Serotype Assignment Research Articles

Phylogenetic and immunological investigations of complete TSA56 ORF of Orientia tsutsugamushi present in acute encephalitis syndrome cases from eastern Uttar Pradesh, India.

Scrub typhus (ST) caused by Orientia tsutsugamushi (OT), has long been known to cause acute encephalitis syndrome (AES) and acute febrile illness (AFI). The immunodominant 56kDa protein of OT, which is encoded by the 56kDa gene (1600bp encoding 516-541 amino acids) is a commonly studied antigen for genotype and serotype assignment. Previous studies from India have utilized partial type specific antigen (TSA) 56kDa sequences for OT strain characterisation. On the other hand, understanding the antigenic diversity of current OT strains, is critical for developing specific diagnostic tests and vaccines against ST. As a result, the current study analyses antigenic variants using the entire TSA56 ORF of OT from AES cases. Phylogenetic investigation using complete TSA56 ORF sequences revealed Karp and Gilliam were the circulating predominant strains of OT. Furthermore, Immuno-informatical analysis demonstrated that the majority of high-binding affinity CD4 TCEs against the most prevalent Indian human leukocyte antigen alleles were present in the S-VDIII/IV and S-VDIV spacer regions of TSA56 ORF. TSA56 conserved spacer is crucial for OT immunological response investigations. Further, the pathophysiological effects of spacer domains in ST require further investigation. Furthermore, the characterization of the TSA56 spacer region of the OT from different parts of India is critical for developing region-specific ST diagnostic assays and vaccines.

ShigaPass: an in silico tool predicting Shigella serotypes from whole-genome sequencing assemblies.

Shigella is one of the commonest causes of diarrhoea worldwide and a major public health problem. Shigella serotyping is based on a standardized scheme that splits Shigella strains into four serogroups and 60 serotypes on the basis of biochemical tests and O-antigen structures. This conventional serotyping method is laborious, time-consuming, impossible to automate, and requires a high level of expertise. Whole-genome sequencing (WGS) is becoming more affordable and is now used for routine surveillance, opening up possibilities for the development of much-needed accurate rapid typing methods. Here, we describe ShigaPass, a new in silico tool for predicting Shigella serotypes from WGS assemblies on the basis of rfb gene cluster DNA sequences, phage and plasmid-encoded O-antigen modification genes, seven housekeeping genes (EnteroBase's MLST scheme), fliC alleles and clustered regularly interspaced short palindromic repeats (CRISPR) spacers. Using 4879 genomes, including 4716 reference strains and clinical isolates of Shigella characterized with a panel of biochemical tests and serotyped by slide agglutination, we show here that ShigaPass outperforms all existing in silico tools, particularly for the identification of Shigella boydii and Shigella dysenteriae serotypes, with a correct serotype assignment rate of 98.5 % and a sensitivity rate (i.e. ability to make any prediction) of 100 %.

Resistance of Streptococcus suis Isolates from the Czech Republic during 2018-2022.

A determination of susceptibility/resistance to antimicrobials via serotype was carried out in 506 field isolates of Streptococcus suis, originating from pig farms in the Czech Republic in the period 2018–2022. A very high level of susceptibility of S. suis isolates was found to amoxicillin, in combination with clavulanic acid and sulfamethoxazole potentiated with trimethoprim. None of the tested isolates were resistant to these antimicrobial substances. Only two isolates were found to be intermediately resistant to enrofloxacin in 2020. With regard to ceftiofur, one isolate was intermediately resistant in 2020 and 2022, and two isolates were intermediately resistant in 2018 and 2021. A low level of resistance was detected to ampicillin (0.6% in 2021) and to florfenicol (1.15% in 2019; 1.3% in 2022). With regard to penicillin, a medium level of resistance was detected in 2018 (10.6%), but a low level of resistance was found in the following years (7.0% in 2019; 3.1% in 2020; 3.3% in 2021; 3.9% in 2022). On the contrary, a high or very high level of resistance was found to tetracycline (66.0% in 2018; 65.1% in 2019; 44.35% in 2020; 46.4% in 2021; 54.0% in 2022). Using molecular and serological methods, serotype 7 (16.4%) was determined to be predominant among S. suis isolates, followed by serotypes 1/2, 2, 9, 4, 3, 1, 29, 16, and 31 (10.7%; 8.5%; 5.7%; 5.5%; 4.5%; 4.3%; 3.6%; 3.4%; 3.4%, respectively). Other serotypes were identified among the investigated strains either rarely (up to 10 cases) or not at all. A relatively high percentage of isolates were detected as non-typeable (79 isolates; 15.6%). Dependence of resistance upon serotype assignment could not be proven in all but serotype 31, wherein all isolates (n = 17) were resistant or intermediately resistant to clindamycin, tilmycosin, tulathromycin, and tetracycline. The resistance to clindamycin and tetracycline may be related to the high consumption of these antibiotics on pig farms at present or in previous years. Macrolides (tilmicosin and tulathromycin) and tiamulin are not suitable for the treatment of streptococcal infections, but are used on pig farms to treat respiratory infections caused by gram-negative bacteria, so they were included in the study.

A new strategy for systematically classifying HLA alleles into serological specificities.

HLA serological specificities were defined by the reactivity of HLA molecules with sets of sera and monoclonal antibodies. Many recently identified alleles defined by molecular typing lack their serotype assignment. We surveyed the literature describing the correlation of the reactivity of serologic reagents with AA residues. 20 - 25 AA residues determining epitopes (DEP) that correlated with 82 WHOserologic specificities were identified for HLA class I loci. Thirteen DEP each located in the beta-1 domains that correlated with 24 WHOserologic specificities were identified for HLA-DRB1 and -DQB1 loci. The designation of possible HLA-DPB1, -DQA1, -DPA1, and additional serological specificities that result from epitopes defined by residues located at both -DQA1 and -DQB1 subunits were also examined. HATS software was developed for automated serotype assignments to HLA alleles in one of the three hierarchical matching criteria: (1) all DEP (FULL); (2) selected DEP specific to each serological specificity (SEROTYPE); (3) one AA mismatch with one or more SEROTYPES (INCOMPLETE). Results were validated by evaluating the alleles whose serotypes do not correspond to the first field of the allele name listed in the HLA dictionary. Additional 85 and 21 DEP patterns that do not correspond to any WHO serologic specificities for common HLA class I and DRB1 alleles were identified, respectively. A comprehensive antibody identification panel would allow for accurate unacceptable antigen listing and compatibility predictions in solid organ transplantation. We propose that antibody-screening panels should include all serologic specificities identified in this study.

Monitoring the Microevolution of Salmonella enterica in Healthy Dairy Cattle Populations at the Individual Farm Level Using Whole-Genome Sequencing.

Livestock represent a possible reservoir for facilitating the transmission of the zoonotic foodborne pathogen Salmonella enterica to humans; there is also concern that strains can acquire resistance to antimicrobials in the farm environment. Here, whole-genome sequencing (WGS) was used to characterize Salmonella strains (n = 128) isolated from healthy dairy cattle and their associated environments on 13 New York State farms to assess the diversity and microevolution of this important pathogen at the level of the individual herd. Additionally, the accuracy and concordance of multiple in silico tools are assessed, including: (i) two in silico serotyping tools, (ii) combinations of five antimicrobial resistance (AMR) determinant detection tools and one to five AMR determinant databases, and (iii) one antimicrobial minimum inhibitory concentration (MIC) prediction tool. For the isolates sequenced here, in silico serotyping methods outperformed traditional serotyping and resolved all un-typable and/or ambiguous serotype assignments. Serotypes assigned in silico showed greater congruency with the Salmonella whole-genome phylogeny than traditional serotype assignments, and in silico methods showed high concordance (99% agreement). In silico AMR determinant detection methods additionally showed a high degree of concordance, regardless of the pipeline or database used (≥98% agreement among susceptible/resistant assignments for all pipeline/database combinations). For AMR detection methods that relied exclusively on nucleotide BLAST, accuracy could be maximized by using a range of minimum nucleotide identity and coverage thresholds, with thresholds of 75% nucleotide identity and 50–60% coverage adequate for most pipeline/database combinations. In silico characterization of the microevolution and AMR dynamics of each of six serotype groups (S. Anatum, Cerro, Kentucky, Meleagridis, Newport, Typhimurium/Typhimurium variant Copenhagen) revealed that some lineages were strongly associated with individual farms, while others were distributed across multiple farms. Numerous AMR determinant acquisition and loss events were identified, including the recent acquisition of cephalosporin resistance-conferring blaCMY- and blaCTX–M-type beta-lactamases. The results presented here provide high-resolution insight into the temporal dynamics of AMR Salmonella at the scale of the individual farm and highlight both the strengths and limitations of WGS in tracking zoonotic pathogens and their associated AMR determinants at the livestock-human interface.

Serotype Assignment by Sero-agglutination, ELISA, and PCR.

For assessing isolates of Listeria monocytogenes, serotype designation is the first subtyping method used. Methodologies used to assign serotype are currently evolving and will eventually be replaced with whole genome sequencing. Traditionally, serotyping has been done with agglutination reactions; however, alternative methods utilizing enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are common. Described here are the three non-genomic methods and the advantages and disadvantages of each.

Salmonella serotype assignment by sequencing analysis of intergenic regions of ribosomal RNA operons

Salmonella laboratorial detection is usually carried out by bacteriological culture and serological methods. Salmonella isolates are then classified into more than 2,650 serotypes with different somatic (O) and flagellar (H) antigenic combinations. More recently, DNA analysis methods were developed and applied for the identification of Salmonella serotypes, including intergenic spacer regions (ISRs) that separates DNA-encoding ribosomal subunits (rRNA gene) in Salmonella genomes. The present study aimed to evaluate the nucleotide diversity of the ISRs in 2 rRNA operons (rrnB and rrnH) for the assignment of Salmonella serotypes. A total of 63 Salmonella isolates (bacterial cultures) from 21 serotypes were obtained in the period of 2014 to 2017. DNA was extracted, and PCRs were used to detect the genus Salmonella and 4 important serotypes: Enteritidis, Gallinarum, Heidelberg, and Typhimurium. ISRs of the operons rrnB and rrnH were amplified by PCR and then sequenced. All sequence results were submitted to BLASTn search and were aligned in comparison to 72 Salmonella reference nucleotide sequences. The results demonstrated that 60 (95.2%) samples returned a sequence of the same serotype (determined by the traditional serological procedure) after searching in BLASTn and/or in the alignment with the reference sequences for both operons (rrnB and rrnH). These PCR-sequencing procedures had a general agreement index of 0.792 based on the Kappa analysis, 98.7% sensitivity value, 100% specificity, and 98.4% accuracy. Three different phylogenetic trees were generated from the alignments with the sequences of rrnH, rrnB, and rrnH plus rrnB and isolates clustered in specific branches according to the serotypes.

An improved DNA array-based classification method for the identification of Salmonella serotypes shows high concordance between traditional and genotypic testing.

Previously we developed and tested the Salmonella GenoSerotyping Array (SGSA), which utilized oligonucleotide probes for O- and H- antigen biomarkers to perform accurate molecular serotyping of 57 Salmonella serotypes. Here we describe the development and validation of the ISO 17025 accredited second version of the SGSA (SGSA v. 2) with reliable and unambiguous molecular serotyping results for 112 serotypes of Salmonella which were verified both in silico and in vitro. Improvements included an expansion of the probe sets along with a new classifier tool for prediction of individual antigens and overall serotype from the array probe intensity results. The array classifier and probe sequences were validated in silico to high concordance using 36,153 draft genomes of diverse Salmonella serotypes assembled from public repositories. We obtained correct and unambiguous serotype assignments for 31,924 (88.30%) of the tested samples and a further 3,916 (10.83%) had fully concordant antigen predictions but could not be assigned to a single serotype. The SGSA v. 2 can directly use bacterial colonies with a limit of detection of 860 CFU/mL or purified DNA template at a concentration of 1.0 x 10−1 ng/μl. The SGSA v. 2 was also validated in the wet laboratory and certified using panel of 406 samples representing 185 different serotypes with correct antigen and serotype determinations for 60.89% of the panel and 18.31% correctly identified but an ambiguous overall serotype determination.

2301. Streptococcus pneumoniae Serotyping: Assessing the Performance of a PCR- and Sequencing-Based Testing Algorithm

Background Streptococcus pneumoniae is a bacterium that causes significant morbidity and mortality worldwide. Its capsular polysaccharides have been used successfully as vaccine antigens, and to characterize S. pneumoniae into 92 different serotypes. Phenotypic (Quellung reaction) or genotopic (PCR or sequencing) methods can be used for serotype assignment, but the performance may vary between methods. This study compared the performance of the Quellung reaction, to an algorithm using PCR- and sequence-based serotyping technologies for vaccine-preventable or closely related serotypes.MethodsA panel of geographically diverse isolates of S. pneumoniae spanning 92 different serotypes was provided by various references laboratories worldwide. Each isolate was subjected to conventional multiplex PCR methods, using previously established methods. Sanger sequencing was performed using genetic signatures defined in the PneumoCaT database. When discrepant, Quellung reaction were repeated, and next-generation sequencing and comparative genomics was used to evaluate the sequence composition of the cps loci.ResultsAs expected, PCR was unable to assign serotype in some cases, and some serotype results were insufficiently discriminatory. Following sequencing, 86.3% (404/468) of isolates were concordant with the Quellung serotyping. Discrepant analyses are underway.ConclusionAn algorithm based on PCR and sequencing, or next-generation sequencing alone, shows much promise for serotyping of S. pneumoniae. However, discrepant results were noted, suggesting either our current understanding of genetic signatures conferring serotype-specificity might not be complete, or the Quellung reaction results were incorrect. Accurate methods for serotyping are essential to monitor the impact of pneumococcal vaccines, and understand the epidemiology of S. pneumoniae diseases.Disclosures S. A. McNeil, GSK: Grant Investigator, Research grant. Pfizer: Grant Investigator, Research grant. Merck: Collaborator and Consultant, Contract clinical trials and Speaker honorarium. Novartis: Collaborator, Contract clinical trials. Sanofi Pasteur: Collaborator, Contract clinical trials.

Genetic Analysis of the Listeria Pathogenicity Island 1 of Listeria monocytogenes 1/2a and 4b Isolates.

The aim of the present study was to apply descriptive, phylogenetic, recombination, and selection analyses on alignments of the Listeria Pathogenicity Island 1 (LIPI-1) of 1/2a and 4b Listeria monocytogenes isolates of different origin in order to gain insights into the evolution of this virulence gene cluster. For that purpose, a total of 19 L. monocytogenes isolates (9 meat isolates, serotype 1/2a; 5 meat isolates, serotype 4b; 5 strawberry isolates, serotype 4b) that have been previously separated at strain level were subjected to sequencing of their LIPI-1. Descriptive analysis revealed extensive nucleotide diversity mostly in the intragenic regions. The actA gene of 1/2a and 4b meat isolates and the hly gene of the 4b strawberry isolates exhibited the higher diversity; limited diversity was observed in prfA and plcA genes of the 4b isolates and mpl gene of the 1/2a isolates. Phylogenetic analysis of the complete island resulted in two major clusters that were consistent with serotype assignment of the isolates. Moreover, effective discrimination between serotypes was obtained by plcA, plcB, mpl, actA and the intergenic regions plcA-prfA and plcA-hly. In all cases but plcB and plcA-prfA 4b isolates were also differentiated according to their source of isolation as well. Selection analysis revealed that the island consisted of randomly evolving DNA with the exception of prfA gene of 1/2a isolates and actA gene of 4b meat isolates for which purifying selection or population expansion was indicated. Finally, no statistically significant evidence for recombination has been observed.

Comparison of molecular serotyping approaches of Streptococcus agalactiae from genomic sequences

BackgroundGroup B streptococcus (GBS) capsular polysaccharide is one of the major virulence factors underlying invasive GBS disease and a component of forthcoming vaccines. Serotype classification of GBS is based on the capsule polysaccharide of which ten variants are known to exist (Ia, Ib, II-IX). Current methods for GBS serotype assignment rely on latex agglutination or PCR while more recently a whole genome sequencing method was reported. In this study, three distinct algorithms for serotype assignment from genomic data were assessed using a panel of 790 clinical isolates.MethodsThe first approach utilised the entire capsular locus coupled with a mapping methodology. The second approach continues from the first and utilised a SNP-based methodology across the conserved cpsD-G region to differentiate serotypes Ia-VII and IX. Finally the third approach used the variable cpsG –K region coupled with a mapping methodology. All three approaches were assessed for typeability (percentage of isolates assigned a serotype) and concordance to the latex agglutination methodology.ResultsFollowing comparisons, the third approach using the variable cpsG-K region demonstrated the best performance with 99.9% typeability and 86.7% concordance. Overall, of the 105 discordant isolates, 71 were resolved following retesting of latex agglutination and whole genome sequencing, 20 failed to assign a serotype using latex agglutination and only 14 were found to be truly discordant on re-testing. Comparison of this final approach with the previously described assembly-based approach returned identical results.ConclusionsThese results demonstrated that molecular capsular typing using whole genome sequencing and a mapping-based approach is a viable alternative to the traditional, latex agglutination-based serotyping method and can be implemented in a public health microbiology setting.

Rapid Identification and Classification of Listeria spp. and Serotype Assignment of Listeria monocytogenes Using Fourier Transform-Infrared Spectroscopy and Artificial Neural Network Analysis.

The use of Fourier Transform-Infrared Spectroscopy (FT-IR) in conjunction with Artificial Neural Network software NeuroDeveloper™ was examined for the rapid identification and classification of Listeria species and serotyping of Listeria monocytogenes. A spectral library was created for 245 strains of Listeria spp. to give a biochemical fingerprint from which identification of unknown samples were made. This technology was able to accurately distinguish the Listeria species with 99.03% accuracy. Eleven serotypes of Listeria monocytogenes including 1/2a, 1/2b, and 4b were identified with 96.58% accuracy. In addition, motile and non-motile forms of Listeria were used to create a more robust model for identification. FT-IR coupled with NeuroDeveloper™ appear to be a more accurate and economic choice for rapid identification of pathogenic Listeria spp. than current methods.

Serotype assignment by sero-agglutination, ELISA, and PCR.

For assessing isolates of Listeria monocytogenes serotype designation is the foremost subtyping method used. Traditionally, serotyping has been done with agglutination reactions. In the last decade, alternative serotyping methods were described using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Herein are described the three methods, and the advantages and disadvantages of each.

Human Adenovirus Type 52: a Type 41 in Disguise?

Recently, Jones et al. described a new human adenovirus (HAdV) (3) and claimed it to be a “new type” and even a “new species”: “human adenovirus 52 or HAdV-52 of species G.” We dispute both claims. By virus neutralization (VN), they showed it to be different from its phylogenetically closest relative, simian adenovirus 1. Surprisingly, they failed to provide additional VN data corroborating their claim of a novel serotype. Consequently, the new adenovirus may later, on the basis of VN data, prove to be a known serotype, e.g., HAdV-40 or HAdV-41. In their attempts to show that the virus was sufficiently distinct from HAdV-40 and HAdV-41 to be considered a new type, they embarked on phylogenetic analyses. These viruses were accepted as separate serotypes by the ICTV (1) on the basis of differences in their DNAs and their antigenic distances as measured by VN (1, 2), complying with the definition of serotypes (or “types”) as follows. Adenovirus serotypes are differentiated on the basis of neutralization assays. A serotype is defined as one which either exhibits no cross-reaction with others, or shows a homologous/heterologous titer ratio greater than 16 (in both directions). For homologous/heterologous titer ratios of 8 or 16, a serotype assignment is made if either the viral haemagglutinins are unrelated (as shown by lack of cross-reaction in haemagglutination-inhibition tests), or if substantial biophysical, biochemical or phylogenetic differences exist (1). Jones et al. also claim that the new adenovirus belongs to a novel species, named “G.” They argue that the phylogenetic distance between “HAdV-52” and HAdV-40 and HAdV-41 is “at least as great as those differentiating the other HAdV species.” This species designation would also be unprecedented. The phylogenetic distance between HAdV-40 and HAdV-41 is also at least as great as those differentiating other HAdV species, and also these two adenoviruses are not considered different species (1). In summary, according to current taxonomical criteria and the data presented, the newly identified adenovirus should not be considered a novel serotype, let alone a novel species.

Frequency and Dynamics of Recombination within Different Species of Human Enteroviruses

Enteroviruses are members of the family Picornaviridae that cause widespread infections in human and other mammalian populations. Enteroviruses are genetically and antigenically highly variable, and recombination within and between serotypes contributes to their genetic diversity. To investigate the dynamics of the recombination process, sequence phylogenies between three regions of the genome (VP4, VP1, and 3Dpol) were compared among species A and B enterovirus variants detected in a human population-based survey in Scotland between 2000 and 2001, along with contemporary virus isolates collected in the same geographical region. This analysis used novel bioinformatic methods to quantify phylogenetic compatibility and correlations with serotype assignments of evolutionary trees constructed for different regions of the enterovirus genome. Species B enteroviruses showed much more frequent, time-correlated recombination events than those found for species A, despite the equivalence in population sampling, concordant with a linkage analysis of previously characterized enterovirus sequences obtained over longer collection periods. An analysis of recombination among complete genome sequences by computation of a phylogenetic compatibility matrix (PCM) demonstrated sharply defined boundaries between the VP2/VP3/VP1 block and sequences to either side in phylogenetic compatibility. The PCM also revealed equivalent or frequently greater degrees of incompatibility between different parts within the nonstructural region (2A-3D), indicating the occurrence of extensive recombination events in the past evolution of this part of the genome. Together, these findings provide new insights into the dynamics of species A and B enterovirus recombination and evolution and into the contribution of structured sampling to documenting reservoirs, emergence, and spread of novel recombinant forms in human populations.

Characterization of Botulinum Progenitor Toxins by Mass Spectrometry

Botulinum toxin analysis has renewed importance. This study included the use of nanochromatography-nanoelectrospray-mass spectrometry/mass spectrometry to characterize the protein composition of botulinum progenitor toxins and to assign botulinum progenitor toxins to their proper serotype and strain by using currently available sequence information. Clostridium botulinum progenitor toxins from strains Hall, Okra, Stockholm, MDPH, Alaska, and Langeland and 89 representing serotypes A through G, respectively, were reduced, alkylated, digested with trypsin, and identified by matching the processed product ion spectra of the tryptic peptides to proteins in accessible databases. All proteins known to be present in progenitor toxins from each serotype were identified. Additional proteins, including flagellins, ORF-X1, and neurotoxin binding protein, not previously reported to be associated with progenitor toxins, were present also in samples from several serotypes. Protein identification was used to assign toxins to a serotype and strain. Serotype assignments were accurate, and strain assignments were best when either sufficient nucleotide or amino acid sequence data were available. Minor difficulties were encountered using neurotoxin-associated protein identification for assigning serotype and strain. This study found that combined nanoscale chromatographic and mass spectrometric techniques can characterize C. botulinum progenitor toxin protein composition and that serotype/strain assignments based upon these proteins can provide accurate serotype and, in most instances, strain assignments using currently available information. Assignment accuracy will continue to improve as more nucleotide/amino acid sequence information becomes available for different botulinum strains.

Characterization of Nontypeable Rotavirus Strains from the United States: Identification of a New Rotavirus Reassortant (P2A[6],G12) and Rare P3[9] Strains Related to Bovine Rotaviruses

Among 1316 rotavirus specimens collected during strain surveillance in the United States from 1996 to 1999, most strains (95%) belonged to the common types (G1 to G4 and G9), while 5% were mixed infections of common serotypes, rare strains, or not completely typeable. In this report, 2 rare (P[9],G3) and 2 partially typeable (P[6],G?; P[9],G?) strains from that study were further characterized. The P[6] strain was virtually indistinguishable by hybridization analysis in 10 of its 11 gene segments with recently isolated P2A[6],G9 strains (e.g., U.S.1205) from the United States, but had a distinct VP7 gene homologous (94.7% aa and 90.2% nt) to the cognate gene from P1B[4],G12 reference strain L26. Thus, this serotype P2A[6],G12 strain represents a previously unrecognized reassortant. Three P3[9] strains were homologous (97.8–98.2% aa) in the VP8 region of VP4 to the P3[9],G3 feline-like reference strain AU-1, but had a high level of genome homology to Italian bovine-like, P3[9],G3 and P3[9],G6 rotavirus strains. Two of the U.S. P3[9] strains were confirmed to be type G3 (97.2–98.2% VP7 aa homology with reference G3 strain AU-1), while the other was most similar to Italian bovine-like strain PA151 (P3[9],G6), sharing 99.0% aa homology in VP7. Cross-neutralization studies confirmed all serotype assignments and represented the first detection of these rotavirus serotypes in the United States. The NSP4 genes of all U.S. P3[9] strains and rotavirus PA151 were most closely related to the bovine and equine branch within the DS-1 lineage, consistent with an animal origin. These results demonstrate that rare strains with P and G serotypes distinct from those of experimental rotavirus vaccines circulate in the United States, making it important to understand whether current vaccine candidates protect against these strains.

High sequence divergence in the 5′ non-coding region of reference Coxsackie B and ECHO viral strains and clinical isolates revealed by restriction fragment length polymorphism analysis

We report the restriction fragment length polymorphism (RFLP) patterns of a 440-bp-long 5′ non-coding region (5′NCR) amplification target of all 34 reference Coxsackie B and ECHO (enteric cytopathic human orphan) enterovirus strains and a total of 42 serotypically pre-assigned clinical isolates, in order to afford meaningful comparisons among these patterns and those of polioviruses. The RFLP patterns of reference Coxsackie B strains differed from one another and from those of polio and ECHO reference enteroviruses except from Coxsackie B1 and B2, which, although they differed from one another, had identical RFLP patterns with ECHO 17 and 13, respectively. The 28 ECHO reference strains formed a more variable viral group including strains with RFLP patterns distinct from one another and from those of polio and Coxsackie B enteroviruses, and others with RFLP pattern identities common to other ECHO viruses and Coxsackie B1 and B2 but not polioviruses. The RFLP patterns of the clinical isolates and their corresponding serotypically assigned reference Coxsackie B and ECHO strains presented the most notable variations. The observed differences between serotype and genotype-dependent assignments within the 440-bp long 5′NCR target sequence of Coxsackie B and ECHO enteroviruses were in sharp contrast to the analogous situation with polioviruses. These findings support the specificity of the described method for clinical diagnostic genotyping of polioviruses and demonstrate that the 440-bp-long target sequence follows a different evolutionary process in polio and non-polio enteroviruses that is particularly prominent between reference non-polio strains and their serotypically assigned clinical isolates.

Emergence of serotype G9 human rotaviruses in Australia.

Emergence of serotype G9 human rotaviruses in Australia.