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.

In-depth Temporal Transcriptome Profiling of Monkeypox and Host Cells using Nanopore Sequencing.

The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level.

A single early introduction governed viral diversity in the second wave of SARS-CoV-2 epidemic in Hungary.

Retrospective evaluation of past waves of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic is key for designing optimal interventions against future waves and novel pandemics. Here, we report on analysing genome sequences of SARS-CoV-2 from the first two waves of the epidemic in 2020 in Hungary, mirroring a suppression and a mitigation strategy, respectively. Our analysis reveals that the two waves markedly differed in viral diversity and transmission patterns. Specifically, unlike in several European areas or in the USA, we have found no evidence for early introduction and cryptic transmission of the virus in the first wave of the pandemic in Hungary. Despite the introduction of multiple viral lineages, extensive community spread was prevented by a timely national lockdown in March 2020. In sharp contrast, the majority of the cases in the much larger second wave can be linked to a single transmission lineage of the pan-European B.1.160 variant. This lineage was introduced unexpectedly early, followed by a 2-month-long cryptic transmission before a soar of detected cases in September 2020. Epidemic analysis has revealed that the dominance of this lineage in the second wave was not associated with an intrinsic transmission advantage. This finding is further supported by the rapid replacement of B.1.160 by the alpha variant (B.1.1.7) that launched the third wave of the epidemic in February 2021. Overall, these results illustrate how the founder effect in combination with the cryptic transmission, instead of repeated international introductions or higher transmissibility, can govern viral diversity.

A Possible Way to Relate the Effects of SARS-CoV-2-Induced Changes in Transferrin to Severe COVID-19-Associated Diseases.

SARS-CoV-2 infections are responsible for the COVID-19 pandemic. Transferrin has been found to explain the link between diseases associated with impaired iron transport and COVID-19 infection. The effect of SARS-CoV-2 on human whole blood was studied by differential scanning calorimetry. The analysis of the thermal transition curves showed that the melting temperature of the transferrin-related peak decreased in the presence of SARS-CoV-2. The ratio of the under-curve area of the two main peaks was greatly affected, while the total enthalpy of the heat denaturation remained nearly unchanged in the presence of the virus. These results indicate that SARS-CoV-2, through binding to transferrin, may influence its Fe3+ uptake by inducing thermodynamic changes. Therefore, transferrin may remain in an iron-free apo-conformational state, which depends on the SARS-CoV-2 concentration. SARS-CoV-2 can induce disturbance in erythropoiesis due to toxicity generated by free iron overload.

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.

Emergence of Hyalomma marginatum and Hyalomma rufipes adults revealed by citizen science tick monitoring in Hungary.

Hyalomma ticks are important vectors of Crimean-Congo haemorrhagic fever virus (CCHFV) and other pathogens. They are frequently carried as immatures from Africa, the Middle East and Mediterranean areas to temperate Europe via migratory birds and emergence of adults has been reported in many countries where it has so far been considered non-endemic. This study aimed to implement the first steps of the DAMA (Document, Assess, Monitor, Act) protocol by monitoring the potential arrival of adult Hyalomma ticks in Hungary applying citizen-science methods. Ticks were collected from April to December 2021 by asking volunteer participants through a self-made website to look for large, quickly moving, striped-legged hard ticks on themselves, their pets and livestock. Owing to an intensive media campaign, the project website had more than 31,000 visitors within 7 months; 137 specimens and several hundred photos of hard ticks were submitted by citizen scientists from all over the country. Beside Ixodes ricinus, Dermacentor reticulatus, Dermacentor marginatus and Haemaphysalis inermis, a specimen from a dog was morphologically identified as a male Hyalomma marginatum and another removed from a cow as a male Hyalomma rufipes. The dog and the cow had never been abroad, lived approximately 280 km apart, so the two Hyalomma observations can be considered separate introductions. Amplification of the partial mitochondrial cytochrome C oxidase subunit I gene was successfully run for both specimens. Sequencing confirmed the morphological identification for both ticks. Based on the phylogenetic analyses, the Hy. marginatum individual most likely belongs to the Eurasian population and the Hy. rufipes tick to a clade of mixed sequences from Europe and Africa. We summarize the scattered historical reports about the occurrence of Hyalomma ticks and CCHFV in Hungary. Our data highlight the effectiveness of citizens science programmes in the monitoring and risk assessment of CCHFV emergence and preparedness in the study area.

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.

Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.

Multi-Approach Investigation Regarding the West Nile Virus Situation in Hungary, 2018.

The West Nile virus is endemic in multiple European countries and responsible for several epidemics throughout the European region. Its evolution into local or even widespread epidemics is driven by multiple factors from genetic diversification of the virus to environmental conditions. The year of 2018 was characterized by an extraordinary increase in human and animal cases in the Central-Eastern European region, including Hungary. In a collaborative effort, we summarized and analyzed the genetic and serologic data of WNV infections from multiple Hungarian public health institutions, universities, and private organizations. We compared human and veterinary serologic data, along with NS5 and NS3 gene sequence data through 2018. Wild birds were excellent indicator species for WNV circulation in each year. Our efforts resulted in documenting the presence of multiple phylogenetic subclades with Balkans and Western-European progenitor sequences of WNV circulating among human and animal populations in Hungary prior to and during the 2018 epidemic. Supported by our sequence and phylogenetic data, the epidemic of 2018 was not caused by recently introduced WNV strains. Unfortunately, Hungary has no country-wide integrated surveillance system which would enable the analysis of related conditions and provide a comprehensive epidemiological picture. The One Health approach, involving multiple institutions and experts, should be implemented in order to fully understand ecological background factors driving the evolution of future epidemics.

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.

Serologic survey of the Crimean-Congo haemorrhagic fever virus infection among wild rodents in Hungary.

Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne pathogen, which causes an increasing number of severe infections in many parts of Africa, Asia and in Europe. The virus is primarily transmitted by ticks, however, the spectrum of natural hosts regarding CCHFV includes a wide variety of domestic and wild animals. Although the presence of CCHFV was hypothesized in Hungary, data in support of CCHFV prevalence has thus far, proven insufficient. In the present study, rodents belonging to four species, the yellow-necked mouse (Apodemus flavicollis), the striped field mouse (A. agrarius), the wood mouse (A. sylvaticus) and the bank vole (Myodes glareolus), were all systematically trapped in the Mecsek Mountain region (Southwest Hungary), from 2011 through 2013. Rodent sera were collected and screened for CCHFV antibodies with dot-blot pre-screening and immunofluorescence assay. Among the 2085 tested rodents, 20 (0.96%) were positive for IgG antibody against CCHFV. Seroprevalence was the highest (1.25%) in A. flavicollis serum samples. Distinctly, we now provide the first data regarding CCHFV occurrence and seroprevalence among wild rodents in Hungary. This observation represents a need for large-scale surveillance to effectively assess the enzootic background and the potential public health risk of CCHFV in Hungary.

First molecular detection of Apis mellifera filamentous virus in honey bees (Apis mellifera) in Hungary.

Western honey bees (Apis mellifera) are important pollinators in the ecosystem and also play a crucial economic role in the honey industry. During the last decades, a continuous decay was registered in honey bee populations worldwide, including Hungary. In our study, we used metagenomic approaches and conventional PCR screening on healthy and winter mortality affected colonies from multiple sites in Hungary. The major goal was to discover presumed bee pathogens with viral metagenomic experiments and gain prevalence and distribution data by targeted PCR screening. We examined 664 honey bee samples that had been collected during winter mortality from three seemingly healthy colonies and from one colony infested heavily by the parasitic mite Varroa destructor in 2016 and 2017. The subsequent PCR screening of honey bee samples revealed the abundant presence of Apis mellifera filamentous virus (AmFV) for the first time in Central Europe. Based on phylogeny reconstruction, the newly-detected virus was found to be most closely related to a Chinese AmFV strain. More sequence data from multiple countries would be needed for studying the detailed phylogeographical patterns and worldwide spreading process of AmFV. Here we report the prevalent presence of this virus in Hungarian honey bee colonies.

Filarial nematode (order: Spirurida) surveillance in urban habitats, in the city of Pécs (Hungary).

As part of the seasonal mosquito control activities in the city of Pécs (Baranya County, Hungary), a total of 1123 adult female mosquitoes belonging to 18 species (including the invasive species Aedes koreicus) were collected from human-inhabited areas, using CO2-baited traps, during two consecutive years. To survey the presence and prevalence of filarial parasites in these mosquitoes, we performed a molecular survey for filarial DNA, attempted by PCR using generic primers (COI), and followed by DNA sequencing. Filaroid nematode DNA was detected in 4% of investigated mosquito pools. Out of 410 pools, 9 pools of mosquitoes were positive for Dirofilaria repens (Aedes vexans, Aedes koreicus, Coquillettidia richiardii), and/or Dirofilaria immitis (Ae. vexans, Cq. richiardii), and further 8 pools were positive for Setaria tundra (Ae. vexans, Cq. richiardii). Our study provides novel insight for prevalence of filaroid nematodes in mosquitoes occurring in close proximity to humans, thereby highlights the possible human and veterinary health importance of these mosquito species, including the recently introduced invasive mosquito Ae. koreicus.

First genetic characterization of Usutu virus from Culex pipiens mosquitoes Serbia, 2014.

Since its first appearance in Europe, Usutu virus (USUV) diverged to several different genetic lineages. The virus was reported to date from multiple countries across Europe (Hungary, Italy, Switzerland, Spain, Germany, Czech Republic and Belgium). Considering the more frequently published impact of the virus on humans it is crucial to investigate locally circulating genetic variants and trace its evolution. We retrospectively analyzed mosquito samples from Serbia Vojvodina region, collected during 2014. In this study we report the results of the screening of 23,753 female mosquitoes (753 pools) for USUV-specific nucleic-acid. Out of the 753 pools sampled, the presence of USUV RNA was confirmed in 3 pools of Culex pipiens mosquitoes, collected in August. Based on their partial NS5 sequence, all strains were identical, therefore we adjusted one representative strain for complete genome sequencing. Based on phylogenetic analysis the Serbian USUV sequences were most closely related to the virus that emerged in Austria in 2001, in Hungary in 2005 and was circulating until 2015 in Hungary. This data presents a wider geographic distribution of this genetic variant and provides the first genetic data from this region.