Identification and relative abundance of naturally presented and cross-reactive influenza A virus MHC class I-restricted T cell epitopes.

Cytotoxic T lymphocytes are key for controlling viral infection. Unravelling CD8+ T cell-mediated immunity to distinct influenza virus strains and subtypes across prominent HLA types is relevant for combating seasonal infections and emerging new variants. Using an immunopeptidomics approach, naturally presented influenza A virus-derived ligands restricted to HLA-A*24:02, HLA-A*68:01, HLA-B*07:02, and HLA-B*51:01 molecules were identified. Functional characterization revealed multifunctional memory CD8+ T cell responses for nine out of sixteen peptides. Peptide presentation kinetics was optimal around 12 h post infection and presentation of immunodominant epitopes shortly after infection was not always persistent. Assessment of immunogenic epitopes revealed that they are highly conserved across the major zoonotic reservoirs and may contain a single substitution in the vicinity of the anchor residues. These findings demonstrate how the identified epitopes promote T cell pools, possibly cross-protective in individuals and can be potential targets for vaccination.

Influenza A virus replication has a stronger dependency on Raf/MEK/ERK signaling pathway activity than SARS-CoV-2.

The recent COVID-19 pandemic again highlighted the urgent need for broad-spectrum antivirals, both for therapeutic use in acute viral infection and for pandemic preparedness in general. The targeting of host cell factors hijacked by viruses during their replication cycle presents one possible strategy for development of broad-spectrum antivirals. By inhibiting the Raf/MEK/ERK signaling pathway, a central kinase cascade of eukaryotic cells, which is being exploited by numerous viruses of different virus phyla, the small-molecule MEK inhibitor zapnometinib has the potential to address this need. We here performed a side-by-side comparison of the antiviral efficacy of zapnometinib against IAV and SARS-CoV-2 to determine the concentration leading to 50% of its effect on the virus (EC50) and the concentration leading to 50% reduction of ERK phosphorylation (IC50) in a comparable manner, using the same experimental conditions. Our results show that the EC50 value and IC50 value of zapnometinib are indeed lower for IAV compared to SARS-CoV-2 using one representative strain for each. The results suggest that IAV's replication has a stronger dependency on an active Raf/MEK/ERK pathway and, thus, that IAV is more susceptible to treatment with zapnometinib than SARS-CoV-2. With zapnometinib's favorable outcome in a recent phase II clinical trial in hospitalized COVID-19 patients, the present results are even more promising for an upcoming phase II clinical trial in severe influenza virus infection.

Improved in vitro Efficacy of Baloxavir Marboxil Against Influenza A Virus Infection by Combination Treatment With the MEK Inhibitor ATR-002.

Currently, all available antiviral drugs against influenza virus (IV) that target the virus proteins directly, like Baloxavir acid (BXA), lead to viral resistance. Therefore, cellular mechanisms and factors essential for IV replication are promising antiviral targets. As IV strongly depends on the virus-induced Raf/MEK/ERK signal pathway for efficient generation of infectious progeny virions, this pathway represents an important target. We aimed to determine whether the MEK inhibitor ATR-002 (PD0184264) is able to impair replication of BXA-resistant influenza A virus (IAV) and whether a treatment combining BXA and ATR-002 improves the therapeutic efficiency in vitro. A549 cells infected with different IAV strains including BXA-resistant variants were treated with ATR-002 or BXA and the effect on virus titer reduction was determined. The synergistic effect of ATR-002 and BXA was also analyzed using different evaluation methods. The data demonstrated that ATR-002 has a significant and dose-dependent inhibitory effect on IAV replication across different strains and subtypes. IAV with the PA-I38T mutation shows resistance against BXA, but is still susceptible toward ATR-002. The combination of ATR-002 and BXA exhibited a synergistic potency reflected by low combination index values. In conclusion, we show that ATR-002 permits to counteract the limitations of BXA against BXA-resistant IAV. Moreover, the results support the use of ATR-002 (i) in a mono-therapy, as well as (ii) in a combined approach together with BXA. These findings might also apply to the treatment of infections with IAV, resistant against other direct-acting antiviral compounds.

Antiviral efficacy against influenza virus and pharmacokinetic analysis of a novel MEK-inhibitor, ATR-002, in cell culture and in the mouse model.

Antiviral therapies against influenza are required, especially for high-risk patients, severe influenza and in case of highly pathogenic influenza virus (IV) strains. However, currently, licensed drugs that target the virus directly are not very effective and often lead to the development of resistant IV variants. This may be overcome by targeting host cell factors that are required for IV propagation. IV induces a variety of host cell signaling cascades, such as the Raf/MEK/ERK kinase pathway. The activation of this pathway is necessary for IV propagation. MEK-inhibitors block the activation of the pathway on the bottleneck of the signaling cascade leading to impaired virus propagation. In the present study, we aimed to compare the antiviral potency and bioavailability of the MEK-inhibitor CI-1040 versus its major active metabolite ATR-002, in vitro as well as in the mouse model. In cell culture assays, an approximately 10-fold higher concentration of ATR-002 is required to generate the same antiviral activity as for CI-1040. Interestingly, we observed that considerably lower concentrations of ATR-002 were required to achieve a reduction of the viral load in vivo. Pharmacokinetic studies with ATR-002 and CI-1040 in mice have found the Cmax and AUC to be far higher for ATR-002 than for CI-1040. Our results thereby demonstrate the in vivo superiority of the active metabolite ATR-002 over CI-1040 as an antiviral agent despite its weaker cell membrane permeability. Therefore, ATR-002 is an attractive candidate for development as an efficient antiviral agent, especially given the fact that a treatment based on cellular pathway inhibition would be far less likely to lead to viral drug resistance.

Pepper mild mottle virus in wastewater in Egypt: a potential indicator of wastewater pollution and the efficiency of the treatment process.

There is increasing evidence that the fecal indicator bacteria that are routinely used for testing water quality are inadequate for ensuring protection of the public health. Pepper mild mottle virus (PMMoV) has recently been suggested as an alternative indicator of human fecal contamination in water; however, in Egypt there are no data available about its occurrence and concentration in aquatic environment. The concentration of PMMoV in the influent and effluent of three wastewater treatment plants was measured using qRT-PCR over a period of one year and compared to that of human adenovirus (HAdV), which is considered an indicator for human fecal contamination. PMMoV was detected in ~ 94% of the influent samples and 78% of the effluent samples, with concentrations ranging from 3.9 × 104 to 3.3 × 108 genome copies/l (GC/l) in the influent and 3.9 × 104 to 1.2 × 107 GC/l in the effluent. Similarly, HAdV was identified in 88% and 78% of the influent and effluent samples, respectively. The HAdV concentration ranged between 1.5 × 104 and 1.5 × 107 GC/l for the influent and 2.6 × 104 and 4.4 × 106 GC/l for the effluent. No significant difference was found between the removal ratio of PMMoV and HAdV. Viral reduction of 0.2-1.9 log10 and 0.2- 2.3 log10 by the treatment process was observed for PMMoV and HAdV, respectively. Both viruses showed no clear seasonality. Our data support the use of PMMoV as a fecal indicator of wastewater contamination and a process indicator for the performance of the treatment process.

Oncogenic papillomavirus and polyomavirus in urban sewage in Egypt.

Recently, the occurrence of oncogenic viruses in contaminated water and their potential for waterborne transmission has been reported. We addressed an environmental surveillance of both HPyVs (JCPyV and BKPyV) and HPVs in three wastewater treatment plants in Egypt. A high level of dissemination was found for both viruses. HPyVs (JCPyV and BKPyV) were found in ~73% of examined samples, while HPVs were detected in 30.5%. Sequence analysis of HPV positive samples revealed a wide variety of circulating genotypes representing both anogenital (HPV-6, HPV-16, HPV-53, HPV-44, HPV-31, HPV-43) and cutaneous (HPV-37, HPV-21, HPV-120, HPV-111, HPV-5) types. In addition, two unclassified sequences were identified, suggesting putative types. The median concentrations of HPyVs in inflow samples were 3.03×1005, 3.9×1005, and 1.44×1005GC/l in the three WWTPs, respectively. Whereas, the viral concentration in outflow reduced by one order of magnitude in WWTP-A and WWTP-C and two orders of magnitude in WWTP-B. On the other hand, the mean concentration of the quantified HPVs positive samples was 1.68×1003GC/l for inflow and a quite similar pattern in the outflow as well. These data provide an evidence about the actual circulation pattern of both viruses in the population. Also, the high abundance of HPyVs supports its potential as a possible fecal indicator. However, further investigations are required for both viruses to elucidate the potential health risk via contaminated water.

Detection and characterization of hepatitis A virus circulating in Egypt.

Hepatitis A virus (HAV) still poses a considerable problem worldwide. In the current study, hepatitis A virus was recovered from wastewater samples collected from three wastewater treatment plants over one year. Using RT-PCR, HAV was detected in 43 out of 68 samples (63.2%) representing both inlet and outlet. Eleven positive samples were subjected to sequencing targeting the VP1-2A junction region. Phylogenetic analysis revealed that all samples belonged to subgenotype IB with few substitutions at the amino acid level. The complete sequence of one isolate (HAV/Egy/BI-11/2015) showed that the similarity at the amino acid level was not reflected at the nucleotide level. However, the deduced amino acid sequence derived from the complete nucleotide sequence showed distinct substitutions in the 2B, 2C, and 3A regions. Recombination analysis revealed a recombination event between X75215 (subgenotype IA) and AF268396 (subgenotype IB) involving a portion of the 2B nonstructural protein coding region (nucleotides 3757-3868) assuming the herein characterized sequence an actual recombinant. Despite the role of recombination in picornaviruses evolution, its involvement in HAV evolution has rarely been reported, and this may be due to the limited available complete HAV sequences. To our knowledge, this represents the first characterized complete sequence of an Egyptian isolate and the described recombination event provides an important update on the circulating HAV strains in Egypt.

Relative Abundance of Human Bocaviruses in Urban Sewage in Greater Cairo, Egypt.

Human bocavirus (HBoV) is predominantly found in the respiratory tract infections and in the stool of patients with gastroenteritis symptoms. However, data on the prevalence of HBoV genotypes in environmental samples are limited. Here we addressed the prevalence of HBoV in sewage collected from three different wastewater treatment plants in Egypt. HBoV-1, HBoV-2, and HBoV-3 were detected, whereas none of the samples were positive for HBoV-4. The median concentration of HBoV in influent samples was 8.5 × 103 GC/l for HBoV-1, 3.0 × 104 GC/l for HBoV-2, and 2.5 × 104 GC/l for HBoV-3. The concentration was reduced but not completely removed in the effluent samples. The median concentration in the outlet samples was 2.9 × 103 GC/l for HBoV-1, 4.1 × 103 GC/l for HBoV-2, and 2.1 × 103 GC/l for HBoV-3. Moreover, no seasonality pattern of HBoVs was found. The high incidence of HBoV in sewage samples provided an evidence of its circulation in the local population. Although the role of HBoV in respiratory or gastro-intestinal infections still remains to be fully elucidated, the risk of infection via contaminated water should be taken into consideration.