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Prevalence and molecular characterization of human group C rotaviruses in Hungary
Journal of Clinical Virology 37 (2006) 317–322
Short communication
Prevalence and molecular characterization of human group C rotaviruses in Hungary ´ Bogd´an a , B. Horv´ath a , F. Jakab a , E. Meleg a,d , K. B´anyai a,d,∗ , B. Jiang b , A. V. Martella c , L. Magyari e , B. Melegh e , G. Sz˝ucs a,d a
Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Szabads´ag u´ t 7., H-7623 P´ecs, Hungary Respiratory and Gastroenteritis Viruses Branch, Division of Viral Diseases, MSG04, Centers for Disease Control and Prevention, Atlanta, 1600 Clifton Road, GA 30333, USA c Department of Animal Health and Well-Being, University of Bari, Sp Casamassima Km 3, 70010 Valenzano, Bari, Italy d Department of Medical Microbiology and Immunology, Faculty of Medicine, University of P´ ecs, Szigeti u´ t 12, H-7624 P´ecs, Hungary e Department of Medical Genetics and Child Development, Faculty of Medicine, University of P´ ecs, Szigeti u´ t 12, H-7624 P´ecs, Hungary b
Received 22 May 2006; received in revised form 8 August 2006; accepted 19 August 2006
Abstract Background: Group C rotaviruses are recognized enteric pathogens of humans and animals. Human group C rotaviruses have been associated with sporadic episodes and large outbreaks of gastroenteritis in children and adults but their epidemiology and ecology are still unexplored. Objectives: To collect epidemiological data on group C rotavirus infections among children with gastroenteritis in Hungary and perform molecular characterization on the identified strains. Study design: Fecal samples were collected during the 2003 surveillance in Baranya County, Hungary. The presence of group C rotavirus RNA was investigated by polyacrylamide gel electrophoresis and by reverse transcription-nested polymerase chain reaction for the VP6 gene. The identified strains were further characterized by sequencing and phylogenetic analysis of the VP7, VP6, VP4, and NSP4 genes. Results: Three of 472 samples (0.6%) tested positive for group C rotavirus. Two samples were selected for molecular analysis. Strains BaC 6104/03 and BaC 11549/03 displayed an overall identity of >99.8% and 99.3% at the nucleotide and amino acid level, respectively. The VP7 of the strain BaC 6104/03 was most closely related (99.5% aa) to the Nigerian strain Jajeri, while the VP4s of strains BaC 6104/03 and BaC 11549/03 were more similar (98.1% aa) to strains Belem and 208, detected in Brazil and China, respectively. Conclusions: Based on this 1-year study, we conclude that group C rotaviruses are not of epidemiological relevance in the etiology of childhood acute gastroenteritis in Hungary. The low sequence divergence between the Hungarian strains suggested that a single group C rotavirus strain circulated in this period in the study area. © 2006 Elsevier B.V. All rights reserved. Keywords: Diarrhea; Laboratory diagnostics; Epidemiology; RNA profile; Phylogenetic analysis
1. Introduction The genus Rotavirus (family Reoviridae) includes antigenically and genetically diverse viruses that cause acute gastroenteritis in birds and mammals (Estes, 2001). Members of the genus are classified into seven (sero)groups (A–G) ∗ Corresponding author at. Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Szabads´ag u´ t 7, H-7623 P´ecs, Hungary. Tel.: +36 72 514971; fax: +36 72 514949. E-mail address: [email protected] (K. B´anyai).
1386-6532/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2006.08.017
based on a lack of cross reactivity of the major capsid protein VP6. Group A–C rotaviruses have been found in association with human and animal diseases, while group D to G rotaviruses have been detected only in animals (Estes, 2001). The Chinese human strain ADRV-N might represent the prototype of an additional serogroup (Yang et al., 2004). Group A rotaviruses are the major cause of childhood diarrhea worldwide, accounting for an estimated 110–130 million episodes and 350,000–710,000 deaths each year (Parashar et al., 2006). Non-group A rotaviruses are also of epidemiologic relevance in humans and are recognized
318
K. B´anyai et al. / Journal of Clinical Virology 37 (2006) 317–322
mostly in outbreaks of gastroenteritis. Human group B rotaviruses were first described in 1982 in China as the cause of nationwide epidemics affecting several hundreds of thousands to more than 1 million people (Saif and Jiang, 1994) and they have been recently detected in India and Bangladesh (Kelkar and Zade, 2004; Ahmed et al., 2004). The first report on human group C rotavirus infection dates back to 1973 in an infant hospitalized with diarrhea in Australia (Rodger et al., 1982). Since then group C rotaviruses have been detected from both sporadic episodes and outbreaks of gastroenteritis throughout the world (Szucs et al., 1987; Arista et al., 1990; Saif and Jiang, 1994; Schnagl et al., 2004; Kuzuya et al., 2005; Rahman et al., 2005). Discovery and early laboratory diagnostics of non-group A rotaviruses (historically called atypical rotaviruses or pararotaviruses) have been based on detection of typical rotavirus morphology by electron microscopy together with unusual genome pattern and/or lack of specific reaction with immunologic reagents prepared against group A rotaviruses. Furthermore, some conservative features in the RNA profile predict rotavirus serogroups by analysis of the rotavirus genome pattern in polyacrylamide gel electrophoresis (PAGE). The 11 double-stranded (ds) RNA segments of the viral genome segregate into four size classes in a characteristic fashion for most serogroups (group A, 4-2-3-2; group B, 4-2-2-3; group C, 4-3-2-2; Saif and Jiang, 1994). However, due to occasional inter- or intra-segmental rearrangement, the dsRNA electropherotype of group A rotavirus strains may be altered and antigenic or genetic confirmatory tests to diagnose non-group A rotavirus infections are recommended. At present, immunologic reagents specific for group C rotaviruses are not widely available for diagnostic purposes. Sensitive and specific alternative tools are amplification of group C rotavirus genes by using reverse transcriptionpolymerase chain reaction (RT-PCR) and nested PCR (RTnPCR), followed by probe hybridization or nucleic acid sequencing (Sanchez-Fauquier et al., 2003). In this study, we detect and characterize group C rotaviruses in children with gastroenteritis in Hungary during a 1-year surveillance using polyacrylamide gel electrophoresis (PAGE) and RT-nPCR.
2. Materials and methods 2.1. Samples Fecal specimens collected from children under 15 years of age with symptoms of gastroenteritis were sent from pediatric hospital wards (87%) and general pediatricians (13%) in Baranya County, located in the south-western part of Hungary. Bulk stool samples were collected in regular stool containers by the hospital staff within 48 h of admission, or, by the care giver of affected children visiting general pediatricians. Samples were immediately tested for the presence of group A rotavirus by latex-agglutination (Rotalex, Orion Diagnostica, Espoo or Slidex, BioMerieux, Marcy l’Etoile)
or kept for no more than 24 h in a refrigerator before testing. Remaining stool samples were kept frozen at −20 ◦ C until the RNA was extracted for further investigation. All samples available in sufficient amount for testing were subjected to molecular analysis. 2.2. RNA extraction Viral RNA was extracted from supernatant of 10–20% stool samples by SDS-proteinase K (Sigma, Saint Louis, MO) digestion and phenol–chloroform–isoamyl alcohol (Sigma) extraction followed by repeated precipitation with alcohol, as described elsewhere (B´anyai et al., 2005). 2.3. PAGE For PAGE, one-third of the RNAs dissolved in nucleasefree water was loaded on 10% polyacrylamide slab gels and run at 17 mA for 20 h at 4 ◦ C. The RNA bands were visualized by silver staining as described by Dolan et al. (1985). For RTPCR, the remaining dsRNA was purified by the GTC/glass milk method in order to eliminate possible enzyme inhibitors (Gentsch et al., 1992). 2.4. RT-nPCR In the presence of 5 M random oligonucleotide hexamer, the RNA specimens were heat-denatured at 97 ◦ C for 5 min in a total volume of 10 l and immediately chilled on a pre-cooled tube rack to prevent the re-annealing of RNA strands. Complementary DNA was synthesized using AMV (avian myeloblastosis virus) reverse transcriptase at 42 ◦ C for 1 h in 30 l reaction volume that consisted of 1× reaction buffer (50 mM Tris–HCl, 50 mM KCl, 10 mM MgCl2 , 0.5 mM spermidine, and 10 mM DTT), 0.33 mM dNTP, 10 U recombinant RNAsin, and 2 U AMV reverse transcriptase (all from Promega, Madison, WI). First round amplification was carried out by adding 2 l cDNA to 48 l PCR reaction mixture that contained a final concentration of 1× reaction PCR buffer (75 mM Tris–HCl, 20 mM (NH4 )2 SO4 , 0.01% Tween 20, 2 mM MgCl2 ; Fermentas Inc., Vilnius, Lithuania), 0.2 mM dNTP (Promega, Madison, WI), 1 M each of the two primers, BMJ44 and BMJ145 (Integrated DNA Technologies, Inc., Coralville, IA) (Sanchez-Fauquier et al., 2003), and 2.5 U Taq thermostable DNA polymerase (Zenon Ltd., Szeged, Hungary). The reaction conditions included a denaturing step at 94 ◦ C for 3 min, followed by 40 cycles with consecutive steps of denaturing (94 ◦ C, 30 s), annealing (50 ◦ C, 30 s), and elongation (72 ◦ C, 1 min) and terminated with a final elongation step at 72 ◦ C for 3 min. In the second round, we used the same scheme with the exception of primers that were BMJ43mod and BMJ144 (Integrated DNA Technologies, Inc.) (Sanchez-Fauquier et al., 2003). The sequence of BMJ43mod is 5 TCA GCG ATG CCA GCT GGA AC 3 . The expected sizes of amplicons were 340 bp and 254 bp for RT-PCR and nested PCR, respectively. Both,
K. B´anyai et al. / Journal of Clinical Virology 37 (2006) 317–322
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first round and second round PCR products were run on 2% NuSieve agarose gel and stained with ethidium-bromide. 2.5. Sequence and phylogenetic analysis The PCR products were electrophoresed and extracted from gel slices using the QIAquick gel extraction kit (QIAGEN, Hilden, Germany). Cycle sequencing was carried out using the BigDye kit (v1.1; Applied Biosystems, Foster City, CA) with consensus primers specific to VP4, VP6, VP7, and NSP4 genes (gene-specific primers with the corresponding orientation and site are indicated; VP4, 5 GGC GTC TTC ACT GTA YGC ACA 3 , + sense, nt 23–43, 5 ACT TCT TGG AAA CTC GTG ATT C 3 , − sense, nt 620–641, VP6, 5 GCA TTT AAA ATC TCA TTC ACA A 3 , + sense, nt 1–23, 5 AGC CAC ATA GTT CAC ATT TCA 3 , − sense, nt 1333–1353; VP7, 5 GTA CAA CAT TGT ACA CTG TTT GC 3 , + sense, nt 56–78, 5 ACT TCA TAA GTT TCT GTA CTA GC 3 , − sense, nt 691–713; NSP4, 5 TCA GAT CAC TTT GCT CTA CGA A 3 , + sense, nt 14–35, 5 TAC ATT GAT CCT CAA CTC AGC 3 , − sense, nt 571–591). Dyelabeled products were run on an automated sequence analyzer (ABI Prism type 3100; Applied Biosystems, Foster City, CA). Phylogenetic reconstruction was carried out with the pdistance and the neighbor joining algorithm, supported with bootstrapping (500 replications; MEGA2 software; Kumar et al., 2001). The sequences are available from the corresponding author upon request.
3. Results and discussion Between January 2003 and December 2003, a total of 708 diarrheic stool samples (one sample per case) collected from children <15 years of age were sent to our laboratory from Baranya County for virological examination. Stools were screened for group A rotaviruses by commercial latex-agglutination test; 179 samples tested positive (25.3%). Subsequently, 679 stool samples (including 170 group A rotavirus positive specimens) were screened by PAGE. As a result, an additional 35 samples were demonstrated to be positive for group A rotaviruses, yielding an overall prevalence of these viruses to 30.2% (205/679). Out of the 679 specimens, 2 displayed a genome pattern consistent with that of group C rotavirus (0.3%; Fig. 1). To increase the sensitivity of group C rotavirus detection, we adapted an RT-nPCR assay using the primers described earlier except for primer BMJ43, which has a mismatch at the 3 -end with the corresponding sequence of Hungarian group C rotaviruses (data not shown). This modified primer (designated BMJ43mod) utilized in our assay was one nucleotide shorter than the original sequence. A subset of samples (n = 472), including 395 negative and 77 positive for group A rotaviruses, were tested with this RT-nPCR. One additional sample was positive for group C rotavirus, resulting in only negligible increase in the overall prevalence of
Fig. 1. Parallel electrophoresis of the 11 genome segments of group C (left) and A (right) rotaviruses. See the group-specific patterns 4-3-2-2 for group C rotavirus and 4-2-3-2 for mammalian group A rotavirus.
group C rotaviruses in the samples examined (3/472; 0.6%). No mixed infections with group A and group C rotaviruses have been identified. Two children were from rural areas, the third child lived in the largest city of Baranya county. Two of the three children with group C rotavirus infection were nonhospitalized outpatients and the third patient was treated at hospital. There was no epidemiological correlation among the three cases (Fig. 2). The three patients with group C rotavirus infection had a mean and median age of 5.0 years and 6.1 years, respectively. Corresponding ages of children infected with group A rotaviruses were 2.4 years (mean) and 1.8 years (median). Our data on the group C rotavirus epidemiology in Hungary is in agreement with findings of studies from other countries (Nilsson et al., 2000; Castello et al., 2002) and with seroepidemiological investigations (Kuzuya et al., 2001; Iturriza-Gomara et al., 2004), suggesting that group
Fig. 2. Seasonal distribution of group A (grey) and C (black) rotavirus cases identified in 2003, Baranya County, Hungary.
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C rotavirus infection may occur more frequently in older children. To study the genetic heterogeneity among group C rotaviruses and their relationships with group C rotavirus strains circulating in other countries, we performed phylogenetic analysis on partial sequences of structural and non-structural genes of two Hungarian group C rotavirus strains, designated BaC 6104/03 and BaC 11549/03. These two strains were selected because sufficient amount of amplicons in a single-round PCR was available for direct nucleotide sequencing. The analysis involved genes for the inner shell protein, VP6, the outer shell proteins, VP4 and VP7, and the enterotoxigenic non-structural protein, NSP4. In the four genes, strains BaC 6104/03 and BaC 11549/03 displayed an overall similarity of >99.8% at nucleotide (nt) level and >99.3% at amino acid (aa) level, suggesting that a single strain circulated in this period in the study area. In comparison with other strains, the VP7 of BaC 6104/03 displayed the closest relatedness (99.5% aa similarity) to the Nigerian strain Jajeri, while the VP4s of strains BaC 6104/03 and BaC 11549/03 were more similar (98.1%) to strains Belem and 208, detected in Brazil and China, respectively. The NSP4s of the Hungarian strains were most closely related to the strain Bristol (99.7%, aa) isolated in England and to the Japanese
isolate, Ehime9301 (99.5%, aa). Future studies should clarify whether these findings can be explained with reassortment events of certain genes between different group C rotavirus strains. At present, the number of strains eligible in a comprehensive multi-gene comparison is too low to be able to do such an analysis. Nonetheless, in the phylogenetic trees of VP4, VP6, and NSP4 genes, human group C rotavirus strains appear to constitute a single statistically significant lineage (supported by high bootstrap values, 100; Fig. 3), irrespective of the strains geographic origin, while two sub-lineages may exist in the VP7 gene (Rahman et al., 2005). Group C rotaviruses have been detected from small (Gabbay et al., 1999; Kuzuya et al., 2005) to massive (Otsu, 1998; Hamano et al., 1999) outbreaks involving schools or restricted communities, while they have been detected sporadically or at low frequencies during more comprehensive epidemiological surveys. The prevalence of group C rotaviruses was about 1% in a 4-year study in Argentina (Castello et al., 2002), 1.4% in a 6-month surveillance in Bangladesh (Rahman et al., 2005) and 2.7% in a 1-year study in Spain (Sanchez-Fauquier et al., 2003). Interestingly, the prevalence was 4.7% in a 6-month survey encompassing 10 prefectures in Japan (Kuzuya et al., 1998) and 10.2% in a 1-year study in Maizuru city, Japan (Phan et al., 2004).
Fig. 3. Phylogenetic analysis of group C rotaviruses. Partial nucleotide sequences of VP4, VP6, VP7, and NSP4 genes of group C rotaviruses were determined and analyzed as described in the text.
K. B´anyai et al. / Journal of Clinical Virology 37 (2006) 317–322
This higher prevalence rates in Japan were conjectured to be accounted for by large-scale outbreaks, as suggested by parallel increase in seroprevalence and by temporal pattern of distribution (Kuzuya et al., 2001; Phan et al., 2004). There are several questions about the ecology of such group C rotaviruses that remain unresolved. The sporadic pattern of detection and the description of localized outbreaks may be interpreted as a spill-over introduction of group C rotavirus strains from an unclear, unidentified source or reservoir. Serological investigations suggest higher incidence in rural than in urban populations, a finding that is consistent with transmission from animal sources (IturrizaGomara et al., 2004). However, in agreement with other studies, we found that all human strains are easily distinguishable from animal (i.e., porcine and bovine) strains by sequence analysis (Tsunemitsu et al., 1996; Jiang et al., 1996). Thus, phylogenetic analyses demonstrate an absence of direct animal ancestors and a lack of genetic reassortment between recent human and animal group C rotavirus strains. This finding implies an early differentiation of human strains from animal strains and different evolutionary pathways when adapted to their recognized hosts, suggesting that human group C rotaviruses might be only descendants of an early strain of animal origin that have spread throughout the world after host switching. Alternatively, the origin of human strains might be associated with several independent zoonotic events. More in-depth investigations in companion and farm animals might rule out or confirm these hypotheses and help obtain insights into the epidemiology and ecology of group C rotaviruses. In conclusion, this study adds further proof that group C rotaviruses are a widespread but relatively rare cause of childhood enteric infections. In agreement with an early report from Hungary published in the 1980s (Szucs et al., 1987), we identified only very few human infections with group C rotaviruses in spite of our use of a sensitive molecular detection method on a large number of samples. These data indicate that group C rotaviruses were epidemiologically minor causes of acute childhood gastroenteritis in Hungary in the surveillance period. Discrepancies between our data and analogous investigations might reflect variations in the geographical or temporal patterns. In addition, small-scale outbreaks in restricted communities might occur and go undetected, particularly if severity of the disease is less and hospitalization is not needed.
Acknowledgements The technical assistance of Gertrud Domonkos, Csilla Br´ada, and Judit Oksai is highly appreciated. The financial support is also acknowledged (Hungarian Research Fund-OTKA, grant no. T049020; Enteric Virus Emergence, New Tools (EVENT), grant no. SP22-CT-2004-502571). K. B´anyai is a recipient of the Bolyai J´anos Fellowship of the Hungarian Academy of Sciences.
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Short communication
Prevalence and molecular characterization of human group C rotaviruses in Hungary ´ Bogd´an a , B. Horv´ath a , F. Jakab a , E. Meleg a,d , K. B´anyai a,d,∗ , B. Jiang b , A. V. Martella c , L. Magyari e , B. Melegh e , G. Sz˝ucs a,d a
Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Szabads´ag u´ t 7., H-7623 P´ecs, Hungary Respiratory and Gastroenteritis Viruses Branch, Division of Viral Diseases, MSG04, Centers for Disease Control and Prevention, Atlanta, 1600 Clifton Road, GA 30333, USA c Department of Animal Health and Well-Being, University of Bari, Sp Casamassima Km 3, 70010 Valenzano, Bari, Italy d Department of Medical Microbiology and Immunology, Faculty of Medicine, University of P´ ecs, Szigeti u´ t 12, H-7624 P´ecs, Hungary e Department of Medical Genetics and Child Development, Faculty of Medicine, University of P´ ecs, Szigeti u´ t 12, H-7624 P´ecs, Hungary b
Received 22 May 2006; received in revised form 8 August 2006; accepted 19 August 2006
Abstract Background: Group C rotaviruses are recognized enteric pathogens of humans and animals. Human group C rotaviruses have been associated with sporadic episodes and large outbreaks of gastroenteritis in children and adults but their epidemiology and ecology are still unexplored. Objectives: To collect epidemiological data on group C rotavirus infections among children with gastroenteritis in Hungary and perform molecular characterization on the identified strains. Study design: Fecal samples were collected during the 2003 surveillance in Baranya County, Hungary. The presence of group C rotavirus RNA was investigated by polyacrylamide gel electrophoresis and by reverse transcription-nested polymerase chain reaction for the VP6 gene. The identified strains were further characterized by sequencing and phylogenetic analysis of the VP7, VP6, VP4, and NSP4 genes. Results: Three of 472 samples (0.6%) tested positive for group C rotavirus. Two samples were selected for molecular analysis. Strains BaC 6104/03 and BaC 11549/03 displayed an overall identity of >99.8% and 99.3% at the nucleotide and amino acid level, respectively. The VP7 of the strain BaC 6104/03 was most closely related (99.5% aa) to the Nigerian strain Jajeri, while the VP4s of strains BaC 6104/03 and BaC 11549/03 were more similar (98.1% aa) to strains Belem and 208, detected in Brazil and China, respectively. Conclusions: Based on this 1-year study, we conclude that group C rotaviruses are not of epidemiological relevance in the etiology of childhood acute gastroenteritis in Hungary. The low sequence divergence between the Hungarian strains suggested that a single group C rotavirus strain circulated in this period in the study area. © 2006 Elsevier B.V. All rights reserved. Keywords: Diarrhea; Laboratory diagnostics; Epidemiology; RNA profile; Phylogenetic analysis
1. Introduction The genus Rotavirus (family Reoviridae) includes antigenically and genetically diverse viruses that cause acute gastroenteritis in birds and mammals (Estes, 2001). Members of the genus are classified into seven (sero)groups (A–G) ∗ Corresponding author at. Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Szabads´ag u´ t 7, H-7623 P´ecs, Hungary. Tel.: +36 72 514971; fax: +36 72 514949. E-mail address: [email protected] (K. B´anyai).
1386-6532/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2006.08.017
based on a lack of cross reactivity of the major capsid protein VP6. Group A–C rotaviruses have been found in association with human and animal diseases, while group D to G rotaviruses have been detected only in animals (Estes, 2001). The Chinese human strain ADRV-N might represent the prototype of an additional serogroup (Yang et al., 2004). Group A rotaviruses are the major cause of childhood diarrhea worldwide, accounting for an estimated 110–130 million episodes and 350,000–710,000 deaths each year (Parashar et al., 2006). Non-group A rotaviruses are also of epidemiologic relevance in humans and are recognized
318
K. B´anyai et al. / Journal of Clinical Virology 37 (2006) 317–322
mostly in outbreaks of gastroenteritis. Human group B rotaviruses were first described in 1982 in China as the cause of nationwide epidemics affecting several hundreds of thousands to more than 1 million people (Saif and Jiang, 1994) and they have been recently detected in India and Bangladesh (Kelkar and Zade, 2004; Ahmed et al., 2004). The first report on human group C rotavirus infection dates back to 1973 in an infant hospitalized with diarrhea in Australia (Rodger et al., 1982). Since then group C rotaviruses have been detected from both sporadic episodes and outbreaks of gastroenteritis throughout the world (Szucs et al., 1987; Arista et al., 1990; Saif and Jiang, 1994; Schnagl et al., 2004; Kuzuya et al., 2005; Rahman et al., 2005). Discovery and early laboratory diagnostics of non-group A rotaviruses (historically called atypical rotaviruses or pararotaviruses) have been based on detection of typical rotavirus morphology by electron microscopy together with unusual genome pattern and/or lack of specific reaction with immunologic reagents prepared against group A rotaviruses. Furthermore, some conservative features in the RNA profile predict rotavirus serogroups by analysis of the rotavirus genome pattern in polyacrylamide gel electrophoresis (PAGE). The 11 double-stranded (ds) RNA segments of the viral genome segregate into four size classes in a characteristic fashion for most serogroups (group A, 4-2-3-2; group B, 4-2-2-3; group C, 4-3-2-2; Saif and Jiang, 1994). However, due to occasional inter- or intra-segmental rearrangement, the dsRNA electropherotype of group A rotavirus strains may be altered and antigenic or genetic confirmatory tests to diagnose non-group A rotavirus infections are recommended. At present, immunologic reagents specific for group C rotaviruses are not widely available for diagnostic purposes. Sensitive and specific alternative tools are amplification of group C rotavirus genes by using reverse transcriptionpolymerase chain reaction (RT-PCR) and nested PCR (RTnPCR), followed by probe hybridization or nucleic acid sequencing (Sanchez-Fauquier et al., 2003). In this study, we detect and characterize group C rotaviruses in children with gastroenteritis in Hungary during a 1-year surveillance using polyacrylamide gel electrophoresis (PAGE) and RT-nPCR.
2. Materials and methods 2.1. Samples Fecal specimens collected from children under 15 years of age with symptoms of gastroenteritis were sent from pediatric hospital wards (87%) and general pediatricians (13%) in Baranya County, located in the south-western part of Hungary. Bulk stool samples were collected in regular stool containers by the hospital staff within 48 h of admission, or, by the care giver of affected children visiting general pediatricians. Samples were immediately tested for the presence of group A rotavirus by latex-agglutination (Rotalex, Orion Diagnostica, Espoo or Slidex, BioMerieux, Marcy l’Etoile)
or kept for no more than 24 h in a refrigerator before testing. Remaining stool samples were kept frozen at −20 ◦ C until the RNA was extracted for further investigation. All samples available in sufficient amount for testing were subjected to molecular analysis. 2.2. RNA extraction Viral RNA was extracted from supernatant of 10–20% stool samples by SDS-proteinase K (Sigma, Saint Louis, MO) digestion and phenol–chloroform–isoamyl alcohol (Sigma) extraction followed by repeated precipitation with alcohol, as described elsewhere (B´anyai et al., 2005). 2.3. PAGE For PAGE, one-third of the RNAs dissolved in nucleasefree water was loaded on 10% polyacrylamide slab gels and run at 17 mA for 20 h at 4 ◦ C. The RNA bands were visualized by silver staining as described by Dolan et al. (1985). For RTPCR, the remaining dsRNA was purified by the GTC/glass milk method in order to eliminate possible enzyme inhibitors (Gentsch et al., 1992). 2.4. RT-nPCR In the presence of 5 M random oligonucleotide hexamer, the RNA specimens were heat-denatured at 97 ◦ C for 5 min in a total volume of 10 l and immediately chilled on a pre-cooled tube rack to prevent the re-annealing of RNA strands. Complementary DNA was synthesized using AMV (avian myeloblastosis virus) reverse transcriptase at 42 ◦ C for 1 h in 30 l reaction volume that consisted of 1× reaction buffer (50 mM Tris–HCl, 50 mM KCl, 10 mM MgCl2 , 0.5 mM spermidine, and 10 mM DTT), 0.33 mM dNTP, 10 U recombinant RNAsin, and 2 U AMV reverse transcriptase (all from Promega, Madison, WI). First round amplification was carried out by adding 2 l cDNA to 48 l PCR reaction mixture that contained a final concentration of 1× reaction PCR buffer (75 mM Tris–HCl, 20 mM (NH4 )2 SO4 , 0.01% Tween 20, 2 mM MgCl2 ; Fermentas Inc., Vilnius, Lithuania), 0.2 mM dNTP (Promega, Madison, WI), 1 M each of the two primers, BMJ44 and BMJ145 (Integrated DNA Technologies, Inc., Coralville, IA) (Sanchez-Fauquier et al., 2003), and 2.5 U Taq thermostable DNA polymerase (Zenon Ltd., Szeged, Hungary). The reaction conditions included a denaturing step at 94 ◦ C for 3 min, followed by 40 cycles with consecutive steps of denaturing (94 ◦ C, 30 s), annealing (50 ◦ C, 30 s), and elongation (72 ◦ C, 1 min) and terminated with a final elongation step at 72 ◦ C for 3 min. In the second round, we used the same scheme with the exception of primers that were BMJ43mod and BMJ144 (Integrated DNA Technologies, Inc.) (Sanchez-Fauquier et al., 2003). The sequence of BMJ43mod is 5 TCA GCG ATG CCA GCT GGA AC 3 . The expected sizes of amplicons were 340 bp and 254 bp for RT-PCR and nested PCR, respectively. Both,
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first round and second round PCR products were run on 2% NuSieve agarose gel and stained with ethidium-bromide. 2.5. Sequence and phylogenetic analysis The PCR products were electrophoresed and extracted from gel slices using the QIAquick gel extraction kit (QIAGEN, Hilden, Germany). Cycle sequencing was carried out using the BigDye kit (v1.1; Applied Biosystems, Foster City, CA) with consensus primers specific to VP4, VP6, VP7, and NSP4 genes (gene-specific primers with the corresponding orientation and site are indicated; VP4, 5 GGC GTC TTC ACT GTA YGC ACA 3 , + sense, nt 23–43, 5 ACT TCT TGG AAA CTC GTG ATT C 3 , − sense, nt 620–641, VP6, 5 GCA TTT AAA ATC TCA TTC ACA A 3 , + sense, nt 1–23, 5 AGC CAC ATA GTT CAC ATT TCA 3 , − sense, nt 1333–1353; VP7, 5 GTA CAA CAT TGT ACA CTG TTT GC 3 , + sense, nt 56–78, 5 ACT TCA TAA GTT TCT GTA CTA GC 3 , − sense, nt 691–713; NSP4, 5 TCA GAT CAC TTT GCT CTA CGA A 3 , + sense, nt 14–35, 5 TAC ATT GAT CCT CAA CTC AGC 3 , − sense, nt 571–591). Dyelabeled products were run on an automated sequence analyzer (ABI Prism type 3100; Applied Biosystems, Foster City, CA). Phylogenetic reconstruction was carried out with the pdistance and the neighbor joining algorithm, supported with bootstrapping (500 replications; MEGA2 software; Kumar et al., 2001). The sequences are available from the corresponding author upon request.
3. Results and discussion Between January 2003 and December 2003, a total of 708 diarrheic stool samples (one sample per case) collected from children <15 years of age were sent to our laboratory from Baranya County for virological examination. Stools were screened for group A rotaviruses by commercial latex-agglutination test; 179 samples tested positive (25.3%). Subsequently, 679 stool samples (including 170 group A rotavirus positive specimens) were screened by PAGE. As a result, an additional 35 samples were demonstrated to be positive for group A rotaviruses, yielding an overall prevalence of these viruses to 30.2% (205/679). Out of the 679 specimens, 2 displayed a genome pattern consistent with that of group C rotavirus (0.3%; Fig. 1). To increase the sensitivity of group C rotavirus detection, we adapted an RT-nPCR assay using the primers described earlier except for primer BMJ43, which has a mismatch at the 3 -end with the corresponding sequence of Hungarian group C rotaviruses (data not shown). This modified primer (designated BMJ43mod) utilized in our assay was one nucleotide shorter than the original sequence. A subset of samples (n = 472), including 395 negative and 77 positive for group A rotaviruses, were tested with this RT-nPCR. One additional sample was positive for group C rotavirus, resulting in only negligible increase in the overall prevalence of
Fig. 1. Parallel electrophoresis of the 11 genome segments of group C (left) and A (right) rotaviruses. See the group-specific patterns 4-3-2-2 for group C rotavirus and 4-2-3-2 for mammalian group A rotavirus.
group C rotaviruses in the samples examined (3/472; 0.6%). No mixed infections with group A and group C rotaviruses have been identified. Two children were from rural areas, the third child lived in the largest city of Baranya county. Two of the three children with group C rotavirus infection were nonhospitalized outpatients and the third patient was treated at hospital. There was no epidemiological correlation among the three cases (Fig. 2). The three patients with group C rotavirus infection had a mean and median age of 5.0 years and 6.1 years, respectively. Corresponding ages of children infected with group A rotaviruses were 2.4 years (mean) and 1.8 years (median). Our data on the group C rotavirus epidemiology in Hungary is in agreement with findings of studies from other countries (Nilsson et al., 2000; Castello et al., 2002) and with seroepidemiological investigations (Kuzuya et al., 2001; Iturriza-Gomara et al., 2004), suggesting that group
Fig. 2. Seasonal distribution of group A (grey) and C (black) rotavirus cases identified in 2003, Baranya County, Hungary.
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C rotavirus infection may occur more frequently in older children. To study the genetic heterogeneity among group C rotaviruses and their relationships with group C rotavirus strains circulating in other countries, we performed phylogenetic analysis on partial sequences of structural and non-structural genes of two Hungarian group C rotavirus strains, designated BaC 6104/03 and BaC 11549/03. These two strains were selected because sufficient amount of amplicons in a single-round PCR was available for direct nucleotide sequencing. The analysis involved genes for the inner shell protein, VP6, the outer shell proteins, VP4 and VP7, and the enterotoxigenic non-structural protein, NSP4. In the four genes, strains BaC 6104/03 and BaC 11549/03 displayed an overall similarity of >99.8% at nucleotide (nt) level and >99.3% at amino acid (aa) level, suggesting that a single strain circulated in this period in the study area. In comparison with other strains, the VP7 of BaC 6104/03 displayed the closest relatedness (99.5% aa similarity) to the Nigerian strain Jajeri, while the VP4s of strains BaC 6104/03 and BaC 11549/03 were more similar (98.1%) to strains Belem and 208, detected in Brazil and China, respectively. The NSP4s of the Hungarian strains were most closely related to the strain Bristol (99.7%, aa) isolated in England and to the Japanese
isolate, Ehime9301 (99.5%, aa). Future studies should clarify whether these findings can be explained with reassortment events of certain genes between different group C rotavirus strains. At present, the number of strains eligible in a comprehensive multi-gene comparison is too low to be able to do such an analysis. Nonetheless, in the phylogenetic trees of VP4, VP6, and NSP4 genes, human group C rotavirus strains appear to constitute a single statistically significant lineage (supported by high bootstrap values, 100; Fig. 3), irrespective of the strains geographic origin, while two sub-lineages may exist in the VP7 gene (Rahman et al., 2005). Group C rotaviruses have been detected from small (Gabbay et al., 1999; Kuzuya et al., 2005) to massive (Otsu, 1998; Hamano et al., 1999) outbreaks involving schools or restricted communities, while they have been detected sporadically or at low frequencies during more comprehensive epidemiological surveys. The prevalence of group C rotaviruses was about 1% in a 4-year study in Argentina (Castello et al., 2002), 1.4% in a 6-month surveillance in Bangladesh (Rahman et al., 2005) and 2.7% in a 1-year study in Spain (Sanchez-Fauquier et al., 2003). Interestingly, the prevalence was 4.7% in a 6-month survey encompassing 10 prefectures in Japan (Kuzuya et al., 1998) and 10.2% in a 1-year study in Maizuru city, Japan (Phan et al., 2004).
Fig. 3. Phylogenetic analysis of group C rotaviruses. Partial nucleotide sequences of VP4, VP6, VP7, and NSP4 genes of group C rotaviruses were determined and analyzed as described in the text.
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This higher prevalence rates in Japan were conjectured to be accounted for by large-scale outbreaks, as suggested by parallel increase in seroprevalence and by temporal pattern of distribution (Kuzuya et al., 2001; Phan et al., 2004). There are several questions about the ecology of such group C rotaviruses that remain unresolved. The sporadic pattern of detection and the description of localized outbreaks may be interpreted as a spill-over introduction of group C rotavirus strains from an unclear, unidentified source or reservoir. Serological investigations suggest higher incidence in rural than in urban populations, a finding that is consistent with transmission from animal sources (IturrizaGomara et al., 2004). However, in agreement with other studies, we found that all human strains are easily distinguishable from animal (i.e., porcine and bovine) strains by sequence analysis (Tsunemitsu et al., 1996; Jiang et al., 1996). Thus, phylogenetic analyses demonstrate an absence of direct animal ancestors and a lack of genetic reassortment between recent human and animal group C rotavirus strains. This finding implies an early differentiation of human strains from animal strains and different evolutionary pathways when adapted to their recognized hosts, suggesting that human group C rotaviruses might be only descendants of an early strain of animal origin that have spread throughout the world after host switching. Alternatively, the origin of human strains might be associated with several independent zoonotic events. More in-depth investigations in companion and farm animals might rule out or confirm these hypotheses and help obtain insights into the epidemiology and ecology of group C rotaviruses. In conclusion, this study adds further proof that group C rotaviruses are a widespread but relatively rare cause of childhood enteric infections. In agreement with an early report from Hungary published in the 1980s (Szucs et al., 1987), we identified only very few human infections with group C rotaviruses in spite of our use of a sensitive molecular detection method on a large number of samples. These data indicate that group C rotaviruses were epidemiologically minor causes of acute childhood gastroenteritis in Hungary in the surveillance period. Discrepancies between our data and analogous investigations might reflect variations in the geographical or temporal patterns. In addition, small-scale outbreaks in restricted communities might occur and go undetected, particularly if severity of the disease is less and hospitalization is not needed.
Acknowledgements The technical assistance of Gertrud Domonkos, Csilla Br´ada, and Judit Oksai is highly appreciated. The financial support is also acknowledged (Hungarian Research Fund-OTKA, grant no. T049020; Enteric Virus Emergence, New Tools (EVENT), grant no. SP22-CT-2004-502571). K. B´anyai is a recipient of the Bolyai J´anos Fellowship of the Hungarian Academy of Sciences.
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