Phylogeography of horseshoe bat sarbecoviruses in Vietnam and neighbouring countries. Implications for the origins of SARS-CoV and SARS-CoV-2.

Previous studies on horseshoe bats (Rhinolophus spp.) have described many coronaviruses related to SARS-CoV (SARSCoVr) in China and only a few coronaviruses related to SARS-CoV-2 (SARSCoV2r) in Yunnan (southern China), Cambodia, Laos and Thailand. Here, we report the results of several field missions carried out in 2017, 2021 and 2022 across Vietnam during which 1218 horseshoe bats were sampled from 19 locations. Sarbecoviruses were detected in 11% of faecal RNA extracts, with much more positives among Rhinolophus thomasi (46%). We assembled 38 Sarbecovirus genomes, including 32 SARSCoVr, four SARSCoV2r, and two recombinants of SARSCoVr and SARSCoV2r (RecSar), one showing a Spike protein very similar to SARS-CoV-2. We detected a bat co-infected with four coronaviruses, including two sarbecoviruses. Our analyses revealed that Sarbecovirus genomes evolve in Vietnam under strong geographical and host constraints. First, we found evidence for a deep separation between viruses from northern Vietnam and those from central and southern Vietnam. Second, we detected only SARSCoVr in Rhinolophus thomasi, both SARSCoVr and SARSCoV2r in Rhinolophus affinis, and only RecSar in Rhinolophus pusillus captured close to the border with China. Third, the bias in favour of Uracil in synonymous third codon positions of SARSCoVr extracted from R. thomasi showed a negative correlation with latitudes. Our results also provided support for an emergence of SARS-CoV in horseshoe bats from northern Yunnan and emergence of SARS-CoV-2 in horseshoe bats from northern Indochina subtropical forests (southern Yunnan, northern Laos and north-western Vietnam).

Detection and sequence analysis of Canine morbillivirus in multiple species of the Mustelidae family.

BACKGROUND: Canine morbillivirus (canine distemper virus, CDV) is a member of the Paramyxoviridae family. Canine distemper is a serious viral disease that affects many mammalian species, including members of the Mustelidae family. These animals have an elusive nature, which makes related virological studies extremely challenging. There is a significant knowledge gap about the evolution of their viruses and about the possible effects of these viruses to the population dynamics of the host animals. Spleen and lung tissue samples of 170 road-killed mustelids belonging to six species were collected between 1997 and 2022 throughout Hungary and tested for CDV with real-time RT-PCR. RESULTS: Three species were positive for viral RNA, 2 out of 64 Steppe polecats (Mustela eversmanii), 1 out of 36 European polecats (Mustela putorius) and 2 out of 36 stone martens (Martes foina); all 18 pine martens (Martes martes), 10 least weasels (Mustela nivalis) and 6 stoats (Mustela erminea) tested negative. The complete CDV genome was sequenced in five samples using pan-genotype CDV-specific, amplicon-based Nanopore sequencing. Based on the phylogenetic analysis, all five viral sequences were grouped to the Europe/South America 1 lineage and the distribution of one sequence among trees indicated recombination of the Hemagglutinin gene. We verified the recombination with SimPlot analysis. CONCLUSIONS: This paper provides the first CDV genome sequences from Steppe polecats and additional complete genomes from European polecats and stone martens. The infected specimens of various species originated from distinct parts of the country over a long time, indicating a wide circulation of CDV among mustelids throughout Hungary. Considering the high virulence of CDV and the presence of the virus in these animals, we highlight the importance of conservation efforts for wild mustelids. In addition, we emphasize the importance of full genomic data acquisition and analysis to better understand the evolution of the virus. Since CDV is prone to recombination, specific genomic segment analyses may provide less representative evolutionary traits than using complete genome sequences.

The updated genome of the Hungarian population of Aedes koreicus.

Vector-borne diseases pose a potential risk to human and animal welfare, and understanding their spread requires genomic resources. The mosquito Aedes koreicus is an emerging vector that has been introduced into Europe more than 15 years ago but only a low quality, fragmented genome was available. In this study, we carried out additional sequencing and assembled and characterized the genome of the species to provide a background for understanding its evolution and biology. The updated genome was 1.1 Gbp long and consisted of 6099 contigs with an N50 value of 329,610 bp and a BUSCO score of 84%. We identified 22,580 genes that could be functionally annotated and paid particular attention to the identification of potential insecticide resistance genes. The assessment of the orthology of the genes indicates a high turnover at the terminal branches of the species tree of mosquitoes with complete genomes, which could contribute to the adaptation and evolutionary success of the species. These results could form the basis for numerous downstream analyzes to develop targets for the control of mosquito populations.

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.

Genomic characterization of Volzhskoe tick virus (Bunyaviricetes) from a Hyalomma marginatum tick, Hungary.

Hyalomma marginatum, a vector for the high-consequence pathogen, the Crimean-Congo hemorrhagic fever virus (CCHFV), needs particular attention due to its impact on public health. Although it is a known vector for CCHFV, its general virome is largely unexplored. Here, we report findings from a citizen science monitoring program aimed to understand the prevalence and diversity of tick-borne pathogens, particularly focusing on Hyalomma ticks in Hungary. In 2021, we identified one adult specimen of Hyalomma marginatum and subjected it to Illumina-based viral metagenomic sequencing. Our analysis revealed sequences of the uncharacterized Volzhskoe tick virus, an unclassified member of the class Bunyaviricetes. The in silico analysis uncovered key genetic regions, including the glycoprotein and the RNA-dependent RNA polymerase (RdRp) coding regions. Phylogenetic analysis indicated a close relationship between our Volzhskoe tick virus sequences and other unclassified Bunyaviricetes species. These related species of unclassified Bunyaviricetes were detected in vastly different geolocations. These findings highlight the remarkable diversity of tick specific viruses and emphasize the need for further research to understand the transmissibility, seroreactivity or the potential pathogenicity of Volzhskoe tick virus and related species.

Snapshot study of canine distemper virus in Bangladesh with on-site PCR detection and nanopore sequencing.

Canine distemper virus (CDV) is a highly contagious virus that affects domestic and wild animals, causing severe illness with high mortality rates. Rapid monitoring and sequencing can provide valuable information about circulating CDV strains, which may foster effective vaccination strategies and the successful integration of these into conservation programs. During two site visits in Bangladesh in 2023, we tested a mobile, deployable genomic surveillance setup to explore the genetic diversity and phylogenetic patterns of locally circulating CDV strains. We collected and analysed 355 oral swab samples from stray dogs in Rajshahi and Chattogram cities, Bangladesh. CDV-specific real-time RT-PCR was performed to screen the samples. Out of the 355 samples, 7.4% (10/135) from Rajshahi city and 0.9% (2/220) from Chattogram city tested positive for CDV. We applied a real-time RT-PCR assay and a pan-genotype CDV-specific amplicon-based Nanopore sequencing technology to obtain the near-completes. Five near-complete genome sequences were generated, with phylogenetic relation to the India-1/Asia-5 lineage previously identified in India. This is the first study to provide genomic data on CDV in Bangladesh and the first demonstration of a mobile laboratory setup as a powerful tool in rapid genomic surveillance and risk assessment for CDV in low resource regions.

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.

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.

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.

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.

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.

Companion animals likely do not spread COVID-19 but may get infected themselves.

Coronavirus disease 2019 (COVID-19) is a highly contagious infectious disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). From the epidemiological data, the picture emerges that the more severe etiopathologies among COVID-19 patients are found in elderly people. The risk of death due to COVID-19 increases exponentially with age. Eight out of 10 COVID-19 related deaths occur in people older than 65 years of age. Older patients with comorbid conditions such as hypertension, heart failure, diabetes mellitus, asthma, chronic obstructive pulmonary disease, and cancer have a much higher case fatality rate. Governments and public health authorities all over the world have realized that protections of vulnerable older adults should be a priority during the COVID-19 pandemic. COVID-19 is a zoonotic disease. The SARS-CoV-2 virus was originally transmitted likely from a bat or a pangolin to humans. Recent evidence suggests that SARS-CoV-2, similar to other coronaviruses, can infect several species of animals, including companion animals such as dogs, cats, and ferrets although their viral loads remain low. While the main source of infection transmission therefore is human to human, there are a few rare cases of pets contracting the infection from a SARS-CoV-2-infected human. Although there is no evidence that pets actively transmit SARS-CoV-2 via animal-to-human transmission, senior pet ownership potentially may pose a small risk to older adults by (1) potentially enabling animal-to-human transmission of SARS-CoV-2 in the most vulnerable population and (2) by increasing the exposition risk for the elderly due to the necessity to care for the pet and, in the case of dogs, to take them outside the house several times per day. In this overview, the available evidence on SARS-CoV-2 infection in pets is considered and the potential for spread of COVID-19 from companion animals to older individuals and the importance of prevention are discussed.

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.

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.

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.

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.

Revising mtDNA haplotypes of the ancient Hungarian conquerors with next generation sequencing.

As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination.