Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

Hantavirus Brno loanvirus is highly specific to the common noctule bat (Nyctalus noctula) and widespread in Central Europe.

Bat-associated hantaviruses have been detected in Asia, Africa and Europe. Recently, a novel hantavirus (Brno loanvirus, BRNV) was identified in common noctule bats (Nyctalus noctula) in the Czech Republic, but nothing is known about its geographical range and prevalence. The objective of this study was to evaluate the distribution and host specificity of BRNV by testing bats from neighbouring countries Germany, Austria and Poland. One thousand forty-seven bats representing 21 species from Germany, 464 bats representing 18 species from Austria and 77 bats representing 12 species from Poland were screened by L segment broad-spectrum nested reverse transcription-polymerase chain reaction (RT-PCR) or by BRNV-specific real-time RT-PCR. Three common noctules from Germany, one common noctule from Austria and three common noctules from Poland were positive in the hantavirus RNA screening. Conventional RT-PCR and primer walking resulted in the amplification of partial L segment and (almost) complete S and M segment coding sequences for samples from Germany and partial L segment sequences for samples from Poland. Phylogenetic analysis of these nucleotide sequences showed highest similarity to BRNV from Czech Republic. The exclusive detection of BRNV in common noctules from different countries suggests high host specificity. The RNA detection rate in common noctules ranged between 1 of 207 (0.5%; Austria), 3 of 245 (1.2%; Germany) and 3 of 20 (15%; Poland). In conclusion, this study demonstrates a broader distribution of BRNV in common noctules in Central Europe, but at low to moderate prevalence. Additional studies are needed to prove the zoonotic potential of this hantavirus and evaluate its transmission within bat populations.

Membrane-Targeting Perylenylethynylphenols Inactivate Medically Important Coronaviruses via the Singlet Oxygen Photogeneration Mechanism.

Perylenylethynyl derivatives have been recognized as broad-spectrum antivirals that target the lipid envelope of enveloped viruses. In this study, we present novel perylenylethynylphenols that exhibit nanomolar or submicromolar antiviral activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and feline infectious peritonitis virus (FIPV) in vitro. Perylenylethynylphenols incorporate into viral and cellular membranes and block the entry of the virus into the host cell. Furthermore, these compounds demonstrate an ability to generate singlet oxygen when exposed to visible light. The rate of singlet oxygen production is positively correlated with antiviral activity, confirming that the inhibition of fusion is primarily due to singlet-oxygen-induced damage to the viral envelope. The unique combination of a shape that affords affinity to the lipid bilayer and the capacity to generate singlet oxygen makes perylenylethynylphenols highly effective scaffolds against enveloped viruses. The anticoronaviral activity of perylenylethynylphenols is strictly light-dependent and disappears in the absence of daylight (under red light). Moreover, these compounds exhibit negligible cytotoxicity, highlighting their significant potential for further exploration of the precise antiviral mechanism and the broader scope and limitations of this compound class.

First detection of Bartonella spp. in bat bugs Cimex pipistrelli (Hemiptera: Cimicidae), Central Europe.

Bats are an important reservoir for many viral pathogens in humans. However, their role in the transmission of bacterial pathogens is neglected, as is that of their ectoparasites. This study focuses on the molecular detection of Bartonella spp. in bat bugs Cimex pipistrelli using partial sequences of gltA (citrate synthase), ssrA (transfer messenger RNA, tmRNA), and the 16S-23S rDNA internal transcribed spacer (ITS) region as targets. Bartonella DNA was detected in 2/112 (1.79% prevalence) samples from bat bugs. Due to the fact that bat bugs can sporadically bite humans, more extensive surveillance and vector competence studies are needed to ascertain zoonotic risk of bat-associated Bartonella spp.

Broad-Spectrum Antiviral Activity of 3'-Deoxy-3'-Fluoroadenosine against Emerging Flaviviruses.

Emerging flaviviruses are causative agents of severe and life-threatening diseases, against which no approved therapies are available. Among the nucleoside analogues, which represent a promising group of potentially therapeutic compounds, fluorine-substituted nucleosides are characterized by unique structural and functional properties. Despite having first been synthesized almost 5 decades ago, they still offer new therapeutic opportunities as inhibitors of essential viral or cellular enzymes active in nucleic acid replication/transcription or nucleoside/nucleotide metabolism. Here, we report evaluation of the antiflaviviral activity of 28 nucleoside analogues, each modified with a fluoro substituent at different positions of the ribose ring and/or heterocyclic nucleobase. Our antiviral screening revealed that 3'-deoxy-3'-fluoroadenosine exerted a low-micromolar antiviral effect against tick-borne encephalitis virus (TBEV), Zika virus, and West Nile virus (WNV) (EC50 values from 1.1 ± 0.1 µM to 4.7 ± 1.5 µM), which was manifested in host cell lines of neural and extraneural origin. The compound did not display any measurable cytotoxicity up to concentrations of 25 µM but had an observable cytostatic effect, resulting in suppression of cell proliferation at concentrations of >12.5 µM. Novel approaches based on quantitative phase imaging using holographic microscopy were developed for advanced characterization of antiviral and cytotoxic profiles of 3'-deoxy-3'-fluoroadenosine in vitro In addition to its antiviral activity in cell cultures, 3'-deoxy-3'-fluoroadenosine was active in vivo in mouse models of TBEV and WNV infection. Our results demonstrate that fluoro-modified nucleosides represent a group of bioactive molecules with excellent potential to serve as prospective broad-spectrum antivirals in antiviral research and drug development.

Excellent option for mass testing during the SARS-CoV-2 pandemic: painless self-collection and direct RT-qPCR.

The early identification of asymptomatic yet infectious cases is vital to curb the 2019 coronavirus (COVID-19) pandemic and to control the disease in the post-pandemic era. In this paper, we propose a fast, inexpensive and high-throughput approach using painless nasal-swab self-collection followed by direct RT-qPCR for the sensitive PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This approach was validated in a large prospective cohort study of 1038 subjects, analysed simultaneously using (1) nasopharyngeal swabs obtained with the assistance of healthcare personnel and analysed by classic two-step RT-qPCR on RNA isolates and (2) nasal swabs obtained by self-collection and analysed with direct RT-qPCR. Of these subjects, 28.6% tested positive for SARS-CoV-2 using nasopharyngeal swab sampling. Our direct RT-qPCR approach for self-collected nasal swabs performed well with results similar to those of the two-step RT-qPCR on RNA isolates, achieving 0.99 positive and 0.98 negative predictive values (cycle threshold [Ct] < 37). Our research also reports on grey-zone viraemia, including samples with near-cut-off Ct values (Ct ≥ 37). In all investigated subjects (n = 20) with grey-zone viraemia, the ultra-small viral load disappeared within hours or days with no symptoms. Overall, this study underscores the importance of painless nasal-swab self-collection and direct RT-qPCR for mass testing during the SARS-CoV-2 pandemic and in the post-pandemic era.

Bartonella species in medically important mosquitoes, Central Europe.

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.

Molecular Epidemiology of Hantaviruses in the Czech Republic.

During 2008-2018, we collected samples from rodents and patients throughout the Czech Republic and characterized hantavirus isolates. We detected Dobrava-Belgrade and Puumala orthohantaviruses in patients and Dobrava-Belgrade, Tula, and Seewis orthohantaviruses in rodents. Increased knowledge of eco-epidemiology of hantaviruses will improve awareness among physicians and better outcomes of patients.

Responses of phenology and biomass production of boreal fens to climate warming under different water-table level regimes.

Climate change affects peatlands directly through increased air temperatures and indirectly through changes in water-table level (WL). The interactions of these two still remain poorly known. We determined experimentally the separate and interactive effects of temperature and WL regime on factors of relevance for the inputs to the carbon cycle: plant community composition, phenology, biomass production, and shoot:root allocation in two wet boreal sedge-dominated fens, "southern" at 62°N and "northern" at 68°Ν. Warming (1.5°C higher average daily air temperature) was induced with open-top chambers and WL drawdown (WLD; 3-7 cm on average) by shallow ditches. Total biomass production varied from 250 to 520 g/m2 , with belowground production comprising 25%-63%. Warming was associated with minor effects on phenology and negligible effects on community composition, biomass production, and allocation. WLD clearly affected the contribution of different plant functional types (PFTs) in the community and the biomass they produced: shrubs benefited while forbs and mosses suffered. These responses did not depend on the warming treatment. Following WLD, aboveground biomass production decreased mainly due to reduced growth of mosses in the southern fen. Aboveground vascular plant biomass production remained unchanged but the contribution of different PFTs changed. The observed changes were also reflected in plant phenology, with different PFTs showing different responses. Belowground production increased following WLD in the northern fen only, but an increase in the contributions of shrubs and forbs was observed in both sites, while sedge contribution decreased. Moderate warming alone seems not able to drive significant changes in plant productivity or community composition in these wet ecosystems. However, if warming is accompanied by even modest WL drawdown, changes should be expected in the relative contribution of PFTs, which could lead to profound changes in the function of fens. Consequently, hydrological scenarios are of utmost importance when estimating their future function.

Puumala Virus in Bank Voles, Lithuania.

Little is known about the presence of human pathogenic Puumala virus (PUUV) in Lithuania. We detected this virus in bank voles (Myodes glareolus) in a region of this country in which previously PUUV-seropositive humans were identified. Our results are consistent with heterogeneous distributions of PUUV in other countries in Europe.

Overwintering of Uranotaenia Unguiculata Adult Females in Central Europe: A Possible Way of Persistence of the Putative New Lineage of West Nile Virus?

We report the overwintering of Uranotaenia unguiculata adult females in Central Europe (Czech Republic, Hungary, Austria). This finding suggests a potential mode of winter persistence of putative novel lineage of West Nile virus in the temperate regions of Europe.

Brno loanvirus (BRNV) in bats inhabiting the urban area of Brno, Czech Republic.

Bats are known reservoirs of various emerging pathogens, and have recently been found to host a novel hantavirus, named Brno loanvirus (BRNV), from the Mammantavirinae subfamily (family Hantaviridae, order Bunyavirales). Here we report BRNV detection in bats from the urban area of Brno, Czech Republic in March 2022. Specifically, we uncovered a high prevalence of BRNV (8.8%, 5/57) among hibernating bats (Nyctalus noctula) in urban area, which poses a risk of human exposure. The positive bats included adult females (3/9 positive), a juvenile female (1/32 positive), and an adult male (1/6 positive). All 10 juvenile males were negative. We used RT-qPCR to quantify the BRNV RNA levels in various bat organs, which yielded positive results for viral RNA in organs, including the kidneys, heart, spleen, brain, liver, lung, and gut, and in body cavity fluid. Among all tested organs, the liver showed the highest levels of viral RNA in 4 out of 5 animals examined (average Ct value of 20.8 ± 7.4).

Multicentric evaluation of sensitivity of eight commercial anti-SARS-CoV-2 antibody assays and their correlation to virus neutralization titers in seropositive subjects.

Diagnosis of SARS-CoV-2 virus is mainly based on direct detection. Determination of specific antibodies has been used mostly for epidemiological reasons. However, select immunoassays showed good correlation to plaque reduction virus neutralization test (PRNT) in smaller patient cohorts, which suggests their potential as predictors of virus neutralization titer. A total of 3,699 samples from Covid-19 patients were included in the multicentric study performed in the Czech Republic. Anti-SARS-CoV-2 antibody levels were evaluated by 8 commercial antibody assays. Simultaneously, PRNT evaluations were performed with the SARS-CoV-2 B.1.258 variant. All immunoassays showed an overall high true positive diagnostic value ranging from 79.17 to 98.04%. Several commercial EIA methods showed highly positive correlation between the assay results and PRNT levels, e.g., Liaison CoV-2 TrimericS IgG DiaSorin (Spearman r = 0.8833; Architect SASRS-CoV-2 IgG Abbott (r = 0.7298); NovaLisa SARS-CoV-2 IgG NovaTec (r = 0.7103) and Anti-SARS-CoV-2 ELISA IgG Euroimmun (r = 0.7094). While this correlation was less positive for other assays, those, conversely, presented higher true positive values. For most immunoassays, the positive percent agreement of the results was ≥ 95% in sera exhibiting PRNT levels of 1:80 and higher. The assays tested have shown variable correlation to PRNT. Those possessing high positive predictive values serve well as qualitative tests, while others can be utilised as quantitative tests highly predictive of neutralization antibody levels.

The structure of immature tick-borne encephalitis virus supports the collapse model of flavivirus maturation.

We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation.

Nanoformulation of the Broad-Spectrum Hydrophobic Antiviral Vacuolar ATPase Inhibitor Diphyllin in Human Recombinant H-ferritin.

Background: As highlighted by recent pandemic outbreaks, antiviral drugs are crucial resources in the global battle against viral diseases. Unfortunately, most antiviral drugs are characterized by a plethora of side effects and low efficiency/poor bioavailability owing to their insolubility. This also applies to the arylnaphthalide lignin family member, diphyllin (Diph). Diph acts as a vacuolar ATPase inhibitor and has been previously identified as a promising candidate with broad-spectrum antiviral activity. However, its physicochemical properties preclude its efficient administration in vivo, complicating preclinical testing. Methods: We produced human recombinant H- ferritin (HsaFtH) and used it as a delivery vehicle for Diph encapsulation through pH-mediated reversible reassembly of HsaFtH. Diph nanoformulation was subsequently thoroughly characterized and tested for its non-target cytotoxicity and antiviral efficiency using a panel of pathogenic viral strain. Results: We revealed that loading into HsaFtH decreased the undesired cytotoxicity of Diph in mammalian host cells. We also confirmed that encapsulated Diph exhibited slightly lower antiviral activity than free Diph, which may be due to the differential uptake mechanism and kinetics of free Diph and Diph@HsaFtH. Furthermore, we confirmed that the antiviral effect was mediated solely by Diph with no contribution from HsaFtH. Conclusion: It was confirmed that HsaFtH is a suitable vehicle that allows easy loading of Diph and production of highly homogeneous nanoparticles dispersion with promising broad-spectrum antiviral activity.

Efficacy and immunogenicity of a veterinary vaccine candidate against tick-borne encephalitis in dogs.

Tick-borne encephalitis (TBE) is a severe neuroinfection of humans. Dogs are also commonly infected with tick-borne encephalitis virus (TBEV). These infections are usually asymptomatic, but sometimes show clinical signs similar to those seen in humans and can be fatal. To date, there is no TBEV vaccine available for use in dogs. To address this need, a TBEV vaccine candidate for dogs based on inactivated whole virus antigen was developed. The safety, immunogenicity, and efficacy of the vaccine candidate were tested in mice as the preclinical model and in dogs as the target organism. The vaccine was well tolerated in both species and elicited the production of specific anti-TBEV antibodies with virus neutralising activity. Vaccination of mice provided complete protection against the development of fatal TBE. Immunisation of dogs prevented the development of viremia after challenge infection. Therefore, the developed vaccine candidate is promising to protect dogs from severe TBEV infections.

The Influence of Adjuvant Type on the Immunogenicity of RBD/N Cocktail Antigens as a Vaccine Candidate against SARS-CoV-2 Virus.

The emerging virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2 virus), agent of COVID-19, appeared in December 2019 in Wuhan, China, and became a serious threat to global health and public safety. Many COVID-19 vaccines have been approved and licensed around the world. Most of the developed vaccines include S protein and induce an antibody-based immune response. Additionally, T-cell response to the SARS-CoV-2 antigens could be beneficial for combating the infection. The type of immune response is greatly dependent not only on the antigen, but also on adjuvants used in vaccine formulation. Here, we compared the effect of four different adjuvants (AddaS03, Alhydrogel/MPLA, Alhydrogel/ODN2395, Quil A) on the immunogenicity of a mixture of recombinant RBD and N SARS-CoV-2 proteins. We have analyzed the antibody and T-cell response specific to RBD and N proteins and assessed the impact of adjuvants on virus neutralization. Our results clearly indicated that Alhydrogel/MPLA and Alhydrogel/ODN2395 adjuvants elicited the higher titers of specific and cross-reactive antibodies to S protein variants from various SARS-CoV-2 and SARS-CoV-1 strains. Moreover, Alhydrogel/ODN2395 stimulated high cellular response to both antigens, as assessed by IFN-γ production. Importantly, sera collected from mice immunized with RBD/N cocktail in combination with these adjuvants exhibited neutralizing activity against the authentic SARS-CoV-2 virus as well as particles pseudotyped with S protein from various virus variants. The results from our study demonstrate the immunogenic potential of RBD and N antigens and point out the importance of adjuvants selection in vaccine formulation in order to enhance the immunological response. IMPORTANCE Although several COVID-19 vaccines have been approved worldwide, continuous emergence of new SARS-CoV-2 variants calls for new efficient vaccines against them, providing long-lasting immunity. As the immune response after vaccination is dependent not only on antigen used, but also on other vaccine components, e.g., adjuvants, the purpose of this work was to study the effect of different adjuvants on the immunogenicity of RBD/N SARS-CoV-2 cocktail proteins. In this work, it has been shown that immunization with both antigens plus the different adjuvants studied elicited higher Th1 and Th2 responses against RBD and N, which contributed to higher neutralization of the virus. The obtained results can be used for design of new vaccines, not only against SARS-CoV-2, but also against other important viral pathogens.

Fine-root biomass production and its contribution to organic matter accumulation in sedge fens under changing climate.

Climate change may affect the carbon sink function of peatlands through warming and drying. Fine-root biomass production (FRBP) of sedge fens, a widespread peatland habitat, is important in this context, since most of the biomass is below ground in these ecosystems. We examined the response of fine-root biomass production, depth distribution (10 cm intervals down to 60 cm), chemical characteristics, and decomposition along with other main litter types (sedge leaves, Sphagnum moss shoots) to an average May-to-October warming of 1.7 °C above ambient daily mean temperature and drying of 2-8 cm below ambient soil water-table level (WL) in two sedge fens situated in Northern and Southern Boreal zones. Warming was induced with open top chambers and drying with shallow ditching. Finally, we simulated short-term organic matter (OM) accumulation using net primary production and mass loss data. Total FRBP, and FRBP in deeper layers, was clearly higher in southern than northern fen. Drying significantly increased, and warming marginally increased, total FRBP, while warming significantly increased, and drying marginally increased, the proportional share of FRBP in deeper layers. Drying, especially, modified root chemistry as the relative proportions of fats, wax, lipids, lignin and other aromatics increased while the proportion of polysaccharides decreased. Warming did not affect the decomposition of any litter types, while drying reduced the decomposition of sedge leaf litter. Although drying increased OM accumulation from root litter at both fens, total OM accumulation decreased at the southern fen, while the northern fen with overall lower values showed no such pattern. Our results suggest that in warmer and/or modestly drier conditions, sedge fen FRBP will increase and/or be allocated to deeper soil layers. These changes along with the altered litter inputs may sustain the soil carbon sink function through OM accumulation, unless the WL falls below a tipping point.

Antiviral activity of singlet oxygen-photogenerating perylene compounds against SARS-CoV-2: Interaction with the viral envelope and photodynamic virion inactivation.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted great interest in novel broad-spectrum antivirals, including perylene-related compounds. In the present study, we performed a structure-activity relationship analysis of a series of perylene derivatives, which comprised a large planar perylene residue, and structurally divergent polar groups connected to the perylene core by a rigid ethynyl or thiophene linker. Most of the tested compounds did not exhibit significant cytotoxicity towards multiple cell types susceptible to SARS-CoV-2 infection, and did not change the expressions of cellular stress-related genes under normal light conditions. These compounds showed nanomolar or sub-micromolar dose-dependent anti-SARS-CoV-2 activity, and also suppressed the in vitro replication of feline coronavirus (FCoV), also termed feline infectious peritonitis virus (FIPV). Perylene compounds exhibited high affinity for liposomal and cellular membranes, and efficiently intercalated into the envelopes of SARS-CoV-2 virions, thereby blocking the viral-cell fusion machinery. Furthermore, the studied compounds were demonstrated to be potent photosensitizers, generating reactive oxygen species (ROS), and their anti-SARS-CoV-2 activities were considerably enhanced after irradiation with blue light. Our results indicated that photosensitization is the major mechanism underlying the anti-SARS-CoV-2 activity of perylene derivatives, with these compounds completely losing their antiviral potency under red light. Overall, perylene-based compounds are broad-spectrum antivirals against multiple enveloped viruses, with antiviral action based on light-induced photochemical damage (ROS-mediated, likely singlet oxygen-mediated), causing impairment of viral membrane rheology.