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Serogrouping of United States and some African serotypes of bluetongue virus using RT-PCR.

The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.

Animals↗

Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models.

A detailed comparison of recombinant adeno-associated viral (rAAV) vectors of serotypes 2, 5, and 8 was performed in mice and nonhuman primates. Differences within the capsid proteins and viral terminal repeats of rAAV-2 and -5 did not significantly influence their ability to transduce murine liver. However, vectors pseudotyped with AAV-8 capsid (rAAV-2/8) mediated transgene expression more rapidly and from lower doses than possible with rAAV-2 and -5, although expression declined from peak values in a distinct dose-dependent manner prior to reaching steady-state levels. Nevertheless, at all time points and vector doses, rAAV-2/8 transgene levels were 17- to 84-fold higher than with rAAV-2 or -5 due to a more rapid conversion of the single-stranded genome to transcriptionally active stable duplex DNA. In nonhuman primates, liver-targeted administration of rAAV-5 and rAAV-2/8 vectors established therapeutic levels of transgene expression. The importance of preexisting serotype immunity was highlighted by the inability to achieve successful transduction in the presence of serotype-specific antibodies, although this impediment was successfully avoided through the use of alternative serotypes. In summary, serotype-specific differences in transduction biology and the appreciation of preexisting immunity will likely influence the selection of the rAAV serotype for future clinical trials.

Animals↗

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes. The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response. In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.

Acridines↗

Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.

Amino Acid Sequence↗

Influence of the sigB gene on the cold stress survival and subsequent recovery of two Listeria monocytogenes serotypes.

The influence of serotype and the role of the sigB gene of Listeria monocytogenes during the survival and recovery on different substrates were determined. Wild-type and sigB mutants of two serotypes of L. monocytogenes were inoculated into buffer and onto beef steaks, and incubated at 4 degrees C for 8 weeks during which samples were removed and Listeria numbers determined. Growth kinetics of stationary phase wild-type and sigB mutant cells were compared, without prechilling and after prechilling at 4 degrees C. The two serotypes had similar survival capabilities under the conditions examined, and the sigB gene was influential in survival of chill stress, but was dependent upon additional nutritional factors. Prechilling cells prior to growth extended the lag phase of both strains, and this lag phase extension was compounded by the absence of a functional sigB gene. In conclusion, the sigB gene is involved in the survival and recovery from chill stress by the two serotypes tested. Additional factors such as previous growth conditions, nutritional requirements and serotype susceptibility are also contributory. This study adds relevant information regarding the influence of the sigB gene, in conjunction with the historical growth conditions and serotype differences. Understanding the significance of these factors may be useful in creating improved recovery systems for the detection of L. monocytogenes from at-risk foods.

Adaptation, Physiological↗

The genetic organization and transcriptional analysis of the short unique region in the genome of nononcogenic Marek's disease virus serotype 2.

Studies on the Marek's disease virus (MDV) serotype 2 (MDV2) genome may be important for understanding the naturally nononcogenic nature of the virus. To determine the complete DNA sequence of MDV2 unique short (Us) region, genomic BamHI fragments F, M1 and R were sequenced. The MDV2 Us region is 12109 bp long and contains 12 potential open reading frames (ORFs) likely to encode for proteins. Seven of them exhibit homologies to herpes simplex virus type 1 (HSV-1) US1 (ICP22), US2, US3 (protein kinase), US6 (gD), US7 (gI), US8 (gE) and US10 genes. These ORFs are conserved in a similar arrangement with those of HSV-1, except for US10 which is transposed in the Us regions of all three MDV serotypes. The predicted amino acid sequence of MDV2 ORF6 is homologous to SORF3 of the other serotypes of MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT) and to infectious laryngotracheitis virus SR1. In addition, four ORFs, which have been identified around the Us and inverted repeat junction regions, have no apparent relation to any other known herpesvirus genes. The identified ORFs in the MDV2 Us region were more colinear with their previously reported locations of MDV1 than with those of HVT and other alphaherpesviruses. Ten of the 12 ORFs in the MDV2 Us region were expressed and transcribed with 3'-coterminal transcripts and/or a unique transcript in the virus-infected cells. Compared to other MDV serotypes, the MDV2 Us-encoded proteins showed 46-70% and 33-59% identities with equivalent of MDV1 and HVT at the amino acid level, respectively. Our present data will be useful to understand the different pathogenicity among serotypes of MDV and to allow precise manipulation of the genes for a possible use in genetically engineered vaccines.

Amino Acid Sequence↗

Occurrence, serotypes and biotypes of thermophilic Campylobacters isolated in Vienna.

During the 1982-1986 period of all bacterial pathogens found to have caused diarrhoea, 35% belonged to the genus Campylobacter (C). Approximately 70% of the strains were isolated from persons under the age of 30 years, with a distinct peak of occurrence in the autumn. Biotyping and serotyping according to Lior yielded the following results: C. jejuni biotype I: 32.9%, C. jejuni biotype II: 48.6%, C. coli biotype I: 10.3%, C. coli biotype II: 8.2%. From the 121 strains serotyped, 118 (97.5%) were typable. The serotypes most frequently encountered were type 1 (15.7%), 4 (9.9%), 2 and 11 (7.4% each). There were 2 familial outbreaks of Campylobacter enteritis which could be completely elucidated by biotyping and serotyping. One outbreak was caused by C. jejuni biotype I serotype 11, the other by C. jejuni biotype II serotype 6. Considering the frequent occurrence of Campylobacter infections, isolates should be routinely typed. The existing typing methods and schemes are highly developed.

Age Factors↗

Serotype H5a5b is a major clone within mosquito-pathogenic strains of Bacillus sphaericus.

Seventy six mosquito pathogenic strains of Bacillus sphaericus and 10 non-pathogens were examined by pulsed field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA. Non-pathogenic strains were clearly distinguished from the entomopathogenic types which were assigned to 21 groups (SmaI restriction patterns; SRPs). Some agreement between SRP based on PFGE and serotyping was noted, in particular all 39 strains of serotype 5a5b examined revealed identical SRPs indicating total conservation of the SmaI restriction site in these bacteria. Serotype 5a5b (SRP 12) strains comprise a widely distributed and abundant clonal lineage. Most serotypes, however, were divided into several SRPs. Seven strains from serotype 2a2b were covered in five SRPs in which toxin synthesis was correlated with chromosomal structure. Similarly, toxicity correlated with SRP in strains from serotypes 3 and 6.

Animals↗

Structure of poliovirus type 2 Lansing complexed with antiviral agent SCH48973: comparison of the structural and biological properties of three poliovirus serotypes.

BACKGROUND: Polioviruses are human pathogens and the causative agents of poliomyelitis. Polioviruses are icosahedral single-stranded RNA viruses, which belong to the picornavirus family, and occur as three distinct serotypes. All three serotypes of poliovirus can infect primates, but only type 2 can infect mice. The crystal structures of a type 1 and a type 3 poliovirus are already known. Structural studies of poliovirus type 2 Lansing (PV2L) were initiated to try to enhance our understanding of the differences in host range specificity, antigenicity and receptor binding among the three serotypes of poliovirus. RESULTS: The crystal structure of the mouse neurovirulent PV2L complexed with a potent antiviral agent, SCH48973, was determined at 2.9 A resolution. Structural differences among the three poliovirus serotypes occur primarily in the loop regions of the viral coat proteins (VPs), most notably in the loops of VP1 that cluster near the fivefold axes of the capsid, where the BC loop of PV2L is disordered. Unlike other known structures of enteroviruses, the entire polypeptide chain of PV2L VP4 is visible in the electron density and RNA bases are observed stacking with conserved aromatic residues (Tyr4020 and Phe4046) of VP4. The broad-spectrum antiviral agent SCH48973 is observed binding in a pocket within the beta-barrel of VP1, in approximately the same location that natural 'pocket factors' bind to polioviruses. SCH48973 forms predominantly hydrophobic interactions with the pocket residues. CONCLUSIONS: Some of the conformational changes required for infectivity and involved in the control of capsid stability and neurovirulence in mice may occur in the vicinity of the fivefold axis of the poliovirus, where there are significant structural differences among the three poliovirus serotypes in the surface exposed loops of VP1 (BC, DE, and HI). A surface depression is located at the fivefold axis of PV2L that is not present in the other two poliovirus serotypes. The observed interaction of RNA with VP4 supports the observation that loss of VP4 ultimately leads to the loss of viral RNA. A model is proposed that suggests dual involvement of the virion fivefold and pseudo-threefold axes in receptor-mediated initiation of infection by picornaviruses.

Adaptation, Physiological↗

HCV serotypes in Brazilian patients.

OBJECTIVE: To investigate the prevalence of the different types of hepatitis C virus (HCV) in a population of chronic HCV carriers using the Murex HCV serotyping 1-6 assay. METHODS: All serum samples from these patients had a positive nested PCR HCV reaction. The sera were submitted to ELISA, modified, for the identification of antibodies against HCV serotypes 1, 2, 3, 4, 5 and 6 (Murex HCV serotyping 1-6 assay). RESULTS: The viral serotype was identified in 166 (75.8%) of the 219 patients, 108 (65.11%) males and 58 (34.9%) females. Patient age ranged from 12 to 73 years, with a mean of 41.1 years. The form of acquisition of the disease most frequently reported was blood transfusion. The results showed a predominance of type 1 (70.0%), followed by type 3 (22.3%) and type 2 (4.2%). CONCLUSION: Samples presenting low and very close optical density readings may lead to discrepant diagnoses concerning HCV serotypes and should be confirmed by genotyping. The serotyping can be useful in clinical practice and can be of help in establishing the prognosis of the disease, also favoring epidemiologic studies independently of the technology required for genotyping tests.

Adolescent↗

Salmonella serotypes isolated from man in Malaysia over the 10-year period 1973-1982.

The results of serotyping of 10 953 salmonella isolates from humans over a 10-year period, 1973-82 at the Bacteriology Division, Institute for Medical Research, Malaysia are presented. A total of 104 serotypes from 22 'O' groups were encountered; 95 isolates were considered untypable. The three most predominant serotypes, namely Salmonella typhi, S. typhimurium and S. weltevreden together accounted for 54.1% of all isolates whilst the 25 most frequent serotypes accounted for 93.6% of the total. Whilst the commoner serotypes occurred regularly throughout the study period, the rarer ones tended to appear only in one year, when they might be associated with an outbreak, and never again. The pattern of serotypes, though quite similar to the one seen in neighbouring Singapore, is different from those experienced in other places such as Hong Kong, Jakarta, Bangladesh and Manchester.

Humans↗

Detection of human rotavirus serotype G6 in Hungary.

During an ongoing survey of human rotavirus serotypes, we demonstrated for the first time the circulation of serotype G6 in two regions of Hungary. Of five rotavirus seasons surveyed to date (1994-9), serotype G6 was found in all seasons except 1994-5 at an overall prevalence of 1.4% (17 of 1252) and ranging from 0.6 to 4.5%. Children infected with G6 strains were older (mean age, 3.3 years) than children infected with the four (G1-G4) globally common serotypes (mean age, 2.1 years; unpaired Student's t test, P<0.001). Our data indicate that rotavirus serotype G6 may be an epidemiologically important G serotype in Hungary.

Adolescent↗

Invasive group B streptococcal disease in infants: a 19-year nationwide study. Serotype distribution, incidence and recurrent infection.

During the period 1984-2002, 472 cases of invasive group B streptococcal (GBS) disease in infants aged 0-90 days in Denmark were registered. The overall incidence was 0.4/1000 live births. Most infants (73%) had early-onset GBS infection with 53% registered within the first day. Serotype III predominated (59%) with other serotypes as follows: Ia (16%), Ib (8%), NT (7%), II (6%), other serotypes (5%). Recurrence of GBS infection was registered in six infants, and the interval with no antibiotic therapy varied from 2 to 39 days. The serotypes of the isolates obtained from first and second episodes were identical (serotype III in five, and serotype Ia in one infant). Paired isolates were indistinguishable by PFGE and antibiotic susceptibility testing. Invasive GBS infections in infants are still a problem in Denmark, and recurrent infections are registered in 1% of these infants.

Denmark↗

The serotype distribution of Campylobacter jejuni and C. coli isolated from patients with diarrhoea and controls at Tikur Anbassa Hospital, Addis Ababa, Ethiopia.

Sixty-eight isolates of Campylobacter jejuni and C. coli isolated from patients with diarrhoea (n = 630) and controls (n = 220) at Tikur Anbassa Hospital, Addis Ababa, Ethiopia were serotyped on the basis of the heat-labile (HL) and the heat-stable (HS) antigens, by using 16 and 34 antisera, respectively, for the two methods. With the antisera against heat labile antigens, 89.3% of the C. jejuni and 75% of the C. coli were typable. The HL serotypes 1, 2, 3, 4, 5, 6 and 7 were the most common among the C. jejuni while HL serotypes 1 and 2 were dominant among the C. coli isolates. These serotypes accounted for 63.2% of all isolates. For the heat-stable antigens, 60% of the C. jejuni and 83.3% of the C. coli isolates were typable. The HS serotypes 1, 3, 8, 26 and 34 were most common among the C. jejuni, while serotypes 3 and 8 were dominant among C. coli isolates. This study shows that the most common HL and HS antigens among campylobacter isolates from Ethiopia correspond to the most frequent antigenic types from other parts of the world. A limited number of antisera were sufficient to identify the majority of the isolates.

Adolescent↗

Molecular variation of meningococcal serotype 4 antigen genes.

Changes in the frequency of serogroup B non serotypable (B:NT) meningococci isolated in England and Wales were investigated by T-track fingerprint analysis, DNA nucleotide sequence determination, and serotyping by whole cell ELISA and dot blot assay. Seventy-three per cent of the isolates designated as B:NT by the Meningococcal Reference Unit (MRU) dot blot assay during 1993-4, expressed variants of the serotyping antigen, PorB, that were serotype 4 by whole cell ELISA. T-track fingerprint patterns of these and other 'serotype 4' isolates revealed five distinct porB alleles which were shown by nucleotide sequence determination to encode different peptide sequences. Differential binding of the 'serotype 4' mAbs MN14G21 and 5DC4C8G8 in whole cell ELISA and dot blot assays was the result, (i) of differences in the peptide sequence of predicted surface loop I and (ii) an amino acid deletion in predicted loop VI of the PorB protein.

Amino Acid Sequence↗

Prevalence of Salmonella serotypes in environmental waters and their relationships with indicator organisms.

The incidence of serotypes of Salmonella in three types of environmental water (sea, river and fresh reservoirs) from north-east Spain was investigated. The study was performed at specific sampling locations during the summer for a period of five years (1992-1996). A total of 823 strains were isolated and 55 different serotypes were identified, 42 were recovered from sea water, 32 from river water and 12 from freshwater reservoirs. The most frequently isolated serotypes coincided with those involved in clinical cases in the area studied. Salmonella enteritidis was the most common (111 isolates), it was found in all types of water, although most predominantly in sea water (16.1% of the isolates). This serotype, together with S. hadar, significantly increased in frequency during the five year study period. The most frequent serotypes in river water and freshwater reservoirs were S. virchow (9.5%) and S. mikawasima (23.8%) respectively. Significant differences were assessed in the indicator organism densities between the samples with serotypes of clinical significance (S. enteritidis, S. infantis, S. typhimurium, S. virchow and S. paratyphi B) and those without clinical significance. Therefore their presence in all environmental waters may be of epidemiological significance.

Fresh Water↗

Serotyping of Cryptococcus neoformans strains isolated from clinical specimens in Thailand and their susceptibility to various antifungal agents.

One hundred and thirty-nine strains of Cryptococcus neoformans were isolated in Thailand from clinical specimens including 97 AIDS patients: 67 from Northern, 48 from Central, 17 from Northeastern and 7 from Southern regional hospitals. Six out of the 139 strains were serotype B and the remaining 133 were A. There was no correlation between serotypes and regional distribution. To our knowledge, this is the first report of serotyping studies on C. neoformans in Thailand. Studies on random amplified polymorphic DNA (RAPD) analysis showed that this method is useful for the differentiation of C. neoformans var. gattii (serotypes B and C) and C. neoformans var. neoformans (serotypes A and D). They also indicated that Thai isolates of C. neoformans var. gattii (serotype B) were a homogeneous group on the basis of their genotypes. Antifungal susceptibility tests using 5 antifungal agents including amphotericin B, fluconazole, flucytosine, itraconazole and micronazole against 50 selected strains of C. neoformans showed that they were sensitive to all of the antifungal agents tested except for one strain that was resistant to flucytosine.

AIDS-Related Opportunistic Infections↗

Heat-stable antigen serotyping of Campylobacter jejuni strains isolated from hospitalized children in Athens, Greece.

A hundred and twentynine Campylobacter jejuni strains isolated from hospitalized children with gastroenteritis were serotyped by the heat-stable antigen scheme (HS, Penner's method). Isolates belonged to two different periods. Group A contained strains isolated in 1987-1988 and group B contained strains which were isolated in 1998-2000. A variety of serotypes was found. Serotype HS:2 was predominant, followed by the HS:4 complex and HS:1,44. Many clinically important Guillain-Barré Syndrome associated serotypes--like HS:19--were identified. There were no significant differences in the distribution of serotypes between the two periods. The present report provides reference data, as this is the first C. jejuni serotyping study ever made in Greece.

Adolescent↗