Correction: Recombinant Lloviu virus as a tool to study viral replication and host responses.

[This corrects the article DOI: 10.1371/journal.ppat.1010268.].

Recombinant Lloviu virus as a tool to study viral replication and host responses.

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

Pathogenicity of Lloviu and Bombali Viruses in Type I Interferon Receptor Knockout Mice.

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV.

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.

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.

Tick-Borne Flaviviruses, with a Focus on Powassan Virus.

The tick-borne pathogen Powassan virus is a rare cause of encephalitis in North America and the Russian Far East. The number of documented cases described since the discovery of Powassan virus in 1958 may be <150, although detection of cases has increased over the past decade. In the United States, the incidence of Powassan virus infections expanded from the estimated 1 case per year prior to 2005 to 10 cases per year during the subsequent decade. The increased detection rate may be associated with several factors, including enhanced surveillance, the availability of improved laboratory diagnostic methods, the expansion of the vector population, and, perhaps, altered human activities that lead to more exposure. Nonetheless, it remains unclear whether Powassan virus is indeed an emerging threat or if enzootic cycles in nature remain more-or-less stable with periodic fluctuations of host and vector population sizes. Despite the low disease incidence, the approximately 10% to 15% case fatality rate of neuroinvasive Powassan virus infection and the temporary or prolonged sequelae in >50% of survivors make Powassan virus a medical concern requiring the attention of public health authorities and clinicians. The medical importance of Powassan virus justifies more research on developing specific and effective treatments and prevention and control measures.

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.

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.

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.

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

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.

Metagenomic analysis of bat guano samples revealed the presence of viruses potentially carried by insects, among others by Apis mellifera in Hungary.

The predominance of dietary viruses in bat guano samples had been described recently, suggesting a new opportunity to survey the prevalence and to detect new viruses of arthropods or even plant-infecting viruses circulating locally in the ecosystem. Here we describe the diversity of viruses belonging to the order Picornavirales in Hungarian insectivorous bat guano samples. The metagenomic analysis conducted on our samples has revealed the significant predominance of aphid lethal paralysis virus (ALPV) and Big Sioux River virus (BSRV) in Hungary for the first time. Phylogenetic analysis was used to clarify the relationship to previously identified ALPV strains infecting honey bees, showing that our strain possesses a close genetic relationship with the strains that have already been described as pathogenic to honey bees. Furthermore, studies have previously confirmed the ability of these viruses to replicate in adult honey bees; however, no signs related to these viruses have been revealed yet. With the identification of two recently described possibly honey bee infecting viruses for the first time in Hungary, our results might have importance for the health conditions of Hungarian honey bee colonies in the future.

Highly divergent cyclo-like virus in a great roundleaf bat (Hipposideros armiger) in Vietnam.

Members of the viral family Circoviridae are increasingly recognized worldwide. Bats seem to be natural reservoirs or dietary-related dispensers of these viruses. Here, we report a distantly related member of the genus Cyclovirus detected in the faeces of a great roundleaf bat (Hipposideros armiger). Interestingly, the novel virus lacks a Circoviridae-specific stem-loop structure, although a Geminiviridae-like nonamer sequence was detected in the large intergenic region. Based on these differences and its phylogenetic position, we propose that our new virus represents a distant and highly divergent member of the genus Cyclovirus. However it is lacking several characteristics of members of the genus, which raises a challenge in its taxonomic classification.

Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (Microtus arvalis).

A novel mammalian orthoreovirus (MRV) strain was isolated from the lung tissue of a common vole (Microtus arvalis) with Tula hantavirus infection. Seven segments (L1-L3, M2-M3, S2, and S4) of the Hungarian MRV isolate MORV/47Ma/06 revealed a high similarity with an MRV strain detected in bank vole (Myodes glareolus) in Germany. The M1 and S3 segment of the Hungarian isolate showed the closest relationship with the sequence of a Slovenian human and a French murine isolate, respectively. The highest nucleotide and amino acid identity values were above 90 and 95% in all of the comparisons to the reference sequences in GenBank, except for the S1 with a maximum of 69.6% nucleotide and 75.4% amino acid identity. As wild rodents are among the main sources of zoonotic infections, the reservoir role of these animals and zoonotic potential of rodent origin MRVs need to be further investigated.

Phylogenetic evidence for a new species of Barbus in the Danube River basin.

Three species of small-sized rheophilic Barbus fishes are endemic to and widely distributed throughout the mountain regions in the Danube River basin. In Hungary, barbels referred to as B. petenyi occur in streams in the foothills of the Carpathians near the borders with Slovakia, Ukraine and Romania. However, up to now, no genetic investigations were carried out on rheophilic barbels in this region. This study aims to clarify the taxonomic identity and distribution of the rheophilic barbels in the Hungarian plain based on molecular and morphological analyses. Two mitochondrial genes (cytochrome b, ATPase 6/8) and one nuclear gene (beta-actin intron 2) were sequenced and several morphometric and meristic characters were recorded. Phylogenetic and morphological analyses revealed that there are four genetically distinct lineages among the rheophilic barbels in the Carpathian Basin. The results demonstrated that North-Hungarian Barbus populations belong to B. carpathicus and that B. petenyi presumably does not occur in Hungary. As expected, B. balcanicus was only recorded in samples from the Balkans analyzed for reference. A distinct species, new to science, was discovered to be present in Sebes-Körös River (Crisul Repede) in eastern Hungary and western Romania and is formally described here as B. biharicus Antal, László, Kotlík - sp. nov.

First molecular identification of Dirofilaria spp. (Onchocercidae) in mosquitoes from Serbia.

Dirofilariosis is a common and widespread veterinary health issue in several European countries with notable zoonotic potential. The causative agents are Dirofilaria immitis and Dirofilaria repens nematoda species which are transmitted by different mosquito vectors. Similar to other mosquito-borne infections, the knowledge about mosquito species involved in disease transmission is crucial for the complex understanding of local transmission cycles. Since there is no available data on mosquito species, potentially involved in disease transmission from Serbia, 6369 female mosquito individuals were retrospectively tested for Dirofilaria nematodes, collected from 13 localities in Vojvodina province, Serbia, in 2013. Altogether, 8.33 % of tested pools showed positivity, composed of five mosquito species, mainly, Culex pipiens and Aedes vexans. D. immitis and D. repens were both detected from multiple localities, during the whole period of mosquito breeding season, which provides the first data on local transmission characteristics regarding mosquitoes from the Balkans.

Emergence of Aedes koreicus (Diptera: Culicidae) in an urban area, Hungary, 2016.

In June 2016, three adult females of Aedes koreicus mosquitoes were trapped in the urban area of Pécs, Southwest Hungary. The introduction of this invasive mosquito species in this region, along with the recent detection in Germany, may indicate the capability of the species to spread across Europe. Along with Aedes albopictus and Aedes japonicus mosquitoes, this is the third invasive mosquito species occurred in Hungary.

Genetic characterization of a novel picornavirus detected in Miniopterus schreibersii bats.

Bats are important reservoirs of many viruses with zoonotic potential worldwide, including Europe. Among bat viruses, members of the Picornaviridae family remain a neglected group. We performed viral metagenomic analyses on Miniopterus schreibersii bat faecal samples, collected in Hungary in 2013. In the present study we report the first molecular data and genomic characterization of a novel picornavirus from the bat species M. schreibersii in Europe. Based on phylogenetic analyses, the novel bat picornaviruses unambiguously belong to the Mischivirus genus and were highly divergent from other bat-derived picornaviruses of the Sapelovirus genus. Although the Hungarian viruses were most closely related to Mischivirus A, they formed a separate monophyletic branch within the genus.

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.

Bacterial Metabarcoding of Raw Palm Sap Samples from Bangladesh with Nanopore Sequencing.

The traditional practice of harvesting and processing raw date palm sap is not only culturally significant but also provides an essential nutritional source in South Asia. However, the potential for bacterial or viral contamination from animals and environmental sources during its collection remains a serious and insufficiently studied risk. Implementing improved food safety measures and collection techniques could mitigate the risk of these infections. Additionally, the adoption of advanced food analytical methods offers the potential to identify pathogens and uncover the natural bacterial diversity of these products. The advancement of next-generation sequencing (NGS) technologies, particularly nanopore sequencing, offers a rapid and highly mobile solution. In this study, we employed nanopore sequencing for the bacterial metabarcoding of a set of raw date palm sap samples collected without protective coverage against animals in Bangladesh in 2021. We identified several bacterial species with importance in the natural fermentation of the product and demonstrated the feasibility of this NGS method in the surveillance of raw palm sap products. We revealed two fermentation directions dominated by either Leuconostoc species or Lactococcus species in these products at the first 6 h from harvest, along with opportunistic human pathogens in the background, represented with lower abundance. Plant pathogens, bacteria with the potential for opportunistic human infection and the sequences of the Exiguobacterium genus are also described, and their potential role is discussed. In this study, we demonstrate the potential of mobile laboratory solutions for food safety purposes in low-resource areas.