The induction of virus-neutralizing antibodies in influenza A-infected mice treated with Oseltamivir phosphate: effect of dosage and scheduling.

Oseltamivir phosphate (OS) is currently the most frequently used influenza antiviral drug. It moderates the course of influenza virus type A (IAV) infection, however, its impact on the induction of virus-neutralizing antibodies (VNAbs) is not understood in details. Here, we examined the influence of low (10 mg/kg) or high (60 mg/kg) doses of OS on the viral titer in lungs of BALB/c mice infected with 0.5 LD50 of IAV and on the level of VNAbs. Prophylactic application of OS (6 h before the infection) delayed the increase of viral titer in lungs with a lower peak in comparison to non-treated control mice. After therapeutic OS application (44 h after the infection), maximum of virus titer did not significantly change. However, the induction of VNAbs strongly decreased, to 16.7%-18.1% of the control, after preventive application of high OS dose. A minimal decrease of VNAbs titers was observed in groups of mice treated with low dose of OS applied therapeutically. They lowered to 91.1% / 14 or to 94.1% / 21 days post infection (p.i.) of VNAbs titers of non-treated control mice. In all other groups, levels of VNAbs titers dropped to 26.5-53.7% of those of non-treated mice. It should be noted that VNAbs titers were in direct proportion to maximal virus titers in mouse lungs of corresponding groups. In summary, after OS application the clinical symptoms of the disease were milder or non-observable in all OS-treated groups, but the lowering of VNAbs titers was dependent on the OS dose and interval between drug app-lication and the start of infection. Keywords: influenza A virus; Oseltamivir; prophylactic treatment; therapeutic treatment; virus-neutralizing antibodies.

Quantification of bacteria by in vivo bioluminescence imaging in comparison with standard spread plate method and reverse transcription quantitative PCR (RT-qPCR).

In vivo bioluminescence imaging (BLI) offers a unique opportunity to analyze ongoing bacterial infections qualitatively and quantitatively in intact animals over time, leading to a reduction in the number of animals needed for a study. Since accurate determination of the bacterial burden plays an essential role in microbiological research, the present study aimed to evaluate the ability to quantify bacteria by non-invasive BLI technique in comparison to standard spread plate method and reverse transcription quantitative PCR (RT-qPCR). For this purpose, BALB/c mice were intranasally infected with 1 × 105 CFU of bioluminescent Streptococcus pneumoniae A66.1. At day 1 post-infection, the presence of S. pneumoniae in lungs was demonstrated by spread plate method and RT-qPCR, but not by in vivo BLI. However, on the second day p.i., the bioluminescent signal was already detectable, and the photon flux values positively correlated with CFU counts and RT-qPCR data within days 2-6. Though in vivo BLI is valuable research tool allowing the continuous monitoring and quantification of pneumococcal infection in living mice, it should be kept in mind that early in the infection, depending on the infective dose, the bioluminescent signal may be below the detection limit.

DNA vaccine targeting the ectodomain of influenza M2 protein to endolysosome pathway enhances anti-M2e protective antibody response in mice.

A promising candidate for developing the universal influenza vaccine is the ectodomain of the M2 protein (M2e). We designed and prepared an experimental DNA vaccine with an improved potential to induce anti-M2e immune response. The sequence for truncated NS1 protein followed by 4xM2e was inserted into the expression vector pTriEx-4 (pEx). M2e repeats were fused to the transmembrane domain and cytoplasmic tail of lysosome-associated membrane glycoprotein  2 isoform A (LAMP-2a) to target the M2e to the endo-lysosome pathway, facilitating increased antigen presentation by MHC II. Using confocal microscope immunofluorescence analysis, we confirmed a strong colocalization of pEx 4M2e-LAMP-2a with early endosomes and a weaker colocalization with late endosomes. BALB/c mice immunized with three doses of pEx 4M2e-LAMP-2a DNA vaccine and challenged with 2LD50 mouse-adapted influenza virus developed significantly (up to 16 times) higher anti-M2e antibody response in comparison to mice immunized with pEx 4M2e vaccine using the same immunization protocol. This was in correlation with the increased survival rate (near to 67% vs 50%) observed in animals immunized with pEx 4M2e-LAMP-2a DNA in comparison to mice immunized with pEx 4M2e. Keywords: influenza A; matrix protein 2 ectodomain; NS1; LAMP-2a; DNA vaccine.

The Contribution of Viral Proteins to the Synergy of Influenza and Bacterial Co-Infection.

A severe course of acute respiratory disease caused by influenza A virus (IAV) infection is often linked with subsequent bacterial superinfection, which is difficult to cure. Thus, synergistic influenza-bacterial co-infection represents a serious medical problem. The pathogenic changes in the infected host are accelerated as a consequence of IAV infection, reflecting its impact on the host immune response. IAV infection triggers a complex process linked with the blocking of innate and adaptive immune mechanisms required for effective antiviral defense. Such disbalance of the immune system allows for easier initiation of bacterial superinfection. Therefore, many new studies have emerged that aim to explain why viral-bacterial co-infection can lead to severe respiratory disease with possible fatal outcomes. In this review, we discuss the key role of several IAV proteins-namely, PB1-F2, hemagglutinin (HA), neuraminidase (NA), and NS1-known to play a role in modulating the immune defense of the host, which consequently escalates the development of secondary bacterial infection, most often caused by Streptococcus pneumoniae. Understanding the mechanisms leading to pathological disorders caused by bacterial superinfection after the previous viral infection is important for the development of more effective means of prevention; for example, by vaccination or through therapy using antiviral drugs targeted at critical viral proteins.

Wastewater-Based Epidemiology as an Early Warning System for the Spreading of SARS-CoV-2 and Its Mutations in the Population.

New methodologies based on the principle of "sewage epidemiology" have been successfully applied before in the detection of illegal drugs. The study describes the idea of early detection of a virus, e.g., SARS-CoV-2, in wastewater in order to focus on the area of virus occurrence and supplement the results obtained from clinical examination. By monitoring temporal variation in viral loads in wastewater in combination with other analysis, a virus outbreak can be detected and its spread can be suppressed early. The use of biosensors for virus detection also seems to be an interesting application. Biosensors are highly sensitive, selective, and portable and offer a way for fast analysis. This manuscript provides an overview of the current situation in the area of wastewater analysis, including genetic sequencing regarding viral detection and the technological solution of an early warning system for wastewater monitoring based on biosensors.

Comparison of infectious influenza A virus quantification methods employing immuno-staining.

Infections caused by highly variable influenza A viruses (IAVs) pose perpetual threat to humans as well as to animals. Their surveillance requires reliable methods for their qualitative and quantitative analysis. The most frequently utilized quantification method is the titration by plaque assay or 50% tissue culture infectious dose estimation by TCID50. However, both methods are time-consuming. Moreover, some IAV strains form hardly visible plaques, and the evaluation of TCID50 is subjective. Employment of immuno-staining into the classic protocols for plaque assay or TCID50 assay enables to avoid these problems and moreover, shorten the time needed for reliable infectious virus quantification. Results obtained by these two alternatives of classic virus titration methods were compared to the newer rapid culture assay (RCA), where titration endpoint of infectious virus was estimated microscopically based on the immuno-staining of infected cells. In our analysis of compared methods, five different IAV strains of H1, H3 and H5 subtypes were used and results were statistically evaluated. We conclude that the RCA proved to be at least as reliable in assessment of infectious viral titer as plaque assay and TCID50, considering the employed immuno-staining.