Accelerating targeted mosquito control efforts through mobile West Nile virus detection.

BACKGROUND: Different mosquito control strategies have been implemented to mitigate or prevent mosquito-related public health situations. Modern mosquito control largely relies on multiple approaches, including targeted, specific treatments. Given this, it is becoming increasingly important to supplement these activities with rapid and mobile diagnostic capacities for mosquito-borne diseases. We aimed to create and test the applicability of a rapid diagnostic system for West Nile virus that can be used under field conditions. METHODS: In this pilot study, various types of adult mosquito traps were applied within the regular mosquito monitoring activity framework for mosquito control. Then, the captured specimens were used for the detection of West Nile virus RNA under field conditions with a portable qRT-PCR approach within 3-4 h. Then, positive samples were subjected to confirmatory RT-PCR or NGS sequencing in the laboratory to obtain genome information of the virus. We implemented phylogenetic analysis to characterize circulating strains. RESULTS: A total of 356 mosquito individuals representing 7 species were processed in 54 pools, each containing up to 20 individuals. These pools were tested for the presence of West Nile virus, and two pools tested positive, containing specimens from the Culex pipiens and Anopheles atroparvus mosquito species. As a result of subsequent sequencing, we present the complete genome of West Nile virus and Bagaza virus. CONCLUSIONS: The rapid identification of infected mosquitoes is the most important component of quick response adulticide or larvicide treatments to prevent human cases. The conceptual framework of real-time surveillance can be optimized for other pathogens and situations not only in relation to West Nile virus. We present an early warning system for mosquito-borne diseases and demonstrate its application to aid rapid-response mosquito control actions.

Isolation and genome characterization of Lloviu virus from Italian Schreibers's bats.

Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers's bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its initial discovery. Recent studies with recombinant and wild-type LLOV isolates confirmed the zoonotic nature of the virus in vitro. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding region of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this study is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection and confirming the previous observation that these bats are effective hosts of the virus in nature. This result further strengthens the role of bats as the natural hosts for zoonotic filoviruses.

The importance of equally accessible genomic surveillance in the age of pandemics.

Genomic epidemiology is now a core component in investigating the spread of a disease during an outbreak and for future preparedness to tackle emerging zoonoses. During the last decades, several viral diseases arose and emphasized the importance of molecular epidemiology in tracking the dispersal route, supporting proper mitigation measures, and appropriate vaccine development. In this perspective article, we summarized what has been done so far in the genomic epidemiology field and what should be considered in the future. We traced back the methods and protocols employed over time for zoonotic disease response. Either to small outbreaks such as the severe acute respiratory syndrome (SARS) outbreak identified first in 2002 in Guangdong, China, or to a global pandemic like the one that we are experiencing now since 2019 when the severe acute respiratory syndrome 2 (SARS-CoV-2) virus emerged in Wuhan, China, following several pneumonia cases, and subsequently spread worldwide. We explored both the benefits and shortages encountered when relying on genomic epidemiology, and we clearly present the disadvantages of inequity in accessing these tools around the world, especially in countries with less developed economies. For effectively addressing future pandemics, it is crucial to work for better sequencing equity around the globe.

Aedes koreicus, a vector on the rise: Pan-European genetic patterns, mitochondrial and draft genome sequencing.

BACKGROUND: The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species' biology, behavior, and dispersal patterns were poorly investigated to date. METHODOLOGY/PRINCIPAL FINDINGS: To understand the species' population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. CONCLUSIONS/SIGNIFICANCE: Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of 'Single Nucleotide Polymorphism' markers. Considering its public health importance, it is crucial to further investigate the species' population genetic dynamic, including a larger sampling and additional genomic markers.

The Anti-Histamine Azelastine, Identified by Computational Drug Repurposing, Inhibits Infection by Major Variants of SARS-CoV-2 in Cell Cultures and Reconstituted Human Nasal Tissue.

Background and purpose: The COVID-19 pandemic continues to pose challenges, especially with the emergence of new SARS-CoV-2 variants that are associated with higher infectivity and/or compromised protection afforded by the current vaccines. There is a high demand for additional preventive and therapeutic strategies effective against this changing virus. Repurposing of approved or clinically tested drugs can provide an immediate solution. Experimental Approach: We applied a novel computational approach to search among approved and commercially available drugs. Antiviral activity of a predicted drug, azelastine, was tested in vitro in SARS-CoV-2 infection assays with Vero E6 cells, Vero cells stably overexpressing the human TMPRSS2 and ACE2 proteins as well as on reconstituted human nasal tissue using the predominant variant circulating in Europe in summer 2020, B.1.177 (D614G variant), and its emerging variants of concern; B.1.1.7 (alpha), B.1.351 (beta) and B.1.617.2 (delta) variants. The effect of azelastine on viral replication was assessed by quantification of viral genomes by droplet digital PCR or qPCR. Key results: The computational approach identified major drug families, such as anti-infective, anti-inflammatory, anti-hypertensive, antihistamine, and neuroactive drugs. Based on its attractive safety profile and availability in nasal formulation, azelastine, a histamine 1 receptor-blocker was selected for experimental testing. Azelastine reduced the virus-induced cytopathic effect and SARS-CoV-2 copy numbers both in preventive and treatment settings upon infection of Vero cells with an EC50 of 2.2-6.5 µM. Comparable potency was observed with the alpha, beta and delta variants. Furthermore, five-fold dilution (containing 0.02% azelastine) of the commercially available nasal spray formulation was highly potent in inhibiting viral propagation in reconstituted human nasal tissue. Conclusion and Implications: Azelastine, an antihistamine available as nasal sprays developed against allergic rhinitis may be considered as a topical prevention or treatment of nasal colonization by SARS-CoV-2. A Phase 2 efficacy indicator study with azelastine-containing nasal spray that was designed based on the findings reported here has been concluded recently, confirming accelerated viral clearance in SARS-CoV-2 positive subjects.

Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology.

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution.

Isolation of infectious Lloviu virus from Schreiber's bats in Hungary.

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics.

Complete genomic sequencing of canine distemper virus with nanopore technology during an epizootic event.

Canine distemper virus (CDV) endangers a wide range of wild animal populations, can cross species barriers and therefore representing a significant conservational and animal health risk around the globe. During spring to autumn 2021, according to our current estimates a minimum of 50 red foxes (Vulpes vulpes) died of CDV in Hungary, with CDV lesions. Oral, nasal and rectal swab samples were RT-PCR screened for Canine Distemper Virus from red fox carcasses. To investigate in more detail the origins of these CDV strains, 19 complete genomes were sequenced with a pan-genotype CDV-specific amplicon-based sequencing method developed by our laboratory and optimized for the Oxford Nanopore Technologies platform. Phylogenetic analysis of the complete genomic sequences and separately the hemagglutinin gene sequences revealed the role of the Europe lineage of CDV as a causative agent for the current epizootic. Here we highlight the growing importance of fast developing rapid sequencing technologies to aid rapid response activities during epidemics or epizootic events. We also emphasize the urgent need for improved surveillance of CDV, considering the epizootic capability of enzootic strains as reported in the current study. For such future efforts, we provide a novel NGS protocol to facilitate future genomic surveillance studies.

The Algerian Chapter of SARS-CoV-2 Pandemic: An Evolutionary, Genetic, and Epidemiological Prospect.

To explore the SARS-CoV-2 pandemic in Algeria, a dataset comprising ninety-five genomes originating from SARS-CoV-2 sampled from Algeria and other countries worldwide, from 24 December 2019, through 4 March 2021, was thoroughly examined. While performing a multi-component analysis regarding the Algerian outbreak, the toolkit of phylogenetic, phylogeographic, haplotype, and genomic analysis were effectively implemented. We estimated the Time to the Most Recent Common Ancestor (TMRCA) in reference to the Algerian pandemic and highlighted the multiple introductions of the disease and the missing data depicted in the transmission loop. In addition, we emphasized the significant role played by local and international travels in disease dissemination. Most importantly, we unveiled mutational patterns, the effect of unique mutations on corresponding proteins, and the relatedness regarding the Algerian sequences to other sequences worldwide. Our results revealed individual amino-acid replacements such as the deleterious replacement A23T in the orf3a gene in Algeria_EPI_ISL_418241. Additionally, a connection between Algeria_EPI_ISL_420037 and sequences originating from the USA was observed through a USA characteristic amino-acid replacement T1004I in the nsp3 gene, found in the aforementioned Algerian sequence. Similarly, successful tracing could be established, such as Algeria/G37318-8849/2020|EPI_ISL_766863, which was imported from Saudi Arabia during the pilgrimage. Lastly, we assessed the Algerian mitigation measures regarding disease containment using statistical analyses.

Effectiveness Regarding Hantavirus Detection in Rodent Tissue Samples and Urine.

The natural hosts of Orthohantaviruses are rodents, soricomorphs and bats, and it is well known that they may cause serious or even fatal diseases among humans worldwide. The virus is persistent among animals and it is shed via urine, saliva and feces throughout the entirety of their lives. We aim to identify the effectiveness of hantavirus detection in rodent tissue samples and urine originating from naturally infected rodents. Initially, animals were trapped at five distinct locations throughout the Transdanubian region in Hungary. Lung, liver, kidney and urine samples were obtained from 163 deceased animals. All organs and urine were tested using nested reverse transcription polymerase chain reaction (nRT-PCR). Furthermore, sera were examined for IgG antibodies against Dobrava-Belgrade virus (DOBV) and Puumala virus (PUUV) by Western blot assay. IgG antibodies against hantaviruses and/or nucleic acid were detected in 25 (15.3%) cases. Among Apodemus, Myodes, and Microtus rodent species, DOBV, PUUV and Tula virus (TULV) were clearly identified. Amid the PCR-positive samples, the nucleic acid of the viruses was detected most effectively in the kidney (100%), while only 55% of screened lung tissues were positive. Interestingly, only three out of 20 rodent urine samples were positive when tested using nRT-PCR. Moreover, five rodents were seropositive without detectable virus nucleic acid in any of the tested organs.

Prolonged Infection of Canine Distemper Virus in a Mixed-Breed Dog.

Canine distemper virus (CDV) is a major viral pathogen in domestic dogs, belonging to the Paramyxoviridae family, in the Morbillivirus genus. It is present worldwide, and a wide range of domestic animals and wild carnivores are at risk. In the absence of vaccination, dogs have a low chance of survival; however, if and when a dog survives, it can take an average of a few weeks to a few months to fully wipe out the virus. In the present study, we traced the course of infection of a 1-year-old mixed-breed male dog. The animal had an unusually long course of persistent CDV infection with a vector-borne heartworm (Dirofilaria immitis) co-infection. The dog excreted the CDV for 17 months with PCR positivity in urine samples collected from February 2019 through June 2020. The sequencing and phylogenetic analysis of the hemagglutinin gene revealed the CDV to be the member of the endemic Arctic-like genetic lineage. To the best of our knowledge, this report represents the longest documented canine infection of CDV. Notably, we highlight the necessity regarding CDV infectivity studies to better comprehend the transmission attributes of the virus.

Effect of An 84-bp Deletion of the Receptor-Binding Domain on the ACE2 Binding Affinity of the SARS-CoV-2 Spike Protein: An In Silico Analysis.

SARS-CoV-2 is a recently emerged, novel human coronavirus responsible for the currently ongoing COVID-19 pandemic. Recombination is a well-known evolutionary strategy of coronaviruses, which may frequently result in significant genetic alterations, such as deletions throughout the genome. In this study we identified a co-infection with two genetically different SARS-CoV-2 viruses within a single patient sample via amplicon-based next generation sequencing in Hungary. The recessive strain contained an 84 base pair deletion in the receptor binding domain of the spike protein gene and was found to be gradually displaced by a dominant non-deleterious variant over-time. We have identified the region of the receptor-binding domain (RBD) that is affected by the mutation, created homology models of the RBDΔ84 mutant, and based on the available experimental data and calculations, we propose that the mutation has a deteriorating effect on the binding of RBD to the angiotensin-converting enzyme 2 (ACE2) receptor, which results in the negative selection of this variant. Extending the sequencing capacity toward the discovery of emerging recombinant or deleterious strains may facilitate the early recognition of novel strains with altered phenotypic attributes and understanding of key elements of spike protein evolution. Such studies may greatly contribute to future therapeutic research and general understanding of genomic processes of the virus.

Multiple SARS-CoV-2 Introductions Shaped the Early Outbreak in Central Eastern Europe: Comparing Hungarian Data to a Worldwide Sequence Data-Matrix.

Severe Acute Respiratory Syndrome Coronavirus 2 is the third highly pathogenic human coronavirus in history. Since the emergence in Hubei province, China, during late 2019, the situation evolved to pandemic level. Following China, Europe was the second epicenter of the pandemic. To better comprehend the detailed founder mechanisms of the epidemic evolution in Central-Eastern Europe, particularly in Hungary, we determined the full-length SARS-CoV-2 genomes from 32 clinical samples collected from laboratory confirmed COVID-19 patients over the first month of disease in Hungary. We applied a haplotype network analysis on all available complete genomic sequences of SARS-CoV-2 from GISAID database as of 21 April 2020. We performed additional phylogenetic and phylogeographic analyses to achieve the recognition of multiple and parallel introductory events into our region. Here, we present a publicly available network imaging of the worldwide haplotype relations of SARS-CoV-2 sequences and conclude the founder mechanisms of the outbreak in Central-Eastern Europe.

Identification of Hepatitis E Virus in the Feces of Red Foxes (Vulpes vulpes).

Orthohepeviruses (HEV) can infect a wide range of animals, showing a relatively strict host specificity; however, its zoonotic potential, natural transmission in the wildlife are less known. Several new HEV-like viruses have been identified in various animal species, including carnivores; however, the phylogenetic relationship among these viruses is poorly resolved, since some of them were known as rodent-related so far. The red fox, the most widespread carnivore worldwide, is a known reservoir of several viruses that transmit from wildlife to humans or domestic animals; they might have a defined role in the circulation of rodent-borne HEV. In this study, we performed a HEV survey by heminested RT-PCR (Reverse Transcription PCR) on red fox fecal samples to investigate the presence of HEV in red foxes living in natural conditions, and to explore the origin of the virus via phylogenetic analysis. Out of the 26 investigated samples, HEV RNA was identified in one sample. Following Sanger sequencing, the novel sequence displayed 91% identity on the nucleotide level with recently published European common vole-HEV derived from Microtus arvalis. In contrast, it shared 85% nucleotide similarity with HEV strains described previously in red foxes. Our results strongly support "the dietary-origin" of unclassified HEV-like strains described from predators that usually prey on rodents.

Nursing homes and the elderly regarding the COVID-19 pandemic: situation report from Hungary.

The global impact of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is significant in terms of public health effects and its long-term socio-economic implications. Among all social groups, the elderly is by far the most affected age group regarding morbidity and mortality. In multiple countries spanning several continents, there are an increasing number of reports referencing the novel coronavirus disease-2019 (COVID-19) spread among nursing homes. These areas are now recognized as potent hotspots regarding the pandemic, which one considers with special regard. Herein, we present currently available data of fatal COVID-19 cases throughout Hungary, along with the analysis of the co-morbidity network. We also report on viral genomic data originating from a nursing home resident. The genomic data was used for viral haplotype network analysis. We emphasize the urgent need for public health authorities to focus on nursing homes and residential service units worldwide, especially in the care of the elderly and infirmed. Our results further emphasize the recent statement released by the World Health Organization (WHO) regarding the vulnerability among seniors and especially the high risk of COVID-19 emergence throughout nursing and social homes.

Crimean-Congo hemorrhagic fever virus infection triggers the upregulation of the Wnt signaling pathway inhibitor genes.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic agent. Thus far, vaccines and specific antiviral therapies are not available against the threat of infection. Our knowledge regarding its pathogenesis is indeed limited, and thus, developing effective antiviral therapies is hampered. Several studies have demonstrated that the CCHFV infection has an impact on numerous signal transduction pathways. In parallel, the Wnt signaling pathway components are responsible for different important biological processes including cell fate determination, cell migration and cell polarity. Moreover, its implication among several virus infections has been proven, yet little is known in reference to which components of the Wnt pathway are being activated/inhibited as a response to the infection. Our aim was to elicit the influence of the CCHFV infection on adenocarcinomic human alveolar basal epithelial cells in vitro regarding the Wnt signaling pathway-related genes. Gene-expression changes of 92 Wnt-associated genes were examined 48 h post-infection. Furthermore, ß-catenin levels were compared in the infected and uninfected cells. Significant changes were observed in the case of 13 genes. The majority of the upregulated genes are associated with the inhibition of the Wnt/ß-catenin signaling. Additionally, infected cells expressed less ß-catenin. Our findings suggest that CCHFV blocks the Wnt/ß-catenin pathway. Our study corroborates the link between CCHFV infection and the Wnt signaling pathways. In addition, it broadens our knowledge in the CCHFV pathomechanism.

Genetic characterization of a novel picornavirus in Algerian bats: co-evolution analysis of bat-related picornaviruses.

Bats are reservoirs of numerous zoonotic viruses. The Picornaviridae family comprises important pathogens which may infect both humans and animals. In this study, a bat-related picornavirus was detected from Algerian Minioptreus schreibersii bats for the first time in the country. Molecular analyses revealed the new virus originates to the Mischivirus genus. In the operational use of the acquired sequence and all available data regarding bat picornaviruses, we performed a co-evolutionary analysis of mischiviruses and their hosts, to authentically reveal evolutionary patterns within this genus. Based on this analysis, we enlarged the dataset, and examined the co-evolutionary history of all bat-related picornaviruses including their hosts, to effectively compile all possible species jumping events during their evolution. Furthermore, we explored the phylogeny association with geographical location, host-genus and host-species in both data sets.

Molecular Identification of a Novel Hantavirus in Malaysian Bronze Tube-Nosed Bats (Murina aenea).

In the past ten years, several novel hantaviruses were discovered in shrews, moles, and bats, suggesting the dispersal of hantaviruses in many animal taxa other than rodents during their evolution. Interestingly, the coevolutionary analyses of most recent studies have raised the possibility that nonrodents may have served as the primordial mammalian host and harboured the ancestors of rodent-borne hantaviruses as well. The aim of our study was to investigate the presence of hantaviruses in bat lung tissue homogenates originally collected for taxonomic purposes in Malaysia in 2015. Hantavirus-specific nested RT-PCR screening of 116 samples targeting the L segment of the virus has revealed the positivity of two lung tissue homogenates originating from two individuals, a female and a male of the Murina aenea bat species collected at the same site and sampling occasion. Nanopore sequencing of hantavirus positive samples resulted in partial genomic data from S, M, and L genome segments. The obtained results indicate molecular evidence for hantaviruses in the M. aenea bat species. Sequence analysis of the PCR amplicon and partial genome segments suggests that the identified virus may represent a novel species in the Mobatvirus genus within the Hantaviridae family. Our results provide additional genomic data to help extend our knowledge about the evolution of these viruses.