Péter Molnár m , Zoltán Nyúl n , Hajnalka Papp b , László Pátri o , Erzsébet Puskás p , Ildikó Sántha p , Ferenc Schneider r , Katalin Szomor d , András Tóth m , ˝ t , Krisztián Bányai b,∗∗ Erzsébet Tóth s , György Szucs a

Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary Veterinary Medical Research Institute, Hungarian Academy of Sciences, Budapest, Hungary Department of Clinical Microbiology, University of Szeged, Szeged, Hungary d Division of Virology National Center for Epidemiology, Budapest, Hungary e Specialist Virology Centre, Microbiology Department, Norfolk and Norwich University Hospital, Norwich, UK f Dr. György Radó Military Medical Centre, Hungarian Defence Forces, Budapest, Hungary g Enteric Virus Unit, Health Protection Agency, London, United Kingdom h Department of Medical Microbiology and Immunology, University of Pécs, Pécs, Hungary i Hungarian National Public Health and Medical Officer Service, North Great Plain Regional Institute, Hajdú-Bihar county, Debrecen, Hungary j Department of Medical Genetics and Child Development, Faculty of Medicine, University of Pécs, Pécs, Hungary k Pediatrics Health Center, Bordány, Hungary l Department of Animal Health and Wellbeing, University of Bari, Bari, Italy m Szent László Hospital for Infectious Diseases, Budapest, Hungary n Kerpel-Fronius Ödön Children’s Hospital, Pécs, Hungary o PÁTRI-MED BT, Pécs, Hungary p Hungarian National Public Health and Medical Officer Service, North Hungarian Regional Institute, Miskolc, Hungary r Markusovszky Hospital, Szombathely, Hungary s Hungarian National Public Health and Medical Officer Service, North Great Plain Regional Institute, Szabolcs-Szatmár-Bereg County, Nyíregyháza, Hungary t Department of Microbiology, University of Kuwait, Kuwait b c

a r t i c l e

i n f o

Article history: Received 28 February 2012 Received in revised form 24 May 2012 Accepted 20 June 2012 Keywords: Gastroenteritis Molecular epidemiology VP7 VP4 Genotype

a b s t r a c t Background: The availability of rotavirus vaccines has resulted in an intensification of post vaccine strain surveillance efforts worldwide to gain information on the impact of vaccines on prevalence of circulating rotavirus strains. Objectives: In this study, the distribution of human rotavirus G and P types in Hungary is reported. In addition, the VP4 and VP7 genes of G1P[8] strains were sequenced to monitor if vaccine-derived strains were introduced and/or some strains/lineages were selected against. Study design: The study was conducted in 8 geographic areas of Hungary between 2007 and 2011. Rotavirus positive stool samples were collected from diarrheic patients mostly <5 years of age. Viral RNA was amplified by multiplex genotyping RT-PCR assay, targeting the medically most important G and P types. When needed, sequencing of the VP7 and VP4 genes was performed. Results: In total, 2380 strains were genotyped. During the 5-year surveillance we observed the dominating prevalence of genotype G1P[8] (44.87%) strains, followed by G4P[8] (23.4%), G2P[4] (14.75%) and G9P[8] (6.81%) genotypes. Uncommon strains were identified in a low percentage of samples (4.12%). Phylogenetic analysis of 318 G1P[8] strains identified 55 strains similar to the Rotarix strain (nt sequence identities; VP7, up to 97.9%; VP4, up to 98.5%) although their vaccine origin was unlikely. Conclusions: Current vaccines would have protected against the majority of identified rotavirus genotypes. A better understanding of the potential long-term effect of vaccine use on epidemiology and evolutionary dynamics of co-circulating wild type strains requires continuous strain surveillance. © 2012 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary. Tel.: +36 52255 425; fax: +36 52255 424. ∗∗ Corresponding author. Tel.: +36 1467 4060; fax: +36 1467 4076. E-mail addresses: [email protected] (B. László), [email protected] (K. Bányai). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.06.016

B. László et al. / Journal of Clinical Virology 55 (2012) 140–146

1. Background Acute gastroenteritis is a public health concern worldwide due to high morbidity and mortality. Among enteric viral pathogens group A rotaviruses (RVA) have been identified as the most important etiological agents in infants and young children. By the age of 5 years virtually all children have been infected by one or more rotaviruses.1,2 Rotaviruses are members of the Rotavirus genus within the Reoviridae family. The virion encapsidates the 11 segments of double-stranded (ds)RNA. The genome encodes six structural (VP1–VP4, VP6, VP7) and six nonstructural proteins (NSP1-6).3 Rotaviruses are classified into groups or species (A–G) according to the antigenic and genetic features of the middle capsid layer protein, VP6. The outer capsid layer is composed of two proteins encoded by the VP4 and VP7 genes.3 VP4 defines P (proteasesensitive) and VP7 defines G (glycoprotein) antigens. To date, 27 G-types and 35 P-types have been described in humans and animals worldwide.4,5 Human rotavirus strains have been shown to carry 12 G and 15 P type specificities.6 Despite the great number of genotype combinations that can be theoretically derived from this large number of VP7 and VP4 genotypes, only a relatively few strains have been reported to have medical significance worldwide, including genotypes G1P[8], G3P[8], G4P[8], G9P[8], and G2P[4].6,7 Other uncommon antigen types and rare combinations of G- and P-types have been identified in different countries and many of them might be locally and temporally important. This spatiotemporal variation in strain prevalence further contributes to the regional strain diversity.6,8 The surface proteins, VP7 and VP4, elicit neutralizing antibodies independently in vivo, therefore they are the main targets of vaccine development.9,10 Two oral live attenuated rotavirus vaccines have been licensed, or, already introduced from 2006 onward in >100 countries worldwide.11 Rotarix and RotaTeq have been available on the private market in Hungary since 2006 and 2007, respectively. Rotarix (GlaxoSmithKline) is a monovalent G1P[8] rotavirus vaccine derived from a human rotavirus strain by serial passage in cell culture.12,13 RotaTeq (Merck) is a pentavalent human–bovine (WC3) reassortant vaccine, with the five most common human type specificities, G1–G4 and P[8], expressed individually on the genetic backbone of a bovine rotavirus strain naturally attenuated for humans.14,15 The role of type-specific versus cross-protective immunity against rotavirus infection is still debated. Multiple doses of both vaccines confer protection against a wide diversity of human rotavirus strains, however, there are concerns regarding the effect of vaccine strains on the dynamics of rotavirus epidemiology and vice versa the efficacy of vaccines against emerging rotavirus strains completely heterotypic to the vaccine strains. This concern has revived several international surveillance networks in parts of the world to assess this interplay between vaccine use and strain prevalence.16–19

2. Objective In this study, the distribution of rotavirus G and P types that have been in circulation between 2007 and 2011 in Hungary is reported. In addition, nucleotide sequencing and phylogenetic analyses of fragments of the VP4 and VP7 genes of G1P[8] strains was performed to monitor the occurrence of vaccine derived strains among field rotavirus strains.

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3. Study design 3.1. Samples and patients data Rotavirus positive fecal samples were collected from inpatients and outpatients with acute gastroenteritis between January 2007 and December 2011. Stool samples were obtained from sporadic, community acquired cases of all age groups through a national network from collaborating laboratories in seven counties plus the capital of Hungary. Viral antigen was detected from stool in different laboratories by various commercial methods: latex agglutination (Rotalex, Orion Diagnostica) and immunchromatography tests (Rota Uni-Strip, Coris BioConcept; Vikia Rota-Adeno, Biomerieux) performed according to manufacturers’ recommendations were the primary detection methods. Information on age and sex of patients, dates of disease onset and sample collection, symptoms, geographical location and setting (community/hospital) were collected as baseline epidemiologic background data. 3.2. Genotyping by multiplex-nested PCR For RT-PCR, 5 ␮l of extracted dsRNA (TriReagent [Sigma–Aldrich, St. Louis, MO, USA], or High Pure Viral Nucleic Acid Kit [Roche, Basel, Switzerland]) was used. First, the genomic RNA was denatured for 5 min at 97 ◦ C in the presence of a cocktail of random-hexamers or gene specific consensus primers and then reverse transcribed.20–22 Amplification and genotyping of the cDNA was performed using a multiplex nested PCR assay. Mixtures of type specific primers targeted to G1–G4, G8–G10, and G12 VP7 specificities23–25 and P[4], P[6] and P[8]-P[11] VP4 specificities20,26,27 were used in the secondround PCR, respectively. Genotypes were determined according to the length of the amplicons after electrophoresis of second-round PCR products in a 1.5% agarose gel stained with ethidium-bromide. The samples, for which no amplified products in the two-step PCR could be detected, were subjected to a VP6 gene specific PCR28 to verify the presence of the rotavirus double-stranded RNA. 3.3. Sequencing and phylogenetic analysis First-round PCR products (VP8* region, nt 11–887; VP7 gene, nt 51–932) were purified from the gel after electrophoresis. The cycle sequencing reaction was carried out with the ABI PRISM BigDye Terminator v1.1 Cycle Sequencing Reaction Kit (Applied Biosystems, Carlsbad, CA) and sequence data were collected by means of an automated DNA analyzer ABIPrism 3100 Avant or ABI3500. Sequence chromatograms were cleaned using the BioEdit software.29 Multiple alignments were manually adjusted using the GeneDoc software.30 Phylogenetic analysis was performed with the MEGA531 ; neighbor-joining trees were generated using the p-distance model and bootstrap resampling was performed. 4. Results During the study period, 2615 rotavirus positive stool samples were collected from patients with acute gastroenteritis. Of these, 2526 samples were available in sufficient quantity for further analysis. A total of 146 (5.8%) samples were omitted from the final analysis after they gave negative results in both nested multiplex genotyping PCR and the subsequent confirmatory VP6 gene specific PCR assay. Baseline epidemiological information was available for 2213 (92.9%) specimens. Of these, 1859 (84.0%) and 354 (16.0%) samples were collected from inpatients and outpatients, respectively. Most samples (79.6%) were obtained from children under 5 years of age, with relative abundance of age 6–24 months (40.5%). A total

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Table 1 Annual prevalence of common and uncommon rotavirus strains from 2007 to 2011. Genotype combination

Common genotypes G1P[8] G2P[4] G3P[8] G4P[8] G9P[8] Subtotal Uncommon genotypes G1P[4] G1P[6] G2P[6] G2P[8] G3P[4] G3P[9] G4P[6] G6P[9] G9P[4] G9P[6] G12P[6] G12P[8] Subtotal NT MIXED Total

No. (%) of strains collected in the following years: 2007

2008

2009

2010

2011

Total

139 (28.60) 95 (19.55) 2 (0.41) 157 (32.30) 50 (10.29) 443 (91.15)

259 (49.81) 75 (14.42) 7 (1.35) 99 (19.04) 43 (8.27) 483 (92.88)

319 (62.92) 15 (2.96) 10 (1.97) 79 (15.58) 10 (1.97) 433 (85.40)

52 (12.53) 112 (26.99) 3 (0.72) 198 (47.71) 9 (2.17) 374 (90.12)

299 (66.15) 54 (11.95) 0 (0.00) 24 (5.31) 50 (11.06) 427 (94.47)

1068 (44.87) 351 (14.75) 22 (0.92) 557 (23.40) 162 (6.81) 2160 (90.75)

2 (0.41) 0 (0.00) 0 (0.00) 6 (1.23) 0 (0.00) 1 (0.21) 4 (0.82) 2 (0.41) 1 (0.21) 4 (0.82) 0 (0.00) 2 (0.41) 22 (4.53)

4 (0.77) 0 (0.00) 1 (0.19) 1 (0.19) 0 (0.00) 1 (0.19) 3 (0.58) 1 (0.19) 1 (0.19) 0 (0.00) 1 (0.19) 3 (0.58) 16 (3.08)

2 (0.39) 1 (0.20) 1 (0.20) 0 (0.00) 2 (0.39) 2 (0.39) 0 (0.00) 3 (0.59) 0 (0.00) 0 (0.00) 14 (2.76) 5 (0.99) 30 (5.92)

1 (0.24) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 2 (0.48) 1 (0.24) 0 (0.00) 0 (0.00) 14 (3.37) 5 (1.20) 23 (5.54)

0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) 5 (1.11) 1 (0.22) 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.22) 0 (0.00) 7 (1.55)

9 (0.38) 1 (0.04) 2 (0.08) 7 (0.29) 2 (0.08) 9 (0.38) 10 (0.42) 7 (0.29) 2 (0.08) 4 (0.17) 30 (1.26) 15 (0.63) 98 (4.12)

16 (3.29) 5 (1.03)

13 (2.50) 8 (1.54)

23 (4.54) 21 (4.14)

15 (3.61) 3 (0.72)

16 (3.54) 2 (0.44)

83 (3.49) 39 (1.64)

486

520

507

415

452

2380

MIXED, presence of multiple genotypes in the same sample; NT, non-typeable G and/or P specificity.

of 465 samples (21.1%) were collected from adolescent and adult patients (max. age 91 years). The peak incidence of rotavirus infections was seen from February to April, except in 2008, when the epidemic peaked in April and May. 4.1. Prevalence of rotavirus strains The distribution of G and P types of rotaviruses detected in the study period are summarized in Table 1. In total, both G and P types could be identified for 2297 (96.5%) strains, representing 17 different genotype combinations. The remainders were only partially typeable, including 41 strains for which the G type was determined and the P type remained untypeable and 41 strains for which only the P type could be determined. In general, almost 91% of the 2380 strains belonged to one of the five globally common genotype combinations (G1P[8], 44.9%; G2P[4], 14.8%; G3P[8], 0.9%; G4P[8], 23.4%; G9P[8], 6.8%). Among the globally important human strains, only G3P[8] strains were not detected in each year. Less common genotypes were found in a small proportion (4%) of strains identified during the study period. These strains were unusual combinations of common human G and P types (G1P[4], 0.38%; G2P[8], 0.30%;G3P[4] 0.08%; and G9P[4] 0.08%;) and some other unusual and rare G–P combinations (G1P[6], 0.04%; G2P[6], 0.08%; G3P[9], 0.38%; G4P[6], 0.42%; G6P[9], 0.30%; and G9P[6], 0.17%). Of interest, an increased activity of G12 strains resulted in higher detection rates for either G12P[8] or G12P[6] genotype than that of G3P[8] in some years (Table 1). 4.2. Spatiotemporal distributions of common rotavirus strains Rotavirus positive samples were collected from eight different geographical regions. Five study sites provided samples in each consecutive season. Because different areas contributed with fluctuating amount of strains to the study in consecutive years, we depicted the spatiotemporal distribution of the five globally

common rotavirus strains for the participating study sites. Differences in prevalence of these common rotavirus strains were found between different regions year by year (Fig. 1). For example, G1P[8] was the most prevalent genotype in Northeast Hungary in 2007 and nearly in all geographical regions in the subsequent 2 years and in 2011 again in line with the increased prevalence of this genotype in this period. G2P[4] emerged to become predominant in Southwest Hungary in 2007 as well as in Northeast Hungary and the capital area in 2010. A higher percentage of G4P[8] strains was observed in West Hungary and in the Budapest area in 2007 and in Southwest-, South- and East Hungary during 2010. The G9P[8] genotype was found at high prevalence in Northeastern Hungary in 2008 and this strain was the second most prevalent genotype combination in Budapest and central Hungary during 2011.

4.3. Sequence analysis of the VP7 and VP4 genes We were eager to know if vaccine strains could have interacted with field strains or vaccine strains may have reverted to become virulent and circulate in the community. A subset of G1P[8] strains were selected from various years and study sites for nucleotide sequencing of the VP7 and VP4 genes. Among 318 successfully sequenced strains we found that the G1 VP7 gene belonged to lineages I and II, while the P[8] VP4 gene belonged to lineages I and III.32,33 None of the selected strains were closely related to the G1 VP7 and P[8] VP4 genes of the pentavalent RotaTeq. The maximum identity between circulating G1P[8] strains and corresponding specificities in the RotaTeq was 93% for the VP7 gene and 94% for the VP4 gene. In contrast, the VP7 gene of four community strains clustered together with the VP7 of strain 89-12, the parental strain of the Rotarix sharing up to 97.9% nucleotide similarity. Furthermore 51 strains clustered together along with the Rotarix strain in the VP4 phylogeny sharing between 98.3% and 98.5% similarity (Fig. 2). However, none of these 55 strains, detected during 2007–2009, shared both the P[8] VP4 and the G1 VP7 lineages with 89-12.

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Fig. 1. Geographical differences in the prevalence of common rotavirus strains detected in Hungary between 2007 and 2011.

5. Discussion Rotavirus surveillance dates back to mid-1990s in Hungary, showing the dominating prevalence of G1–G4 strains in the early period when MAb-EIA typing assays targeting the major human serotypes was utilized.34 Later, we documented the circulation of unusual strains and the emergence of globally spreading strains in line with the introduction of sophisticated methods into our typing protocol whereby we were able to reduce the proportion of untypeable strains.35–40 In the pre rotavirus vaccine era, two sentinel regions were included in the rotavirus surveillance in Hungary and approximately 7000 strains were sero- or genotyped during a 22 years period from mid-1980s to 2006.27,34,37,38,41–44 Between 2007 and 2011 we genotyped over 2400 strains from eight study areas using the typing protocol recommended by the EuroRotaNet, the first European rotavirus strain surveillance network.16,17 The most important strains were G1P[8], G2P[4] and G4P[8], with marked annual fluctuations observed at different study sites. In addition, we identified several hitherto undetected antigen combinations in Hungary. The G9P[6] strain was probably introduced from India,45 although evidence for subsequent spread in Hungary was not obtained. The relative importance of the G12P[6] strains exceeded over the last few years the frequency of G12P[8] strains, which emerged during the mid-2000s in Hungary.39 In addition,

some of the unusual combinations of common strains, such as G1P[6], G2P[6], and G9P[4], were described in this study for the first time in Hungary. The improved and standardized typing protocol of the EuroRotaNet targeted the eight and six most common G type and P type specificities, respectively.16,17 In Hungary, all but few strains belonged to one of these common genotypes. The G6 VP7 specificity, which appears endemic in Hungary is,27,35–37,43 however, not targeted in the assay. We detected this rare genotype in a few cases when we tried to confirm the VP7 specificity of two untypeable strains and five suspected G12P[9] strains by sequencing. In these cases we detected only one nucleotide substitution between the middle region of the G12 primer and the homologous region of the G6 VP7 gene. Another mistyping was the VP7 gene of a G8P[8] strain,46 whose G type proved to be G4 by nucleotide sequencing. Collectively, random selection of common and untypeable strains and systematic confirmation of uncommon strains by nucleotide sequencing showed that the standardized protocol worked well in our hands; however, examples of mistyping by standard methods emphasizes the need for confirmatory assays when unexpected strains are identified. During 2006–2007 two rotavirus vaccines were licensed and introduced in Hungary, the pentavalent RotaTeq and the monovalent Rotarix. When vaccine sales data for each vaccine was

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Fig. 2. Phylogenetic analysis of partial nucleotide sequences of VP4 and VP7 genes of selected G1P[8] human rotavirus strains identified between 2007 and 2011 in Hungary. Phylogenetic trees were generated based on the neighbor-joining method using the Mega5 program. DS-1 rotavirus strain was used as an outgroup. Positions of the vaccine strains are indicated on the tree. Roman numbers indicate the lineage specificity of strains belonging to a particular cluster. Adopted from 32,33.

combined we estimated that the overall vaccine coverage, among infants, increased from 4% to 18% between 2007 and 2010 (unpublished data). This low nationwide coverage is largely attributable to the exclusive private market availability and the high per dose costs of the vaccines. In countries with high vaccine coverage it has been observed that G2P[4] strains are very common in countries where Rotarix is used.47–49 In other regions where RotaTeq is included in the vaccination schedule, the resurgence of G3P[8] strains has been documented.50,51 At present, no definitive conclusion can be made to declare if such re-emergences of different strains in areas where different vaccines are used are associated with vaccination or merely reflect the natural spatiotemporal fluctuation of common co-circulating strains.8 Consequently, it is challenging to assess if vaccine strains could have any impact on locally circulating strains (or vice versa) in our country. However, concerns that vaccine use may influence local strain distribution remain. As information on potential vaccination status could not be collected during this study, we concluded that the only way to measure any possible effect of vaccine use on local strain prevalence and diversity was to determine the genetic content of field strains through DNA sequencing and compare them to the vaccine strains. During the study period the molecular analysis of the genes encoding the surface antigens of a large number of G1P[8] field strains was undertaken as Rotarix was purchased more often (∼90% relative

use). The majority of selected field strains did not share the antigens with vaccine strains. However, 55 strains were closely related but not identical to the Rotarix parental strain, indicating that the possibility of the vaccine strain circulating in the population, even if appeared unlikely, could not be excluded. Nonetheless, point mutations other than those indicating relatedness to the vaccine strain were present in these strains. The presence of these additional point mutations would suggest that these strains had been circulating in the population for some time. 6. Conclusions Enhanced nationwide rotavirus strain surveillance identified large diversity in Hungary after rotavirus vaccines became available for use during 2006 and 2007. The genotype specificities of field strains are partly or completely shared with strains included in both commercially available human rotavirus vaccines. We found no solid evidence for an impact of vaccine use on natural strain dynamics, raising questions if reassortment between field and vaccine strain is a common event and even if reversion of vaccine strains into pathogenic virus occurs, what is the frequency of such events. Future studies based on whole genome sequencing and more in-depth evolutionary analyses will certainly shed light on this issue.

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Funding 18.

Funding was provided, in equal parts, by Glaxo-SmithKline Biologicals and Sanofi Pasteur MSD in the form of unrestricted collaborative grants and administered through the Health Protection Agency (London, UK). Additional funding was given by the Hungarian Scientific Research Fund (OTKA, K 100727). B. L.’s Ph.D. stipend was given by the Sanofi-Aventis Hungary and then was supported by the Hungarian Academy of Sciences. K.B. was supported by the Momentum Initiative.

21.

Competing interests

22.

There is no conflict of interest. Ethical approval

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20.

23.

24.

Not required. 25.

Acknowledgments The technical assistance is acknowledged to Maria Almasi-Lohr, Eniko Lakone-Toth, Bernadette Nagy, Nora Garamvolgyi, and Csilla Weber for technical assistance.

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