Molecular prophage typing of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.

Optimized clade 2.3.2.1c H5N1 recombinant-vaccine strains against highly pathogenic avian influenza

A/Puerto Rico/8/34 (PR8)-derived recombinant viruses have been used for seasonal flu vaccines; however, they are insufficient for vaccines against some human-fatal H5N1 highly pathogenic avian influenza (HPAI) viruses (HPAIV) due to low productivity. Additionally, the polymerase basic 2 (PB2) protein, an important mammalian-pathogenicity determinant, of PR8 possesses several mammalian-pathogenic mutations. We previously reported two avian PB2 genes (01310 and 0028) related to efficient replication in embryonated chicken eggs (ECEs) and nonpathogenicity in BALB/c mice. In this study, we generated PR8-derived H5N1 recombinant viruses harboring hemagglutinin (attenuated) and neuraminidase genes of a clade 2.3.2.1c H5N1 HPAIV (K10-483), as well as the 01310 or 0028 PB2 genes, and investigated their replication and immunogenicity. Compared with a control virus harboring six internal PR8 genes (rK10-483), the recombinant viruses possessing the 01310 and 0028 PB2 genes showed significantly higher replication efficiency in ECEs and higher antibody titers in chickens. In contrast to rK10-483, none of the viruses replicated in BALB/c mice, and all showed low titers in Madin-Darby canine kidney cells. Additionally, the recombinant viruses did not induce a neutralization antibody but elicited decreased protective immune responses against K10-483 in mice. Thus, the highly replicative and mammalian nonpathogenic recombinant H5N1 strains might be promising vaccine candidates against HPAI in poultry.

Effect of the fourth nucleotide at the 3′ end of neuraminidase and matrix viral genomic RNA on the pathogenicity of influenza virus A/PR/8/34

Twelve nucleotides located at the 3′ end of viral genomic RNA (vRNA) are conserved among influenza A viruses (IAV) and have a promoter function. Hoffmann's 8-plasmid reverse genetics vector system introduced mutations at position 4, C nucleotide (C4) to U nucleotide (U4), of the 3′ ends of neuraminidase (NA) and matrix (M) vRNAs of wild-type A/PR/8/34 (PR8). This resulted in a constellation of C4 and U4 vRNAs coding for low (polymerases) and relatively high (all others) copy number proteins, respectively. U4 has been reported to increase promoter activity in comparison to C4, but the constellation effect on the replication efficiency and pathogenicity of reverse genetics PR8 (rgPR8) has not been fully elucidated. In the present study, we generated 3 recombinant viruses with C4 in the NA and/or M vRNAs and rgPR8 by using reverse genetics and compared their pathobiological traits. The mutant viruses showed lower replication efficiency than rgPR8 due to the low transcription levels of NA and/or M genes. Furthermore, C4 in the NA and/or M vRNAs induced lower PR8 virus pathogenicity in BALB/c mice. The results suggest that the constellation of C4 and U4 among vRNAs may be one of the multigenic determinants of IAV pathogenicity.

ABO gene analysis of cis-AB and B subgroup in blood donors / 대한수혈학회지

BACKGROUDS: Based on elucidation of genetic basis of ABO blood group, the genetic mutation of blood subgroup has been investigated. Especially, the discovery of base substitution such as C467T, G803C in cis-AB have made the efforts to determine cis-AB blood group by molecular method. This study was performed to investigate the ABO gene structure and usefulness of genotyping method in blood donors whose blood group are suspected as cis-AB and B subgroup. METHODS: Genotyping for ABO was performed in peripheral blood DNA samples from eight cis-AB donors and five B subgroup donors at red cross blood center. DNA sequencing was performed on Bint, B3 and two cis-AB samples. RESLUTS: All eight cis-AB donors showed that they have cis-AB allele and C467T substitution. Through DNA sequencing it was confirmed that cis-AB allele was derived from A allele mutation and two B subgroups showed no base sequence difference with B blood group. CONCLUSIONS: The genotyping method would be useful tool to determine blood group variants in blood donors. And more investigation is required for elucidation of genetic structure and gene expression of ABO blood subgroup.

Evaluation of V Form Microplate Method for Irregular Antibody Screening of Blood Donors / 대한수혈학회지

BACKGROUND: Clinically significant hemolytic transfusion reaction by warm-reacting irregular antibodies are tested up to antiglobulin phase. It is known that microplate method show similar or better than conventional tube method. Authors examined the irregular antibody screening of blood donors using V form microplate method at Korean Red Cross Blood Centers and evaluated its usefulness. METHOD: Total 200,246 sera from blood donors were performed antibody screening using the automated blood testing system IBG (IBG Co, UK) and V form microplate method. For the identification in antibody screening positive sera, two kinds of panel cell (Search cyte I,II, Data cyte, DADE Co, USA, and Central Red Cross Blood Center, Korea) were used. RESLUTS: Irregular antibodies were screened in 1,581 cases out of 200,246 samples (positive rate;0.79%). However, the actually identified antibodies were 1,086 (0.54%). The identified antibodies were mostly cold antibody anti-Lea (681 cases), anti-Leb (271 cases), anti-P1 (53 cases) and anti-S, anti-Lua, anti-Fya detected only one case, respectively. The incidence of warm antibody was very low. For antibody screening, the reactivity of V form microplate was much higher than IBG system. CONCLUSION: It would be useful to apply V form microplate method for large number of donor screening. An advantage of this method is its possible automation with substantial time and cost effectiveness. Consequently, the adaptation of this method for antibody screening is suitable for Korean Red Cross Blood Centers.

Evaluation of Treponema pallidum antibody test in blood donors / 대한수혈학회지

BACKGROUND: Current serologic tests for Syphilis(STS) in the blood donors are Veneral Disease Reseach Laboratory(VDRL), Rapid Plasma Reagin(RPR) test or Groupamatic Automated Syphilis Test(GAST) using modified VDRL antigen as screening method, and Treponema Pallidum Hemagglutination(TPHA) test as a confirmatory method in Korean Red Cross Blood Centers. This study was carried out to evaluate the usefulness of Enzyme Immunoassay(EIA) as STS in blood donors. METHODS: A total of 11,335 donors s serum samples were tested by RPR and GAST. We analyzed 138 samples including 6 samples of anti-treponema pallidum panel with TPHA and EIA to compare as a confirmatory test. RESULTS: The positive rate of RPR and GAST in 11,335 samples were 0.68%, 0.24%, respectively. Confirmed positive rates by TPHA was 0.26%, and by EIA was 0.27%. False negative results of GAST were 0.11% and 0.13%, respectively according to the results of TPHA and EIA. The agreement between TPHA and EIA was 98.5%(130/132). CONCLUSION: The EIA results were comparable with RPR, GAST and TPHA test. It is considered that EIA method for STS would be alternative one for TPHA as a conformative test because there was excellent agreement between TPHA and EIA method, and EIA method showed almost same results as that of TPHA test.

Relationship Between Anti-HCV with ALT Level and Follow up Study in Anti-HCV Positive Donors / 대한수혈학회지

A prevalence of anti-HCV and ALT value was analyzed in 89,995 healthy Korean blood donors. The positive rate of anti-HCV was 0.43% (386) and increased with age. And the positive rates of anti-HCV was statistically significant higher in group having elevated ALT level than in group having normal ALT level(P<0.005). The mixed-infection rates of the hepatitis B and C was 0.02%(25/89,995), and statistically the positivity of anti-HCV was higher in HBs Ag positive group than in HBs Ag negative group(P<0.01). On follow up study from 51 donors of the anti-HCV positivity in initial test, 15(29.4 %) cases were continuously positive by follow up test in 5-20 months. But these results were independent of transfusional history and intervals of follow up. The positive rates of anti-HCV during the follow up with reagents of Ortho- I and Ortho-II were 24% and 33% respectively. The positivity of anti-HCV was higher in group had continuously elevated serum ALT level than in group with normal serum ALT level.