Comparaison of rabies virus purification using different methods

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Infection causes tens of thousands of deaths every year, mainly in Asia and Africa. Most of these victims are children under the age of 15. Vaccination against rabies is still the sole way to fight against the disease. The aim of this work is to compare the purity of rabies vaccine purified by zonal centrifugation and chromatographic methods, in terms of residual DNA level, host cell protein (HCP) level and the overall recovery yield. For this purpose, Vero cells were grown under animal component free conditions, on Cytodex 1 microcarriers in VP-SFM medium. Vero cell growth and virus production were previously optimized; studies were conducted in a 7-L bioreactor. Virus replication phase was conducted using perfusion culture mode, viral harvests obtained through the culture were clarified, inactivated by BPL (Beta-propiolactone) and then pooled. The pooled harvests were purified by zonal centrifugation on a sucrose density gradient. The fractions of interest (11 in total) were pooled and checked for their antigenic activity according to the NIH potency test. They showed an activity of 61 UI/ml. The yield obtained was around 60%. To improve the overall yield, we have tested during a previous work several chromatography matrices (Sephacryl S200, Sephacryl S300, Sepharose 4FF,...). However the yield obtained was not high, around 40% in the best case. To improve this performance, we tested the Capto Core 700 (GE Healthcare Life Sciences) which a matrice that had a dual-functionality: size separation and binding chromatography, and was specially designed for the purification of large biological products such as viruses. The use of this matrice to purify rabies virus resulted in a yield of 84.5%, which was 2-fold and 1.5 fold higher than that obtained using chromatographic columns or zonal centrifugation, respectively. We also tested Monolytic chromatographic (CIMmultus™ QA-8 Advanced Composite Column) from BIA Separation. Such kind of media represents a new generation of chromatographic matrices with efficient mass transfer and better hydrodynamic properties. This allows fast and efficient separation of large molecules such as DNA and viruses. In our case, we were able to increase the purification yield to values close to 94%. It was the highest yield obtained compared to other methods used. Currently the content of the purified fractions collected using the different methods is analyzed to estimate the efficiency of DNA and HCP removal

Optimization of rAd5 vectored Newcastle vaccine production in HEK293 at high cell densities

Newcastle disease (ND) is a highly contagious and often severe global spread syndrome that affects birds including domestic poultry. It is caused by a virus belonging to the paramyxoviridae family. Indeed, an outbreak of ND can be quite severe, and is considered as a constant threat to the industry and food security worldwide. The disease can be controlled through the administration of effective vaccines. Immunizations with inactivated or live vaccines, although protective, have some eminent disadvantages. The aims of this work is the development of a vectored vaccine using a non-replicative human adenovirus vector, expressing the F antigen from Newcastle Disease Virus (rAd-F-ND) in bioreactor. The recombinant vaccine is produced using the HEK293 cell line. HEK293 cultures were carried out in suspension, first in shake flasks and then in stirred bioreactor at 37°C, 5% CO2 and 150 rpm in chemically defined media. The virus titers were determined by qPCR. To improve rAd-F-ND virus productions in HEK-293 cells, we studied the effects of the following parameters in shake flask cultures: culture media (Hycell Trans FX-H and Xell-GM), cell density, multiplicity of infection (MOI) and feed (Xell-FS , Xell-GM and cell Boost 5). Please click Download on the upper right corner to see the full abstract

Molecular characterization of a multidrug resistance IncF plasmid from the globally disseminated Escherichia coli ST131 clone.

Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections. Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131. In this study, we determined the complete sequence and performed a comprehensive annotation of pEC958, an IncF plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb in size, harbours two replicons (RepFIA and RepFII) and contains 12 antibiotic resistance genes (including the blaCTX-M-15 gene). We also carried out hyper-saturated transposon mutagenesis and multiplexed transposon directed insertion-site sequencing (TraDIS) to investigate the biology of pEC958. TraDIS data showed that while only the RepFII replicon was required for pEC958 replication, the RepFIA replicon contains genes essential for its partitioning. Thus, our data provides direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance. The gene encoding the antitoxin component (ccdA) of the post-segregational killing system CcdAB was also protected from mutagenesis, demonstrating this system is active. Sequence comparison with a global collection of ST131 strains suggest that IncF represents the most common type of plasmid in this clone, and underscores the need to understand its evolution and contribution to the spread of antibiotic resistance genes in E. coli ST131

Complete Genome Sequence of Serotype III Streptococcus agalactiae Sequence Type 17 Strain 874391.

Here we report the complete genome sequence of Streptococcus agalactiae strain 874391. This serotype III isolate is a member of the hypervirulent sequence type 17 (ST-17) lineage that causes a disproportionate number of cases of invasive disease in humans and mammals. A brief historical context of the strain is discussed

Global distribution and diversity of ovine-associated Staphylococcus aureus

Staphylococcus aureus is an important pathogen of many species, including sheep, and impacts on both human and animal health, animal welfare, and farm productivity. Here we present the widest global diversity study of ovine-associated S. aureus to date. We analysed 97 S. aureus isolates from sheep and sheep products from the UK, Turkey, France, Norway, Australia, Canada and the USA using multilocus sequence typing (MLST) and spa typing. These were compared with 196 sheep isolates from Europe (n = 153), Africa (n = 28), South America (n = 14) and Australia (n = 1); 172 bovine, 68 caprine and 433 human S. aureus profiles. Overall there were 59 STs and 87 spa types in the 293 ovine isolates; in the 97 new ovine isolates there were 22 STs and 37 spa types, including three novel MLST alleles, four novel STs and eight novel spa types. Three main CCs (CC133, CC522 and CC700) were detected in sheep and these contained 61% of all isolates. Four spa types (t002, t1534, t2678 and t3576) contained 31% of all isolates and were associated with CC5, CC522, CC133 and CC522 respectively. spa types were consistent with MLST CCs, only one spa type (t1403) was present in multiple CCs. The three main ovine CCs have different but overlapping patterns of geographical dissemination that appear to match the location and timing of sheep domestication and selection for meat and wool production. CC133, CC522 and CC700 remained ovine-associated following the inclusion of additional host species. Ovine isolates clustered separately from human and bovine isolates and those from sheep cheeses, but closely with caprine isolates. As with cattle isolates, patterns of clonal diversification of sheep isolates differ from humans, indicative of their relatively recent host-jump

Concordance of vaccination status and associated factors with incomplete vaccination: a household survey in the health district of Segou, Mali, 2019

Introduction: the region of Segou recorded 36.8% of children were incompletely vaccinated in 2018. In 2019, the district of Segou was one of the districts with the lowest vaccination coverage in the region, with 85.1% coverage for the three doses of the pentavalent vaccine and 85.4% for the measles vaccine. This study was initiated to better understand this low vaccination coverage, in the absence of specific studies on vaccination coverage in the district of Segou. Methods: a prospective cross-sectional study was conducted from May to August 2020 with 30 clusters. We performed Kappa coefficient, bivariate, and multiple logistic regression analysis. Results: findings showed that 18.46% (101/547) [15.44-21.93] of children were incompletely vaccinated. Mothers correctly reported the vaccination status of their children in 67.30% of cases (Kappa coefficient). Uneducated (OR[IC95%]=2.13[1.30-3.50]), living in rural area (OR[IC95%]=2.07[1.23-3.47]), lack of knowledge of Expanded Program on Immunization (EPI) target diseases (OR[IC95%]=2.37[1.52-3.68]), lack of knowledge of vaccination schedule (OR[IC95%]=3.33[1.90-5.81]) and lack of knowledge of the importance of vaccination (OR[IC95%]=3.6[2.35-6.32]) were associated with incomplete vaccination. In multivariate analysis, uneducated (ORa[IC95%>]=1.68[1.004-2.810]) and lack of knowledge of the importance of vaccination were associated with incomplete vaccination (ORa[IC95%]=3.40[2.049-5.649]). Conclusion: findings showed a good concordance of the vaccination status. Living in a rural area, no education, lack of the knowledge of EPI target diseases, lack of the knowledge of vaccination schedule and lack of knowledge of the importance of vaccination were associated with incomplete vaccination

Mutual exclusivity of hyaluronan and hyaluronidase in invasive group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen

Analysis of a Streptococcus pyogenes puerperal sepsis cluster using whole-genome sequencing

Between June and November 2010, a concerning rise in the number of cases of puerperal sepsis, a postpartum pelvic bacterial infection contracted by women after childbirth, was observed in the New South Wales, Australia, hospital system. Group A streptococcus (GAS; Streptococcus pyogenes) isolates PS001 to PS011 were recovered from nine patients. Pulsed-field gel electrophoresis and emm sequence typing revealed that GAS of emm1.40, emm75.0, emm77.0, emm89.0, and emm89.9 were each recovered from a single patient, ruling out a single source of infection. However, emm28.8 GAS were recovered from four different patients. To investigate the relatedness of these emm28 isolates, whole-genome sequencing was undertaken and the genome sequences were compared to the genome sequence of the emm28.4 reference strain, MGAS6180. A total of 186 single nucleotide polymorphisms were identified, for which the phylogenetic reconstruction indicated an outbreak of a polyclonal nature. While two isolates collected from different hospitals were not closely related, isolates from two puerperal sepsis patients from the same hospital were indistinguishable, suggesting patient-to-patient transmission or infection from a common source. The results of this study indicate that traditional typing protocols, such as pulsed-field gel electrophoresis, may not be sensitive enough to allow fine epidemiological discrimination of closely related bacterial isolates. Whole-genome sequencing presents a valid alternative that allows accurate fine-scale epidemiological investigation of bacterial infectious disease