Development of Lentiviral Vectors Pseudotyped With Influenza B Hemagglutinins: Application in Vaccine Immunogenicity, mAb Potency, and Sero-Surveillance Studies.

Influenza B viruses (IBV) cause respiratory disease epidemics in humans and are therefore components of seasonal influenza vaccines. Serological methods are employed to evaluate vaccine immunogenicity prior to licensure. However, classical methods to assess influenza vaccine immunogenicity such as the hemagglutination inhibition assay (HI) and the serial radial hemolysis assay (SRH), have been proven to have many limitations. As such, there is a need to develop innovative methods that can improve on these traditional assays and provide advantages such as ease of production and access, safety, reproducibility, and specificity. It has been previously demonstrated that the use of replication-defective viruses, such as lentiviral vectors pseudotyped with influenza A hemagglutinins in microneutralization assays (pMN) is a safe and sensitive alternative to study antibody responses elicited by natural influenza infection or vaccination. Consequently, we have produced Influenza B hemagglutinin-pseudotypes (IBV PV) using plasmid-directed transfection. To activate influenza B hemagglutinin, we have explored the use of proteases in increasing PV titers via their co-transfection during pseudotype virus production. When tested for their ability to transduce target cells, the influenza B pseudotypes produced exhibit tropism for different cell lines. The pseudotypes were evaluated as alternatives to live virus in microneutralization assays using reference sera standards, mouse and human sera collected during vaccine immunogenicity studies, surveillance sera from seals, and monoclonal antibodies (mAbs) against IBV. The influenza B pseudotype pMN was found to effectively detect neutralizing and cross-reactive responses in all assays and shows promise as an effective and versatile tool in influenza research

Highly Pathogenic Avian Influenza Virus Infection of Mallards with Homo- and Heterosubtypic Immunity Induced by Low Pathogenic Avian Influenza Viruses

The potential role of wild birds as carriers of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is still a matter of debate. Consecutive or simultaneous infections with different subtypes of influenza viruses of low pathogenicity (LPAIV) are very common in wild duck populations. To better understand the epidemiology and pathogenesis of HPAIV H5N1 infections in natural ecosystems, we investigated the influence of prior infection of mallards with homo- (H5N2) and heterosubtypic (H4N6) LPAIV on exposure to HPAIV H5N1. In mallards with homosubtypic immunity induced by LPAIV infection, clinical disease was absent and shedding of HPAIV from respiratory and intestinal tracts was grossly reduced compared to the heterosubtypic and control groups (mean GEC/100 µl at 3 dpi: 3.0×102 vs. 2.3×104 vs. 8.7×104; p<0.05). Heterosubtypic immunity induced by an H4N6 infection mediated a similar but less pronounced effect. We conclude that the epidemiology of HPAIV H5N1 in mallards and probably other aquatic wild bird species is massively influenced by interfering immunity induced by prior homo- and heterosubtypic LPAIV infections

Root inoculation of strawberry with the entomopathogenic fungi Metarhizium robertsii and Beauveria bassiana reduces incidence of the twospotted spider mite and selected insect pests and plant diseases in the field

The effect of inoculation of strawberry roots by two entomopathogenic fungal isolates, Metarhizium robertsii (ESALQ 1622) and Beauveria bassiana (ESALQ 3375), on naturally occurring arthropod pests and plant diseases was investigated in four commercial strawberry fields during two growing seasons in Brazil. Three locations represented open-field production while strawberries were grown in low tunnels at the fourth location. Population responses of predatory mites to the fungal treatments were also assessed. Plants inoculated by the fungal isolates resulted in significantly fewer Tetranychus urticae adults compared to control plants at all four locations. The mean cumulative numbers ± SE of T. urticae per leaflet were: M. robertsii (225.6 ± 59.32), B. bassiana (206.5 ± 51.48) and control (534.1 ± 115.55) at the three open-field locations, while at the location with tunnels numbers were: M. robertsii (79.7 ± 10.02), B. bassiana (107.7 ± 26.85) and control (207.4 ± 49.90). Plants treated with B. bassiana had 50% fewer leaves damaged by Coleoptera, while there were no effects on numbers of whiteflies and thrips. Further, lower proportions of leaflets with symptoms of the foliar plant pathogenic fungi Mycosphaerella fragariae and Pestalotia longisetula were observed in the M. robertsii (4.6% and 1.3%)- and B. bassiana (6.1% and 1.3%)-treated plots compared to control plots (9.8% and 3.7%). No effect was seen on numbers of naturally occurring predatory mites. Our results suggest that both isolates tested may be used as root inoculants of strawberries to protect against foliar pests, particularly spider mites, and also against foliar plant pathogenic fungi without harming naturally occurring and beneficial predatory mites.acceptedVersio