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.

Serum and cerebrospinal fluid phosphorylated neurofilament heavy subunit as a marker of neuroaxonal damage in tick-borne encephalitis.

Extensive axonal and neuronal loss is the main cause of severe manifestations and poor outcomes in tick-borne encephalitis (TBE). Phosphorylated neurofilament heavy subunit (pNF-H) is an essential component of axons, and its detection in cerebrospinal fluid (CSF) or serum can indicate the degree of neuroaxonal damage. We examined the use of pNF-H as a biomarker of neuroaxonal injury in TBE. In 89 patients with acute TBE, we measured CSF levels of pNF-H and 3 other markers of brain injury (glial fibrillary acidic protein, S100B and ubiquitin C-terminal hydrolase L1) and compared the results to those for patients with meningitis of other aetiology and controls. Serum pNF-H levels were measured in 80 patients and compared with findings for 90 healthy blood donors. TBE patients had significantly (P<0.001) higher CSF pNF-H levels than controls as early as hospital admission. Serum pNF-H concentrations were significantly higher in samples from TBE patients collected at hospital discharge (P<0.0001) than in controls. TBE patients with the highest peak values of serum pNF-H, exceeding 10 000 pg ml-1, had a very severe disease course, with coma or tetraplegia. Patients requiring intensive care had significantly higher serum pNF-H levels than other TBE patients (P<0.01). Elevated serum pNF-H values were also observed in patients with incomplete recovery (P<0.05). Peak serum pNF-H levels correlated positively with the duration of hospitalization (P=0.005). Measurement of pNF-H levels in TBE patients might be useful for assessing disease severity and determining prognosis.

The Role of Peridomestic Animals in the Eco-Epidemiology of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an important tick-borne zoonotic agent of human granulocytic anaplasmosis (HGA). In Europe, the Ixodes ticks are the main vector responsible for A. phagocytophilum transmission. A wide range of wild animals is involved in the circulation of this pathogen in the environment. Changes in populations of vertebrates living in different ecosystems impact the ecology of ticks and the epidemiology of tick-borne diseases. In this study, we investigated four species, Western European hedgehog (Erinaceus europaeus), northern white-breasted hedgehog (Erinaceus roumanicus), Eurasian red squirrel (Sciurus vulgaris), and the common blackbird (Turdus merula), to describe their role in the circulation of A. phagocytophilum in urban and periurban ecosystems. Ten different tissues were collected from cadavers of the four species, and blood and ear/skin samples from live blackbirds and hedgehogs. Using qPCR, we detected a high rate of A. phagocytophilum: Western European hedgehogs (96.4%), northern white-breasted hedgehogs (92.9%), Eurasian red squirrels (60%), and common blackbirds (33.8%). In the groEL gene, we found nine genotypes belonging to three ecotypes; seven of the genotypes are associated with HGA symptoms. Our findings underline the role of peridomestic animals in the ecology of A. phagocytophilum and indicate that cadavers are an important source of material for monitoring zoonotic pathogens. Concerning the high prevalence rate, all investigated species play an important role in the circulation of A. phagocytophilum in municipal areas; however, hedgehogs present the greatest anaplasmosis risk for humans. Common blackbirds and squirrels carry different A. phagocytophilum variants some of which are responsible for HGA.

Hepatozoon in Eurasian red squirrels Sciurus vulgaris, its taxonomic identity, and phylogenetic placement.

Adeleorid apicomplexan parasites of the genus Hepatozoon Miller, 1908 are broadly distributed among the rodents. Broader molecular data on Hepatozoon from Palaearctic squirrels are necessary for evaluation of diversity and origin of Hepatozoon in Eurasian red squirrel Sciurus vulgaris populations, considering ongoing invasion by Gray squirrel S. carolinensis. Our report brings a set of molecular data from a population of S. vulgaris in the Czech Republic, non-invaded by any invasive squirrel species. Cadavers of 41 Eurasian red squirrels were examined using nested PCR targeting 18S rRNA gene; 30 animals tested positive for the presence of Hepatozoon spp. DNA in at least one tissue. Phylogenetic analysis of obtained sequence types revealed relatedness to sequences of Hepatozoon sp. from S. vulgaris from Spain and the Netherlands, forming a sister clade to Hepatozoon isolates from other European rodents. The fact that all available 18S rRNA gene sequences form a monophyletic clade is interpreted as a presence of a single Hepatozoon species in S. vulgaris in continental Europe, most probably Hepatozoon sciuri. The presented molecular data on the Hepatozoon from European squirrels provides a basis for future studies on possible exchange of Hepatozoon species between Eurasian red and gray squirrels.

Changes in cytokine and chemokine profiles in mouse serum and brain, and in human neural cells, upon tick-borne encephalitis virus infection.

BACKGROUND: Tick-borne encephalitis (TBE) is a severe neuropathological disorder caused by tick-borne encephalitis virus (TBEV). Brain TBEV infection is characterized by extensive pathological neuroinflammation. The mechanism by which TBEV causes CNS destruction remains unclear, but growing evidence suggests that it involves both direct neuronal damage by the virus infection and indirect damage caused by the immune response. Here, we aimed to examine the TBEV-infection-induced innate immune response in mice and in human neural cells. We also compared cytokine/chemokine communication between naïve and infected neuronal cells and astrocytes. METHODS: We used a multiplexed Luminex system to measure multiple cytokines/chemokines and growth factors in mouse serum samples and brain tissue, and in human neuroblastoma cells (SK-N-SH) and primary cortical astrocytes (HBCA), which were infected with the highly pathogenic TBEV strain Hypr. We also investigated changes in cytokine/chemokine production in naïve HBCA cells treated with virus-free supernatants from TBEV-infected SK-N-SH cells and in naïve SK-N-SH cells treated with virus-free supernatants from TBEV-infected HBCA cells. Additionally, a plaque assay was performed to assess how cytokine/chemokine treatment influenced viral growth following TBEV infection. RESULTS: TBEV-infected mice exhibited time-dependent increases in serum and brain tissue concentrations of multiple cytokines/chemokines (mainly CXCL10/IP-10, and also CXCL1, G-CSF, IL-6, and others). TBEV-infected SK-N-SH cells exhibited increased production of IL-8 and RANTES and downregulated MCP-1 and HGF. TBEV infection of HBCA cells activated production of a broad spectrum of pro-inflammatory cytokines, chemokines, and growth factors (mainly IL-6, IL-8, CXCL10, RANTES, and G-CSF) and downregulated the expression of VEGF. Treatment of SK-N-SH with supernatants from infected HBCA induced expression of a variety of chemokines and pro-inflammatory cytokines, reduced SK-N-SH mortality after TBEV infection, and decreased virus growth in these cells. Treatment of HBCA with supernatants from infected SK-N-SH had little effect on cytokine/chemokine/growth factor expression but reduced TBEV growth in these cells after infection. CONCLUSIONS: Our results indicated that both neurons and astrocytes are potential sources of pro-inflammatory cytokines in TBEV-infected brain tissue. Infected/activated astrocytes produce cytokines/chemokines that stimulate the innate neuronal immune response, limiting virus replication, and increasing survival of infected neurons.

Europe-Wide Meta-Analysis of Borrelia burgdorferi Sensu Lato Prevalence in Questing Ixodes ricinus Ticks.

Lyme borreliosis is the most common zoonotic disease transmitted by ticks in Europe and North America. Despite having multiple tick vectors, the causative agent, Borrelia burgdorferisensu lato, is vectored mainly by Ixodes ricinus in Europe. In the present study, we aimed to review and summarize the existing data published from 2010 to 2016 concerning the prevalence of B. burgdorferi sensu lato spirochetes in questing I. ricinus ticks. The primary focus was to evaluate the infection rate of these bacteria in ticks, accounting for tick stage, adult tick gender, region, and detection method, as well as to investigate any changes in prevalence over time. The data obtained were compared to the findings of a previous metastudy. The literature search identified data from 23 countries, with 115,028 ticks, in total, inspected for infection with B. burgdorferi sensu lato We showed that the infection rate was significantly higher in adults than in nymphs and in females than in males. We found significant differences between European regions, with the highest infection rates in Central Europe. The most common genospecies were B. afzelii and B. garinii, despite a negative correlation of their prevalence rates. No statistically significant differences were found among the prevalence rates determined by conventional PCR, nested PCR, and real-time PCR.IMPORTANCEBorrelia burgdorferisensu lato is a pathogenic bacterium whose clinical manifestations are associated with Lyme borreliosis. This vector-borne disease is a major public health concern in Europe and North America and may lead to severe arthritic, cardiovascular, and neurological complications if left untreated. Although pathogen prevalence is considered an important predictor of infection risk, solitary isolated data have only limited value. Here we provide summarized information about the prevalence of B. burgdorferi sensu lato spirochetes among host-seeking Ixodes ricinus ticks, the principal tick vector of borreliae in Europe. We compare the new results with previously published data in order to evaluate any changing trends in tick infection.

Prevalence of Borrelia miyamotoi in Ixodes ticks in Europe and the United States.

Borrelia miyamotoi, a relapsing fever-related spirochete transmitted by Ixodes ticks, has been recently shown to be a human pathogen. To characterize the prevalence of this organism in questing Ixodes ticks, we tested 2,754 ticks for a variety of tickborne pathogens by PCR and electrospray-ionization mass spectrometry. Ticks were collected from California, New York, Connecticut, Pennsylvania, and Indiana in the United States and from Germany and the Czech Republic in Europe from 2008 through 2012. In addition, an isolate from Japan was characterized. We found 3 distinct genotypes, 1 for North America, 1 for Europe, and 1 for Japan. We found B. miyamotoi infection in ticks in 16 of the 26 sites surveyed, with infection prevalence as high as 15.4%. These results show the widespread distribution of the pathogen, indicating an exposure risk to humans in areas where Ixodes ticks reside.

Detection of Borrelia burgdorferi sensu stricto ospC alleles associated with human lyme borreliosis worldwide in non-human-biting tick Ixodes affinis and rodent hosts in Southeastern United States.

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in European and North American regions where Lyme disease is endemic and where it is not endemic revealed a close relatedness of geographically distinct populations. ospC alleles A, B, and L were detected on both continents in vectors and hosts, including humans. Six ospC alleles, A, B, L, Q, R, and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3, and M, were identified in the far-western United States. Four ospC alleles, B, G, H, and L, were abundant in the southeastern United States. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern United States with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in European and North American regions where the disease is endemic were detected in B. burgdorferi strains isolated from the non-human-biting tick Ixodes affinis and rodent hosts in the southeastern United States. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern United States, a finding that confirms the hypothesis of transoceanic migration of Borrelia species.

Distant genetic variants of Anaplasma phagocytophilum from Ixodes ricinus attached to people.

BACKGROUND: Although the tick-borne pathogen Anaplasma phagocytophilum is currently described as a single species, studies using genetic markers can distinguish groups of variants associated with different hosts, pathogenicity, zoonotic potential and biotic and geographic niches. The objective of our study was to investigate the genetic diversity of A. phagocytophilum and Ixodes ricinus ticks attached to people. METHODS: In collaboration with a commercial diagnostic company, a total of 52 DNA samples were obtained from ticks that tested positive for A. phagocytophilum by quantitative PCR. The genetic profile of each sample was determined using the groEL and ankA genes. Identification of the tick species was confirmed by partial sequencing of the COI subunit and a portion of the TROSPA gene. RESULTS: All 52 ticks were identified as I. ricinus. Two protocols of nested PCR amplifying 1293- and 407-bp fragments of groEL of A. phagocytophilum yielded amplicons of the expected size for all 52 samples. Among all sequences, we identified 10 unique genetic variants of groEL belonging to ecotype I and ecotype II. The analysis targeting ankA was successful in 46 of 52 ticks. Among all sequences, we identified 21 unique genetic variants phylogenetically belonging to three clusters. CONCLUSIONS: Our results indicate that ticks attached to people harbor distant genetic variants of A. phagocytophilum, some of which are not recognized as zoonotic. Further studies are needed to determine the risk of human infection by genetic variants other than those designated as zoonotic.

Serum matrix metalloproteinase-9 (MMP-9) as a biomarker in paediatric and adult tick-borne encephalitis patients.

Matrix metalloproteinases (MMPs) play an important role in central nervous system infections. We analysed the levels of 8 different MMPs in the cerebrospinal fluid (CSF) of 89 adult patients infected with tick-borne encephalitis (TBE) virus and compared them with the levels in a control group. MMP-9 was the only MMP that showed significantly increased CSF levels in TBE patients. Serum MMP-9 levels were subsequently measured in 101 adult TBE patients at various time points during the neurological phase of TBE and at follow-up. In addition, serum MMP-9 was analysed in 37 paediatric TBE patients. Compared with control levels, both paediatric and adult TBE patients had significantly elevated serum MMP-9 levels. In most adult patients, serum MMP-9 levels peaked at hospital admission, with higher serum MMP-9 levels observed in patients with encephalitis than in patients with meningitis. Elevated serum MMP-9 levels were observed throughout hospitalisation but decreased to normal levels at follow-up. Serum MMP-9 levels correlated with clinical course, especially in patients heterozygous for the single-nucleotide polymorphism rs17576 (A/G; Gln279Arg) in the MMP9 gene. The results highlight the importance of MMP-9 in the pathogenesis of TBE and suggest that serum MMP-9 may serve as a promising bioindicator of TBE in both paediatric and adult TBE patients.

Negligible risk of surface transmission of SARS-CoV-2 in public transportation.

BACKGROUND: Exposure to pathogens in public transport systems is a common means of spreading infection, mainly by inhaling aerosol or droplets from infected individuals. Such particles also contaminate surfaces, creating a potential surface-transmission pathway. METHODS: A fast acoustic biosensor with an antifouling nano-coating was introduced to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on exposed surfaces in the Prague Public Transport System. Samples were measured directly without pre-treatment. Results with the sensor gave excellent agreement with parallel quantitative reverse-transcription polymerase chain reaction (qRT-PCR) measurements on 482 surface samples taken from actively used trams, buses, metro trains and platforms between 7 and 9 April 2021, in the middle of the lineage Alpha SARS-CoV-2 epidemic wave when 1 in 240 people were COVID-19 positive in Prague. RESULTS: Only ten of the 482 surface swabs produced positive results and none of them contained virus particles capable of replication, indicating that positive samples contained inactive virus particles and/or fragments. Measurements of the rate of decay of SARS-CoV-2 on frequently touched surface materials showed that the virus did not remain viable longer than 1-4 h. The rate of inactivation was the fastest on rubber handrails in metro escalators and the slowest on hard-plastic seats, window glasses and stainless-steel grab rails. As a result of this study, Prague Public Transport Systems revised their cleaning protocols and the lengths of parking times during the pandemic. CONCLUSIONS: Our findings suggest that surface transmission played no or negligible role in spreading SARS-CoV-2 in Prague. The results also demonstrate the potential of the new biosensor to serve as a complementary screening tool in epidemic monitoring and prognosis.

Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein.

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

Orthohantaviruses in Reservoir and Atypical Hosts in the Czech Republic: Spillover Infection and Indication of Virus-Specific Tissue Tropism.

Orthohantaviruses (genus Orthohantavirus) are a diverse group of viruses that are closely associated with their natural hosts (rodents, shrews, and moles). Several orthohantaviruses cause severe disease in humans. Central and western Europe are areas with emerging orthohantavirus occurrences. In our study, several orthohantaviruses, including the pathogenic Kurkino virus (KURV), were detected in their natural hosts trapped at several study sites in the Czech Republic. KURV was detected mainly in its typical host, the striped field mouse (Apodemus agrarius). Nevertheless, spillover infections were also detected in wood mice (Apodemus sylvaticus) and common voles (Microtus arvalis). Similarly, Tula virus (TULV) was found primarily in common voles, and events of spillover to rodents of other host species, including Apodemus spp., were recorded. In addition, unlike most previous studies, different tissues were sampled and compared to assess their suitability for orthohantavirus screening and possible tissue tropism. Our data suggest possible virus-specific tissue tropism in rodent hosts. TULV was most commonly detected in the lung tissue, whereas KURV was more common in the liver, spleen, and brain. Moreover, Seewis and Asikkala viruses were detected in randomly found common shrews (Sorex araneus). In conclusion, we have demonstrated the presence of human-pathogenic KURV and the potentially pathogenic TULV in their typical hosts as well as their spillover to atypical host species belonging to another family. Furthermore, we suggest the possibility of virus-specific tissue tropism of orthohantaviruses in their natural hosts. IMPORTANCE Orthohantaviruses (genus Orthohantavirus, family Hantaviridae) are a diverse group of globally distributed viruses that are closely associated with their natural hosts. Some orthohantaviruses are capable of infecting humans and causing severe disease. Orthohantaviruses are considered emerging pathogens due to their ever-increasing diversity and increasing numbers of disease cases. We report the detection of four different orthohantaviruses in rodents and shrews in the Czech Republic. Most viruses were found in their typical hosts, Kurkino virus (KURV) in striped field mice (Apodemus agrarius), Tula virus (TULV) in common voles (Microtus arvalis), and Seewis virus in common shrews (Sorex araneus). Nevertheless, spillover infections of atypical host species were also recorded for KURV, TULV, and another shrew-borne orthohantavirus, Asikkala virus. In addition, indications of virus-specific patterns of tissue tropism were observed. Our results highlight the circulation of several orthohantaviruses, including KURV, which is pathogenic to humans, among rodents and shrews in the Czech Republic.

Fitness of mCherry Reporter Tick-Borne Encephalitis Virus in Tick Experimental Models.

The tick-borne encephalitis virus (TBEV) causes a most important viral life-threatening illness transmitted by ticks. The interactions between the virus and ticks are largely unexplored, indicating a lack of experimental tools and systematic studies. One such tool is recombinant reporter TBEV, offering antibody-free visualization to facilitate studies of transmission and interactions between a tick vector and a virus. In this paper, we utilized a recently developed recombinant TBEV expressing the reporter gene mCherry to study its fitness in various tick-derived in vitro cell cultures and live unfed nymphal Ixodes ricinus ticks. The reporter virus was successfully replicated in tick cell lines and live ticks as confirmed by the plaque assay and the mCherry-specific polymerase chain reaction (PCR). Although a strong mCherry signal determined by fluorescence microscopy was detected in several tick cell lines, the fluorescence of the reporter was not observed in the live ticks, corroborated also by immunoblotting. Our data indicate that the mCherry reporter TBEV might be an excellent tool for studying TBEV-tick interactions using a tick in vitro model. However, physiological attributes of a live tick, likely contributing to the inactivity of the reporter, warrant further development of reporter-tagged viruses to study TBEV in ticks in vivo.

Evaluation of two artificial infection methods of live ticks as tools for studying interactions between tick-borne viruses and their tick vectors.

Up to 170 tick-borne viruses (TBVs) have been identified to date. However, there is a paucity of information regarding TBVs and their interaction with respective vectors, limiting the development of new effective and urgently needed control methods. To overcome this gap of knowledge, it is essential to reproduce transmission cycles under controlled laboratory conditions. In this study we assessed an artificial feeding system (AFS) and an immersion technique (IT) to infect Ixodes ricinus ticks with tick-borne encephalitis (TBE) and Kemerovo (KEM) virus, both known to be transmitted predominantly by ixodid ticks. Both methods permitted TBEV acquisition by ticks and we further confirmed virus trans-stadial transmission and onward transmission to a vertebrate host. However, only artificial feeding system allowed to demonstrate both acquisition by ticks and trans-stadial transmission for KEMV. Yet we did not observe transmission of KEMV to mice (IFNAR-/- or BALB/c). Artificial infection methods of ticks are important tools to study tick-virus interactions. When optimally used under laboratory settings, they provide important insights into tick-borne virus transmission cycles.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2.

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary: Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern.

BACKGROUND: The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. METHODS: We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. FINDINGS: AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. INTERPRETATION: The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. FUNDING: The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

Spiroplasma Isolated From Third-Generation Laboratory Colony Ixodes persulcatus Ticks.

Spiroplasma are vertically-transmitted endosymbionts of ticks and other arthropods. Field-collected Ixodes persulcatus have been reported to harbour Spiroplasma, but nothing is known about their persistence during laboratory colonisation of this tick species. We successfully isolated Spiroplasma from internal organs of 6/10 unfed adult ticks, belonging to the third generation of an I. persulcatus laboratory colony, into tick cell culture. We screened a further 51 adult male and female ticks from the same colony for presence of Spiroplasma by genus-specific PCR amplification of fragments of the 16S rRNA and rpoB genes; 100% of these ticks were infected and the 16S rRNA sequence showed 99.8% similarity to that of a previously-published Spiroplasma isolated from field-collected I. persulcatus. Our study shows that Spiroplasma endosymbionts persist at high prevalence in colonised I. persulcatus through at least three generations, and confirms the usefulness of tick cell lines for isolation and cultivation of this bacterium.

Hedgehogs and Squirrels as Hosts of Zoonotic Bartonella Species.

Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by 'Candidatus B. rudakovii' and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.