A novel attenuated enterovirus A71 mutant with VP1-V238A,K244R exhibits reduced efficiency of cell entry/exit and augmented binding affinity to sulfated glycans

Enterovirus A71 (EV-A71) is one of the major etiological agents of hand, foot, and mouth disease, and its infection occasionally leads to fatal neurological complications in children. However, only inactivated whole virus vaccines against EV-A71 are commercially available in Mainland China. Furthermore, the mechanisms underlying the infectivity and pathogenesis of EV-A71 remain to be better understood. By adaptation of an EV-A71 B5 strain in monkey Vero cells in the presence of brilliant black BN (E151), an anti-EV-A71 agent, a double mutant with VP1-V238A,K244R emerged to be E151 enhanced infection. The growth of reverse genetics (RG) mutant RG/B5-VP1-V238A,K244R (RG/B5-AR) was promoted by E151 in Vero cells, but inhibited in other human and murine cells; while its parental wild type RG/B5-wt was strongly prevented by E151 from infection in all tested cells. In the absence of E151, RG/B5-AR exhibited defective cell entry/exit, resulting in reduced viral transmission and growth in vitro . It had augmented binding affinity to sulfated glycans, cells and tissue/organs, which probably functioned as decoys to restrict viral dissemination and infection. RG/B5-AR was also attenuated with 355 times higher 50% lethal dose and shorter timing of virus clearance than RG/B5-wt in suckling AG129 mice. It, however, remained highly immunogenic in adult AG129 mice to protect their suckling mice from lethal EV-A71 challenges through maternal neutralizing antibodies. Overall, discovery of the attenuated mutant RG/B5-AR contributes to better understanding of virulence determinants of EV-A71 and further development of novel vaccines against EV-A71. Importance Enterovirus A71 (EV-A71) is highly contagious in children and has been responsible for thousands of deaths in Asia-Pacific region since 1990s. Unfortunately, the virulence determinants and pathogenesis of EV-A71 are not fully clear. We discovered that a novel EV-A71 mutant VP1-V238A,K244R showed growth attenuation with reduced efficiency of cell entry/exit. In Vero cell line which has been approved for manufacturing EV-A71 vaccines, the growth defects of the mutant were compensated by a food dye brilliant black BN. The mutant was also with augmented binding affinity to sulfated glycans and other cellular components which probably restricted the viral infection and dissemination. Therefore, it was virulence attenuated in a mouse model but still retained its immunogenicity. Our findings suggest the mutant as a promising vaccine candidate against EV-A71 infection.

Knowledge Gaps in Taxonomy, Ecology, Population Distribution Drivers and Genetic Diversity of African Sandalwood (Osyris lanceolata Hochst. & Steud.): A Scoping Review for Conservation

The increasing demand for ornamental, cosmetic and pharmaceutical products is driving exploitation of plant species globally. Sub-Saharan Africa harbours unique and valuable plant resources and is now a target of plant resource depletion. African Sandalwood (Osyris lanceolata), a multi-purpose and drought-tolerant species, has seen increased exploitation for the last thirty years and is now declared endangered. Initiatives to conserve O. lanceolata are not yet successful in Africa due to poor understanding of the species. This review surveys relevant research on the ecology, taxonomy, population dynamics, genetic diversity and ethnobotany of O. lanceolata, and highlights gaps in the literature for further research. A scoping review of grey literature, scholarly papers and reports was applied with pre-determined criteria to screen relevant information. Review findings indicate O. lanceolata is a globally distributed species with no identified center of origin. In Africa, it ranges from Algeria to Ethiopia and south to South Africa; in Europe it occurs in the Iberian Peninsula and Balearic Islands; in Asia from India to China, and also on Socotra. The species has a confusing taxonomy, with unresolved issues in nomenclature, country range distribution, extensive synonymisation and variation in growth form (shrub or tree). The species population is reported to be declining in Africa, but information on population dynamics across its entire range of distribution is anecdotal. Additionally, ecological factors influencing spatial distribution and survival of the species remain unknown. A variety of uses are reported for O. lanceolata globally, including: cultural; medicinal and food; dye; perfumery; timber; ethnoveterinary and phytoremediation. Key research areas and implications for conservation of O. lanceolata in Sub-Saharan Africa are proposed.

Identification of Cat (Felis catus) Blood Splatter on Cotton Fabric After Periods of Drying Using Leucomalachite Green and Takayama Reagent

Blood-stain or blood splatter analysis when used properly can assist in establishing a chain of events linked to violent crimes (Bevel and Gardner, 2008). The methods used in detecting blood splatters in the field are chemical methods. Leucomalachite green is a colorimetric test which is used to test the presence of blood (Castro and Coyle, 2008). Takayama reagent is a confirmatory test for blood (Strassman, 1922). The aim of this research is to detect the blood splatter on cotton fabric after it has been dried for 1 day, 3 days and 5 days using Leucomalachite green and Takayama reagent. Cotton fabric was specifically chosen for this experiment with 3 different periods of drying. The unstained cotton fabric was cut into squares, and a blood sample was splattered on each piece. The fabrics splattered with blood were then dried for 1 day, 3 days and 5 days. The blood splatter was then tested using Leucomalachite green and Takayama reagent, and the results were noted afterwards. For the control, red food dye was dried for 1 day then tested with Leucomalachite green and Takayama reagent. The image results of the Leucomalachite green test are analyzed using ImageJ software 1.8.0_112 where the red, green and blue pixels are converted to grayscale. The image results of the Takayama test are graded based on the number and pattern of crystal. In conclusion, Leucomalachite green and Takayama reagent are able to detect cat blood splatter on the cotton fabric. Leucomalachite green produced a higher intensity/ darker colour as a result of an older sample, and the lower intensity/ lighter colour as a result of a fresher sample of the Leucomalachite green test. Takayama reagent produced a densely packed pattern of crystals as a result of an older sample, and the loosely packed pattern of crystals as a result of a fresher sample of the Takayama test.