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Microbiological Laboratory for Health Protection
Diagnostic Laboratory for Infectious Diseases and Perinatal Screening
Department of Infectious Diseases Epidemiology, National Institute for Public Health and the Environment, Bilthoven
Veterinary Microbiological Diagnostic Centre, Utrecht University, Utrecht, The Netherlands
ABSTRACT
To gain more insight into interspecies transmission of rotavirus group A, human and animal fecal samples were collected between 1997 and 2001 in The Netherlands. A total of 110 human stool samples were successfully P and G genotyped by reverse transcriptase PCR. All strains belonged to the main human rotavirus genotypes G1 to G4, G9, [P4], [P6], [P8], and [P9]. [P8]G1 was predominant, and 5.5% belonged to the G9 genotype. Eleven percent of all P[8] genotypes could be genotyped only by a recently published modified primer. Rotavirus-positive fecal samples from 28 calf herds were genotyped by DNA sequencing. Genotypes G6 and G10 predominated; G6 and G10 were detected in 22 (78.6%) and 16 (57.1%) of the rotavirus-positive calf herds, respectively. In 12 (42.9%) calf herds, we found mixed infections. Genotype G8 was not found. Genotype G6 bovine rotaviruses were divided into three clusters: UK-like, VMRI-29-like, and Hun4-like. DNA sequencing of a part of the VP7 gene was shown to be useful as a quick determination of uncommon or novel strains of which the genotyping cannot be done by genotyping PCR. Of equine strains, both VP4 and VP7 genes could be used for genotyping: two [P12]G3 and four [P12]G14 equine rotaviruses were determined. We did not find indications for rotavirus interspecies transmissions, although the recently published human G6-Hun4 is genetically related to our G6 bovine isolates. All bovine, porcine, and equine rotaviruses were within genotypes previously reported for these animal species.
INTRODUCTION
Group A rotaviruses (family Reoviridae) are the most common cause of gastroenteritis in young children and young animals (27). Rotavirus particles consist of three layers: the central core (enclosing 11 segments of double-stranded genomic RNA), the inner capsid, and the outer capsid (13). The outer capsid is studded with VP7 and VP4 proteins, which elicit neutralizing antibody responses and form the basis of the present dual classification system of G (VP7) and P (VP4) types (27). The VP7 protein expresses the major neutralization antigen and is distinguishable by means of both serological and genomic techniques in 14 G types, with good correlation between the serological and genomic classifications (21, 27). VP4 expresses a minor neutralization antigen, and the serological classification of the P types is much more difficult than genomic classification. To date, 13 P serotypes, including subtypes, and 20 P genotypes have been defined, but a precise correlation between the serological and genomic classifications has not been made (27).
Globally, viruses carrying genotypes G1 to G4 and [P4] or [P8] have consistently been found to be the most common cause of rotavirus disease in humans, and different surveys indicate that [P8]G1, [P4]G2, [P8]G3, and [P8]G4 are the most common G and P types (9, 29). However, rotavirus strains with other G and P types have increasingly been reported in different parts of the world, like the predominance of [P6]G9 strains in India (34), [P6]G8 strains in Malawi (6), and [P8]G5 strains in Brazil (2). Some strains, like G9 rotaviruses, have been found commonly across the world (9).
These new rotavirus genotypes can also cause serious outbreaks of diarrhea in humans and may have a high attack rate, like the [P6]G9 strain in The Netherlands (41). Understanding rotavirus diversity is important, and G1- to G4-based vaccines will not confer optimal protection against the new strains, such as G9 (33).
Several genotypes, like G3, G6, and G8, are shared between humans and animals (9), but direct transmissions among different animal species and between humans and animal species have not really been observed. However, the increased number of reports of new human rotavirus genotypes which are more commonly found in animals suggest the possibility of interspecies transmission or genetic reassortment of rotavirus strains (7, 9, 15, 22, 31).
To discover new emerging strains, intensive surveillance of circulating wild-type human and animal rotavirus strains is necessary. The aim of the present study is to provide information on the relative distribution of G and P genotype combinations of rotaviruses found in humans and animals in The Netherlands.
MATERIALS AND METHODS
Samples and sample preparation. Human fecal samples were collected in a physician-based case control and population-based study between 1996 and 2001. Stool specimens were screened for a broad range of enteric pathogens, including rotavirus (10, 11, 12). All 110 fecal samples, which tested positive by antigen detection enzyme-linked immunosorbent assay (ELISA) (Rotaclone; Meridian Diagnostics Europe, Boxtel, The Netherlands), were used for the rotavirus genotyping. From 1999 to 2002, 10 outbreaks of rotavirus were reported from ongoing surveillance of outbreaks of gastroenteritis (30, 39). An outbreak was labeled rotavirus associated if 50% of the fecal specimens from patients were found positive for rotavirus by ELISA, with a minimum number of 5 specimens analyzed per outbreak and no other pathogen detected. In 1999, individual fecal samples (9 bovine and 14 porcine) were collected by the Dutch Animal Health Service. In the years 2000 and 2001, a large number of individual animal fecal samples (264 equine, 104 bovine, and 53 porcine) were collected by the Veterinary Microbiological Diagnostic Center (VMDC), Utrecht University. All specimens were collected from animals younger than 1 year of age that had shown clinical symptoms of diarrhea. Samples positive by antigen detection ELISA (Rotaclone; Meridian Diagnostics Europe) were used for rotavirus genotyping. This assay has been validated for human samples; sensitivity for other species is not exactly known.
Pooled fecal specimens from calf herds were collected as part of an ongoing surveillance study for potential zoonotic microorganisms associated with gastroenteritis in farm animals (38). All farms were situated in different regions of The Netherlands, pooled samples with a minimum of 20 and a maximum of 60 were assigned as the farm sample, and these were analyzed. Calf herd samples were collected from 1- to 52-week-old veal calves (average age, 12 weeks) at farms of 38 to 930 calves in 1997 (KA6 to KA119), 1998 (KA120 to KA272), and 1999 (KA273 to KA332). Reverse transcriptase (RT) PCR-positive fecal samples were used for genotyping based on sequencing of the VP4 and VP7 coding regions.
For ELISA and extraction of viral RNA, all fecal samples were resuspended in Hanks balanced salt solution (Gibco BRL, Breda, The Netherlands) to a final concentration of approximately 10%. Before use, suspensions were centrifuged at 3,000 x g for 20 min. Samples were stored at 4 or –70°C (long-term storage) in a 1:1 suspension with medium containing 30 g of tryptone soy broth (Oxoid CM 129) and 200 g of glycerol per liter of distilled water.
Prototype rotaviruses. The human rotavirus prototype strains WA, DS1, P, ST3, WI161, K8, and 69M and animal rotavirus prototype strains OSU, YM, NCDV, B37, B223, UK, and WC3 used in this study were kindly provided by J. R. Gentsch (Viral Gastroenteritis Section, Centers for Disease Control and Prevention, Atlanta, Ga.). These strains were propagated in MA104 cells.
RNA extraction. For extraction of viral double-stranded genomic RNA, 100 μl of a 10% fecal sample suspension or virus-infected cell culture was added to a highly concentrated (5.25 M) solution of guanidinium isothiocyanate. Silica-bound RNA was washed and eluted as previously described (4). To control for contamination, one negative-control sample was included for every two samples and designated rooms and equipment were used for each step in the PCR procedure. Human rotavirus WA, propagated in MA104 cells, was included as a positive control.
RT-PCR detection. All fecal specimens from calf herds were tested by rotavirus RT-PCR detection and hybridization. We used a single-round RT-PCR assay with generic primers targeting the VP7 gene (23). For RT, 10 μl of RNA was mixed with 5 μl of 75 pM Rota 1 and Rota 2 primers. The solution was heated to 94°C for 5 min and cooled, and 10 μl of RT buffer was added. The RT reaction was performed in a final volume of 25 μl consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.25 mM concentrations of deoxynucleoside triphosphates, 1 mM dithiothreitol, and 2.5 U of avian myeloblastosis virus RT (Promega, Leiden, The Netherlands). The mixture was incubated for 1 h at 42°C, heated for 5 min at 94°C, and then cooled. Ten microliters of the RT mixture was transferred to the PCR mix. The PCR was performed in a final volume of 50 μl consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.0 mM MgCl2, 0.2 mM concentrations of the deoxynucleoside triphosphates, and 1.5 U of AmpliTaq (Perkin Elmer, Nieuwerkerk a/d IJssel, The Netherlands), and the mixture was covered with mineral oil. After denaturation at 94°C for 5 min, the amplification consisted of 40 amplification cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, followed by a final incubation at 72°C for 10 min. The amplification products were analyzed by a 2% agarose gel electrophoresis and visualized with UV after ethidium bromide staining.
For Southern blots, the RT-PCR products in the agarose gel were denaturated by incubation in 0.5 M NaOH for 30 min and transferred to a positively charged nylon membrane (Boehringer, Almere, The Netherlands) by vacuum blotting (Biometra, Leusden, The Netherlands). Hybridization of rotavirus RT-PCR products was performed as previously described (39) with biotin-labeled probe Rota-VP7 (5'-CRAAYAARTGGATATCRATGGG-3', where Y is C or T and R is A or G) and a hybridization temperature of 42°C.
Rotavirus genotyping by RT-PCR. All human and individual animal fecal samples, positive by rotavirus-specific ELISA, were genotyped by RT-PCR. G and P typing were performed by a modification of previously published RT-PCR genotyping methods (7, 16, 19, 20). The PCR protocol was provided by J. R. Gentsch (Viral Gastroenteritis Section, Centers for Disease Control and Prevention). Primers to amplify full-length copies of the rotavirus VP7 and VP4 genes are shown in Table 1.
The upstream generic primer and different pools of specific G- and P-typing primers were used for characterization in a second-round PCR amplification. Typing primers are shown in Table 1.
The amplification products were analyzed by a 2% agarose gel electrophoresis and visualized with UV after ethidium bromide staining. The G and P genotypes of the fecal samples were analyzed by comparing the size of the second-round PCR product with the amplification products of the concerning prototype reference strains.
Rotavirus genotyping by sequencing of the VP4 and VP7 genes. All mixed fecal specimens from farms, equine rotavirus (no genotyping primer available)- and ELISA-positive specimens that were negative in the genotyping PCR, were typed by sequencing.
We used two single-round RT-PCR assays with generic primer pairs on RNA of the VP4 and VP7 genes. The generic RT-PCR on VP7 was described previously as a detection RT-PCR (23). For the RT-PCR assay of the VP4 gene, primer pair con1b (modified with one nucleotide for the B223 bovine strain, 5'-TTGCCACCAATTCAAAATAC-3') and con2 (Table 1) were used under the same conditions as the VP7 RT-PCR except for the PCR with an MgCl2 concentration of 2 mM (1).
The rotavirus RT-PCR products of the expected sizes, 304 bp (VP7) and 211 bp (VP4), were excised from a 2% agarose gel and purified with the QIAquick gel extraction kit (QIAGEN, Hilden, Germany). Purified PCR products were cloned by using a pGEM T easy vector system (Promega). Ten clones per sample were sequenced with an ABI PRISM BigDye terminator cycle sequencing reaction kit (Perkin-Elmer, Applied Biosystems) on an automated sequencer (model 3700; Applied Biosystems). Nucleotide sequences were edited by using Kodon (version 1.5; Applied Maths, Kortrijk, Belgium) and aligned in BioNumerics (version 3.0; Applied Maths). For phylogenetic analysis, distance calculations were done by using the Kimura-2 correction for evolutionary rate. The confidence values of the internal rods were calculated by performing 1,000 bootstrap analyses. Evolutionary trees for nucleotide sequences were drawn by using the neighbor-joining method.
RESULTS
G and P genotyping of human rotavirus strains collected from 1996 to 2001. In this study, we used 110 samples collected from humans between 1996 and 2001 in The Netherlands that have been found positive for group A rotavirus by ELISA and were further characterized by a second-round RT-PCR assay. Reference strains were tested in parallel (Table 2).
Eighty-seven percent of all human rotavirus strains belonged to the common P and G combinations: [P8]G1 (69.1%), [P4]G2 (6.4%), [P8]G3 (4.5%), and [P8]G4 (7.3%). We also found the [P8]G9 combination in 5.5% of the samples, one strain of [P9]G1, and one strain of [P6]G4. Six strains (5.4%) were not completely typed: five were negative in the G-typing assay (4.5%) and one was negative in the P-typing assay (0.9%). Mixed infections were not found. Eleven of the [P8] strains (11%) could be typed with the new primer 1T-1D, described by Iturriza-Gomara et al. (25); nine of these were [P8]G1 and two were [P8]G3. Rotavirus G9 strains were first detected in 1998 in The Netherlands.
Between 1999 and 2002, 10 rotavirus outbreaks in The Netherlands were reported (Table 3). Six outbreaks were caused by the common rotavirus strain [P8]G1, five of which could be typed only with the modified 1T-1D primer. We found one outbreak with the [P4]G2 strain and one with the [P8]G9 strain. The two outbreaks caused by the [P6]G9 strain were from related cases and were reported previously (41) as the cause of a protracted hospital outbreak of neonatal diarrhea in The Netherlands. The outbreak lasted for 5 months, with 52 cases and an average attack rate of 40%. We did not find this genotype in the 110 fecal samples from the two surveillance studies.
Genotyping rotavirus isolates from individual animals. In 1999, 9 bovine and 14 porcine fecal samples were tested by ELISA. In 2000 and 2001, 264 equine, 104 bovine, and 53 porcine samples were tested by ELISA. All fecal samples were collected from animals showing diarrhea. Seven bovine (2.7%), 3 porcine (4.5%), and 6 equine (2.3%) fecal samples were positive for rotavirus.
The bovine and porcine strains were genotyped by RT-PCR as well as DNA sequencing of a part of the VP7 and VP4 gene. Equine strains were genotyped only by DNA sequencing because genotype-specific primers were not available. To type the individual bovine and porcine strains by RT-PCR, several different typing primers were needed; one porcine strain was not typeable by RT-PCR (Table 4).
Full-length rotavirus VP7 genes were amplified from bovine isolates VMDC 9464, VMDC 11373, and GD14 with the primers BovCom5 and BovCom3 (24). In none of the porcine and equine isolates could a visible PCR product be generated by using full-length primer sets.
Genotyping of rotavirus VP7 clones from calf herds in The Netherlands. A total of 28 rotavirus-positive pooled calf herd samples were G genotyped after cloning and DNA sequencing. From each calf herd, a maximum of 10 clones of the VP7 gene have been sequenced and compared with sequences in GenBank (Table 5). Genotypes G6 and G10 predominated; G6 and G10 were detected in 22 (78.6%) and 16 (57.1%) of the rotavirus calf herds. In 12 (42.9%) calf herds, we found mixed infections. Genotype G8 was not found.
After phylogenetic analysis, the bovine rotavirus G6 could be divided into three groups: UK-like, VMRI-29-like, and Hun4-like. All G10 strains, however, belonged to one group (Fig. 1).
DISCUSSION
The objective of this study was to determine the P and G genotypes of circulating rotaviruses of human and animal origin in The Netherlands. Genotyping by RT-PCR is a well-established and recognized epidemiological tool for examining strain diversity (9). Correlation between rotavirus G genotypes and serotypes is now well understood, and PCR is often used for determining general genotypes (17, 19, 20). In recent years, RT-PCR has been shown to be the most useful assay for the common rotavirus types (9), but the method was less optimal for uncommon rotavirus genotypes and mixed fecal samples. For mixed samples, an explanation may be the low concentrations of virus. Alternatively, however, changes in the primer binding site may explain the lack of signals, as even for the common rotaviruses, several primers were needed. DNA sequencing of a part of the VP7 and VP4 gene enabled us to genotype mixed stool samples with low concentrations of rotaviruses or strains for which no typing primer was available, like the equine isolates.
In this study, we have mainly been focusing on the genotyping of the human and bovine rotaviruses because most of the isolates originated from these species. Because of genotyping of human and animal rotaviruses from different national surveillance studies, we have been able to produce a picture of the common G and P rotavirus genotypes in The Netherlands for the last few years. G9 strains, which have been associated with serious neonatal diarrhea (40), have been reported frequently in recent years in Europe. Although the prevalence of G9 was low in The Netherlands and in the United Kingdom (26), in Germany and Belgium, G9 has recently become the second-most-commonly detected rotavirus G type after G1 (32, 37). For development of a new rotavirus vaccine, the apparent emergence of G9 rotaviruses should be taken into account. This illustrates that a continued surveillance of rotavirus genotypes is needed to collect information on emerging new strains.
Previous reports have shown that a large majority of bovine rotavirus strains are of the G6, G10, [P5], and [P11] genotypes (14, 28). The results obtained in this study also show that the diversity of rotavirus is limited to a relatively restricted number of bovine rotavirus genotypes and that G6 and G10 strains are predominant among cattle populations in The Netherlands as well. G8 bovine rotaviruses represent the third-most-common G genotype among field isolates in Europe. In Italy, 4.7% of strains were G8 (14); in Sweden, this was 1% (28). P genotyping of these calf herd samples was not performed. The number of rotavirus-positive herds (28) was probably too small to find G8 strains. In Swedish beef herds and dairy herds, often just one G type was circulating, which could be explained by the fact that the exchange of animals between dairy herds and suckled beef herds is limited in Sweden (28). In The Netherlands, dairy calves showed various G types (43%), which may be due to more interfarm contacts among this group of animals.
Sequence analysis of the 304 bp of the VP7 gene revealed that there is enough diversity to differentiate G1 to G14. Distinct G genotypes cluster together. Others have also typed rotavirus genotypes by VP7 sequencing (35), and some used other regions (21). Since we found enough diversity on the region we amplified, we did not search for other targets. However, for the P genotypes, neither the 211 bp on the VP4 gene nor the entire genome sequences could be used to differentiate all P genotypes from each other. On several occasions, different P genotypes clustered together, like the bovine rotavirus NCDV [P1] within the genogroup [P5] rotaviruses and human rotavirus DS1 [P4] within [P6] isolates. The hypervariable region on the VP4 gene also gave the same picture. Despite these results, we were able to genotype the six equine rotavirus isolates by sequencing: two [P12]G3 isolates and four [P12]G14 isolates. Also, the six bovine rotavirus isolates could be confirmed by sequencing; because differentiation between [P1] and [P6] was not possible for the bovine isolates by phylogenetic analysis, the calf herd rotaviruses were not tested for P genotypes. Also the two porcine strains, which were genotyped by RT-PCR as [P6]G4, were confirmed. One porcine isolate negative by genotyping by PCR could be genotyped by sequencing as [P7]G3. For a good picture of the porcine rotavirus genotypes prevalent in The Netherlands, further research will be needed.
Interspecies transmissions of group A rotaviruses have been suggested, especially between humans and cattle (8). Several publications reported G6 (3, 5, 18) and G10 (36) genotype strains from humans. The human Hun4 G6 strain isolated from a patient in Hungary reported by Banyai et al. (3) was closely related to bovine strains isolated from calf herds in The Netherlands. This finding again confirms that rotavirus remains a potential zoonotic infection and that animal rotaviruses could be a reservoir for human infection.
From this study, it can be concluded that the usual human and bovine P and G genotype rotaviruses are circulating in The Netherlands. Interspecies transmission was not determined, but this may be due to the relatively small number of samples tested. Therefore, it is suggested that the study of rotavirus P and G genotyping of human and animal rotaviruses of different species be continued and that methods for early detection of interspecies transmissions be developed.
ACKNOWLEDGMENTS
This research was financially supported and approved by the Dutch Food and Consumer Product Safety Authority (VWA), project number V/224920/01/VZ.
We thank A. W. van der Giessen and Ing W. D. C. Deisz for volunteering fecal specimens of calf herds from the monitoring study for zoonotic enteric pathogens. We greatly appreciate the cooperation of the Veterinary Microbiological Diagnostic Center (VMDC), Utrecht University, for collecting individual stool samples, particularly Ing M. Stins. We also thank the Dutch Animal Health Services for providing stool samples.
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