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Journal of Clinical Microbiology, March 2007, p. 972-978, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01696-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Diversity in Indian Equine Rotaviruses: Identification of Genotype G10,P6[1] and G1 Strains and a New VP7 Genotype (G16) Strain in Specimens from Diarrheic Foals in India

B. R. Gulati,^1, R. Deepa,^2, B. K. Singh,¹ and C. Durga Rao^2*

National Research Centre on Equines, Sirsa Road, Hisar, Haryana, India,¹ Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India²

Received 16 August 2006/ Returned for modification 29 October 2006/ Accepted 17 November 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rotaviruses causing severe diarrhea in foals in two organized farms in northern India, during the period from 2003 to 2005, were characterized by electropherotyping, serotyping, and sequence analysis of the genes encoding the outer capsid proteins. Of 137 specimens, 47 (34.31%) were positive for rotavirus and exhibited at least five different electropherotypes (E), E1 to E5. Strains belonging to different electropherotypes exhibited either a different serotype/genotype specificity or a lack of reactivity to typing monoclonal antibodies (MAbs) used in this study. Strains belonging to E1, E2, and E5 exhibited genotype G10,P6[1], G3, and G1 specificities and accounted for 19.0, 42.9, and 9.5% of the isolates, respectively. Though they possessed G10-type VP7, the E1 strains exhibited high reactivity with the G6-specific MAb, suggesting that the uncommon combination of the outer capsid proteins altered the specificity of the conformation-dependent antigenic epitopes on VP7. E3 and E4 strains accounted for 28.6% of the isolates and were untypeable. Sequence analysis of VP7 from E4 strains (Erv92 and Erv99) revealed that they represent a new VP7 genotype, G16. The detection of unexpected bovine rotavirus-derived G10,P6[1] reassortants, G1 serotype strains, and a new genotype (G16) strain in two distant farms reveals an interesting epidemiological situation and diversity of equine rotaviruses in India.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rotaviruses, members of the family Reoviridae, are the major etiologic agents of severe, acute dehydrating diarrhea in the young of many mammalian species, including humans, calves, and foals (43, 55). Recent estimates indicate an annual death toll of approximately 600,000 infants due to rotavirus in addition to a staggering economic burden worldwide (54). In 1975, Flewett et al. (22) first detected and described the presence of group A rotavirus in stools of foals. Equine group A rotavirus is the main cause of diarrhea in foals up to 3 months of age, causing severe economic loss due to morbidity and mortality in studs (4, 36, 38).

The rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA) and encodes six structural and six nonstructural proteins. The genome is enclosed in a triple-layered protein capsid (21). VP4 and VP7, the two proteins comprising the outer capsid, are encoded by gene segments 4 and 7, 8, or 9 (depending on the strain), respectively (20, 43). VP6, encoded by gene segment 6, constitutes the intermediate capsid, and VP2, encoded by RNA segment 2, forms the inner capsid (21, 26).

Based on the antigenic epitopes present on the intermediate capsid protein VP6, rotaviruses are classified into groups and subgroups. Seven groups termed A to G have been identified, of which group A rotaviruses are the major pathogens of humans and animals (43). Four subgroups, I, II, I and II, and non I/II, have been identified among group A rotaviruses (26). Rotaviruses can also be classified as "long" or "short" electropherotypes (E types) based on the "fast" or "slow" electrophoretic mobility, respectively, of gene segment 11 in polyacrylamide gels. While human rotaviruses with a "short" RNA pattern generally exhibit subgroup I specificity, those with a "long" RNA pattern possess subgroup II VP6. In contrast, a long RNA pattern and subgroup I specificity are commonly associated with animal rotaviruses (43). Rotaviruses have been classified into G and P serotypes based on the antigenic specificity of outer capsid proteins VP7 (glycoprotein) and VP4 (protease sensitive), respectively (20). So far, 15 G serotypes/genotypes have been recognized, and of these, several serotypes are shared between humans and animals (43, 53). Serotypes G1 to G4 are most widespread in humans (43). Serotypes G6, G8, and G10 are major pathogens in cattle (27, 58, 63), but strains belonging to these serotypes have frequently been detected in humans in recent years (3, 8, 14, 15, 17, 23, 24, 30, 41, 43, 57). Serotype G3 strains appear to have the broadest host range and were observed in humans and many animal species (43). Three widely separated regions, A (amino acids [aa] 87 to 101), B (amino acids 143 to 152), and C (amino acids 208 to 223), have been identified as major antigenic determinants on VP7 and were suggested to form complex, functionally related, and operationally overlapping conformational epitopes that determine the serotype and neutralization specificities of rotaviruses (16, 18-20, 34, 44, 47, 61). Due to a lack of appropriate antibody reagents, a dual-P typing system (P serotypes and P genotypes) is being used to characterize rotaviruses (20). However, genotypes do not always correlate with serotypes (45, 46). So far, only 11 P serotypes have been characterized, and at least 26 P genotypes have been identified (21, 43). Strains sharing 89% aa sequence identities are considered to belong to the same genotype (21). In this typing system, the P serotype is represented by a number immediately after the letter P and the genotype is denoted by a number in square brackets (20).

Foal rotaviruses exhibit RNA electropherotypes that are distinct from those of strains from calves, pigs, mice, deer, and humans (59). Most of the equine rotaviruses exhibit an RNA electrophoretic migration pattern of 4:2:3:2 characteristic of group A rotavirus (13). A reverse transcriptase PCR assay (64) and an enzyme-linked immunosorbent assay (ELISA) (7) have been developed for G typing, and hybridization assays with specific probes have been used in P typing of equine group A rotaviruses (35, 39). Strains belonging to the G3,P4[12] serotype are most prevalent in diarrheic foals, followed by those belonging to the G14,P4[12] serotype (4, 5, 7, 13, 28, 33, 36, 37, 39, 50, 64). Single isolates, each belonging to G5,P9[7] (H-1) (12, 28, 32), G8,P6[1] (Eq/26/94) (39), G10,P8[11] (Eq/R-22) (35), and G13,P[18] (L338) (6) genotypes/serotypes, have been reported for young horses suffering from diarrhea. G14 equine rotavirus strains had an overall genomic RNA constellation that was highly conserved not only with contemporary and earlier G3 strains isolated in Japan but also with prototype G3 and G14 strains identified in the United States (50). An inactivated vaccine (HO-5 strain; G3,P[12]) was recently developed in Japan to immunize mares and thereby protect foals against rotavirus diarrhea through passive immunity (38).

To date, there exists no information on equine group A rotaviruses in India. This limited epidemiological study was undertaken to determine the serotypic/genotypic nature of rotaviruses circulating in diarrheic foals. Here, we describe for the first time the identification and characterization of G10,P6[1] rotaviruses that are likely to be reassortants between serotype G10 and P6[1] bovine strains, G1 strains, and strains representing a new VP7 genotype (G16) in specimens from diarrheic foals in two equine farms from northern India.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Viruses, virus isolation, and adaptation to cell culture. Fecal samples from young horses below the age of 3 months, suffering from severe diarrhea, were collected from two equine farms in Hisar (Haryana) and Hapur (Uttar Pradesh) in northern India between March and July during the years 2003 to 2005. A total of 137 stool specimens were processed as described earlier (60). Some of the samples that were positive for rotavirus and showed good-quality RNA are Erv2, Erv3, Erv25, Erv28, Erv58, Erv64, Erv75, Erv77, Erv80, Erv92, Erv94, Erv95, Erv96, Erv97, Erv101, Erv104, Erv105, Erv108, Erv155, and Erv165. Of these isolates, Erv2 to Erv28 were collected during the year 2003 from the equine farm in Hisar and Erv58 to Erv165 were collected during 2004 and 2005 from the Hapur farm. The established prototype rotavirus strains Wa (SGII, P1A[8], G1), S2 (SGI, P1B[4], G2), RRV (SGI, P5[3], G3), ST3 (SGII, P2A[6], G4), NCDV (SGI, P6[1], G6), and I321 (SGI, P8[11], G10) were grown in MA104 cells for use as controls in subgrouping and serotyping ELISAs.

Electropherotype analysis. Rotavirus in the specimens was first detected by polyacrylamide gel electrophoresis of genomic dsRNA as previously described (15, 29, 60). Two or three isolates representing different E types were adapted to growth in MA104 cells (67). Differences in RNA patterns were confirmed by coelectrophoresis of the genomic RNAs from two strains belonging to different E types.

Subgroup and serotype analysis. Subgrouping and serotyping ELISAs were carried out as described earlier (1, 25, 51, 60). In the subgrouping ELISA, hyperimmune anti-RRV antiserum R2, the SGI-specific monoclonal antibody (MAb) 255/60, and the SGII-specific MAb 631/9 were used (25). The MAbs specific for G1 (5E8), G2 (2F1), G3 (4F8), G4 (ST-2G7), G6 (1C3), and G10 (B223/N27), used in the serotyping ELISA, were reported earlier (1, 51) and were generously provided by H. B. Greenberg (Stanford University School of Medicine, Stanford, CA).

cDNA cloning and comparative sequence and phylogenetic analysis of VP4 and VP7 genes of equine isolates. Viral genomic dsRNA purified from fecal samples was used for reverse transcriptase PCR using avian myeloblastosis virus reverse transcriptase and Taq DNA polymerase as described previously (53). Gene-specific primers were used for cDNA synthesis and PCR amplification. The VP4 gene-specific 5' and 3' primers were 5'-CTAAGCTTCCCGGGCTATAAAATG(C/G)(C/G)TTC-3' and 5'-CTAAGCTTCCCGGGTCACATC(C/T)T-3', respectively. The respective sequences for VP7 gene primers were 5'-CTTCCCGGGCTTTAAAAG(A/C)GAGAAT-3' and 5'-CTTCCCGGGTCAC(A/G)(T/A)(C/G)ATACA-3'. Primers contained sites for specific restriction enzymes at the 5' ends. Rotavirus gene-specific nucleotide sequences in the primers are underlined. The PCR-amplified DNAs were digested with appropriate restriction endonucleases and cloned into either pUC18 or pBluescript (KS+) vector. Sequencing of the cloned VP4 and VP7 genes was carried out by Macrogen, Korea. To rule out PCR-mediated nucleotide substitutions, sequences of both strands of at least two clones for each gene from Erv2, Erv101, Erv80, Erv105, Erv92, and Erv99 strains, obtained from two independent PCR products (a total of four clones), were determined using vector-specific as well as gene-specific internal primers. The sequence of only about 776 nucleotides (nt) from the 5' and 3' ends of the VP4 gene from Erv2 and Erv101 was determined. The nucleotide and deduced amino acid sequences of VP4 and VP7 genes were analyzed and compared with the previously published corresponding rotavirus gene sequences representing all the established serotypes/genotypes. A phylogram was constructed by the MEGA 3.1 program using the p distances and the neighbor-joining method (56). The distance is the proportion of amino acid differences to the total number of sites compared.

Nucleotide sequence accession numbers. The GenBank accession numbers for the equine VP7 gene sequences are as follows: Erv2/Erv28/Erv105, DQ981476; Erv80, DQ981477; Erv92/Erv99, DQ981478; and Erv105, DQ981479.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Electropherotypes and serotypes. Of 137 samples, 47 (34.31%) were found to be positive for rotavirus by both RNA-polyacrylamide gel electrophoresis and ELISA. All the equine strains, without exception, showed long RNA patterns and subgroup I specificity (Fig. 1). Among the 47 isolates, at least 5 distinct electropherotype patterns (E1 to E5) were observed. Among the five E types, major differences were observed in the migration patterns of RNA segments 2, 3 and 4, 5 and 6, 7, 8 and 9, and 11 (Fig. 1). Coelectrophoresis of the RNAs from strains representing different E types clearly established the differences among the five electropherotypes (data not shown). The E5 pattern is not shown due to poor quality of RNA. Of the 47 rotavirus-positive samples, 21 isolates that exhibited good-quality RNA and that represent each of the five electropherotypes were serotyped using available MAbs specific for serotypes G1 to G4, G6, and G10. As shown in Table 1, strains belonging to different electropherotype patterns and showing differences in the migration of the RNA segments 7, 8, and 9 exhibited either different serotype specificities or a lack of reactivity with the typing MAbs. Of note, E1 strains, represented by Erv2 (Tables 1 and 2), showed high reactivity with the MAb specific for G6 (1C3) compared to what was observed with the G10 MAb B223/N7 and these strains accounted for 19.0% of the characterized isolates. Thus, by serotype analysis, the E1 strains appear to belong to the G6 serotype. E2 strains, represented by Erv80 (Tables 1 and 3), accounted for 42.9% of the isolates and reacted only with the G3-specific MAb 4F8, indicating that this group of strains belongs to the G3 serotype. As reported in previous studies, the G3 serotype is most prevalent among equine strains (4, 5, 28, 36, 37, 39, 64). Strains belonging to E3 and E4, representing 28.6% of the isolates, did not show any reactivity with the typing MAbs, though a good amount of viral RNA was detectable in these samples. E5 strains, represented by Erv155 and Erv165, accounted for 9.5% of the isolates and exhibited high reactivity only with the G1-specific MAb 5E8, suggesting that these isolates represent the G1 serotype.

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FIG. 1. Electropherotype analysis of the genomic dsRNA of the equine rotavirus strains.

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TABLE 1. Serotype analysis of equine isolates by ELISA using serotype-specific MAbs

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TABLE 2. Comparison of the nucleotide and deduced amino acid sequence identities of the VP7 gene from equine strains with that from strains representing the established 15 G serotypes/genotypesa

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TABLE 3. Summary of electropherotypes and serotypes/genotypes of the equine rotavirus isolatesa

Nucleotide sequence analysis of VP7 and VP4 genes. The VP7 gene from all the strains was 1,062 nt in length and encoded a protein of 326 aa. Comparison of the gene sequences from the two E1 strains Erv2 and Erv28 with each other revealed complete identity, indicating that the isolates from two different farms belonging to E1 represent a single strain. Comparative analysis of the nucleotide and deduced amino acid sequences of the VP7 gene from Erv2 and Erv28 with the corresponding gene sequences from the prototype strains representing the known 15 G serotypes/genotypes revealed 86.4% and 96.3% identities, respectively, with the VP7 gene from the G10 serotype bovine strain B223. The nucleotide and amino acid sequence identities with VP7 from other serotypes ranged between 65.5 and 77.7% and 62.1 and 84.4%, respectively (Table 2). These results indicate that the E1 strains, though they appeared to represent the G6 serotype based on reactivity with the G6-specific MAb, in fact belong to the G10 genotype. To determine the VP4 genotype of the E1 strains, the sequence of 776 nt from the 5' and 3' ends was compared with the corresponding gene sequences from all the known VP4 serotypes/genotypes. Of significance, both the 5'- and 3'-end sequences of the VP4 gene from Erv2 and Erv28 showed 96.3% identity with the P6[1]-type VP4 gene from the bovine NCDV strain (data not shown), indicating that the E1 strains represented by Erv2 and Erv28 possess G10,P6[1] serotype/genotype specificities.

Comparison of the VP7 gene sequences from E2 strains Erv80 and Erv105 showed 87.3% nt and 95.6% aa identities between themselves, suggesting that they belong to the same genotype but exhibit limited genetic diversity. Comparison with VP7 sequences from other serotypes revealed that both strains shared greatest homology to the prototype serotype G3 simian strain SA11, exhibiting 95.4% aa identity. The nucleotide sequence identities were 82.4 and 83.1%, respectively. The amino acid sequence identity of VP7 from Erv80 with that from other G3 equine strains ranged between 92.9 and 93.6%. However, VP7 from Erv105 showed slightly higher amino acid identity with other equine G3 strains, ranging between 94.8 and 95.4%. Of significance, the VP7 genes from both strains also showed 87.4 to 89% aa identity with those from G9, G11, and G14 strains BA201, YM, and JE91, respectively (Table 2). The amino acid identities with VP7 from other serotypes ranged from 59.8 to 85.0%. These results indicate that the E2 strains, represented by Erv80 and Erv105, belong to serotype G3.

The VP7 gene sequences from the E4 strains Erv92 and Erv99 were identical, suggesting that both isolates represent a single strain. Comparison with the VP7 sequences representing all the 15 G serotypes/genotypes showed amino acid sequence identities ranging between 59.5 and 85.3% (Table 2). The predicted 85.3% aa identity with G3 strain SA11 is less than the required 89% identity for assigning a strain to a specific G serotype/genotype. The major antigenic determinant regions A, B, and C of the E4 strains also showed very high sequence diversity compared to those of all the established 15 G serotypes/genotypes (Fig. 2), suggesting that strains Erv92 and Erv99 represent a new G genotype. Phylogenetic analysis also revealed Erv99 as a new genotype that is distinct from the known G genotypes (Fig. 3). The VP7 gene from the E3 strains, represented by Erv25, Erv58, and Erv64, could not be PCR amplified with the primers used. Although the E5 strains, represented by Erv155 and Erv165, were assigned to the G1 serotype based on their high reactivity with the G1-specific MAb, the sequences of VP7 and VP4 genes need to be determined. Efforts are directed towards cloning the VP7 gene from E3 and E5 strains as well as the VP4 gene from strains belonging to E2 to E5. Serotype and nucleotide sequence analyses revealed a general correspondence between electropherotypes and serotypes/genotypes, as summarized in Table 3.

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FIG. 2. Comparison of the deduced amino acid sequences of the major antigenic variable regions A, B, and C of VP7 from equine rotavirus strains with those from strains representing all the established G serotypes/genotypes. Note that the antigenic regions of the E4 strains, represented by Erv99, are highly divergent from those of all other G serotypes. The accession numbers for the gene sequences compared in this study are as follows: Wa, P03532; HU5, P04328; SA11, P03533; Hochi, BAB032864; OSU, P08406; NCDV, Q65699; Ch2, P29821; R291, AY855064; BA201, AY695811; B223, P17700; YM, P17466; L26, M58290; L338, D13549; JE91, BAB40366; and Hg18, AF237666.

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FIG. 3. Phylogenetic tree of the deduced amino acid sequences of VP7 representing all the established G serotypes/genotypes and the equine strains representative of the three electropherotypes E1, E2, and E4. The phylogram was constructed by the MEGA 3.1 program using the p distances and the neighbor-joining method.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study on characterization of a limited number of strains revealed high diversity of electropherotypes and serotypes/genotypes in equine rotaviruses isolated from two farms in northern India. Prior to this study, there was no information on the serotypic and genotypic nature of rotaviruses circulating in diarrheic foals in India. At least five distinct electropherotypes, each of which represented a distinct G serotype/genotype, have been identified. Among the isolates characterized from diarrheic foals, G3, G10,P6[1], and G1 serotypes/genotypes represented 42.9, 19.0, and 9.5%, respectively. E3 and E4 strains, accounting for 28.6% of the isolates, did not react with the serotyping MAbs used in this study. In contrast to the G3 and G10 serotype strains, VP7 from the E4 strains Erv92 and Erv99 exhibited highly divergent antigenic regions A, B, and C (Fig. 2) and less than 85.3% aa identity with that of strains representing the established 15 G serotypes/genotypes (Table 2), suggesting that these strains represent a new G genotype, G16. The serotypic/genotypic nature of the E3 strains needs to be determined.

Another observation of significance is the identification of strains that exhibited high reactivity with the typing MAb specific for the G1 serotype in specimens from diarrheic foals. To date, G1 strains have not been reported to be associated with diarrhea in young horses. It may be noted that though strains belonging to G3, G1, and the new G16 genotypes were detected only in the Hapur farm, their presence in the Hisar farm cannot be precluded because of the limited number of samples characterized from the latter.

The observation that the unusual G10,P6[1] and the E3 untypeable strains are detected in diarrheic foals from two distant farms in India is of epidemiological importance. Recently, we reported very high prevalence of G10,P8[11] strains in diarrheic calves which accounted for 80 to 85% of the bovine isolates in some farms in different regions of India (66). Age-old traditions, the extensive use of cattle waste as manure and firewood, and the close proximity of the majority of the Indian population with cattle appear to have played a facilitating role in the evolution and persistence of genotype G10,P[11] and G9,P[11] reassortant asymptomatic/symptomatic strains in newborn children in India (15, 23, 41, 66). Strains that are reassortants between animal-human or animal-animal strains have also been reported for both humans and cattle in India (15, 17, 40, 65, 66). This is the first report of genotype G10,P6[1] strains in equines, and considering the unique epidemiological scenario in India, it is likely that the genotype G10,P6[1] strains evolved by reassortment in nature between genotype G10,P8[11] and G6,P6[1] bovine strains and adapted to growth in foals, causing diarrhea.

It is of importance to note that the E1 strains, though they possessed G10 VP7, exhibited high reactivity with the G6-specific MAb compared to what was observed with the G10-specific MAb (Table 1). In this context, it should be noted that the natural partner of G10-type VP7 in the outer capsid of bovine strains is P8[11]-type VP4 and that of G6 VP7 is P6[1]-type VP4. Independent segregation of VP4 and VP7 has also been reported for several strains (31). Further, a single VP4 (P2A[6]) is associated with four VP7 serotypes, 1, 2, 3, and 4, in neonatal asymptomatic strains (43). In recent years, reassortants with a wide variety of gene constellations that encode proteins of heterologous parental origin have been reported (43). Though VP4 and VP7 can function as independent protective antigens, several reports indicate that an uncommon combination of VP4 and VP7 in some reassortants affects the expression of important viral phenotypes, such as virion stability (11), receptor binding (49), protease sensitivity of VP4 and plaque morphology (9, 62), and expression of conformation-specific neutralization epitopes on both the proteins (9, 10, 16, 52). The neutralizing epitopes on VP7 and VP4 appear to require association and highly specific interactions between VP4 and VP7 (10, 52). The neutralizing epitopes on VP7 are conformation dependent, appear to be complex and unstable, and require calcium for stability (16). This could explain the failure of purified recombinant VP7 to function as an effective protective antigen in animals immunized against rotavirus (2, 42, 48). In this context, it is possible that the association of G10 VP7 with P6[1] VP4, instead of the P8[11] VP4 that is commonly found in the G10 bovine strains, could have resulted in altered antigenic properties leading to high reactivity of the G10 VP7 with the G6-specific MAb. In contrast, the G10,P8[11] asymptomatic neonatal strain I321 showed reactivity only with the G10-specific MAb, suggesting that the high reactivity of all the E1 strains with the G6-specific MAb is indeed due to the antigenic property of the strains and not due to a nonspecific reaction. The present study, though limited, unraveled an interesting epidemiology of equine rotaviruses, with novel genotypes/serotypes circulating in large numbers in diarrheic foals in two different locations in India. The identification of two new rotavirus genotypes (G15 and G16) in succession from India (reference 53 and this study) signifies the need to characterize untypeable strains to further identify novel genotypes/serotypes. Taking into account the unique genetic/antigenic repertoire of rotaviruses, these findings are of significance in the context of the development of an effective vaccine against equine rotavirus diarrhea in India.

 
This work was supported in part by financial support from the Indian Council of Agricultural Research, Government of India, to B. R. Gulati.

The use of the DBT bioinformatics facility at the Indian Institute of Science is acknowledged.

 
* Corresponding author. Mailing address: Department of Microbiology & Cell Biology, Indian Institute of Science, Bangalore 560012, India. Phone: 91-80-23602149. Fax: 91-80-23602697. E-mail: cdr{at}mcbl.iisc.ernet.in.

Published ahead of print on 29 November 2006.

These authors contributed equally to this work.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, March 2007, p. 972-978, Vol. 45, No. 3
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