Microbead-based extracorporeal immuno-affinity virus capture: a feasibility study to address the SARS-CoV-2 pandemic.

In this paper, we report on the utilization of micro-technology based tools to fight viral infections. Inspired by various hemoperfusion and immune-affinity capture systems, a blood virus depletion device has been developed that offers highly efficient capture and removal of the targeted virus from the circulation, thus decreasing virus load. Single-domain antibodies against the Wuhan (VHH-72) virus strain produced by recombinant DNA technology were immobilized on the surface of glass micro-beads, which were then utilized as stationary phase. For feasibility testing, the virus suspension was flown through the prototype immune-affinity device that captured the viruses and the filtered media left the column. The feasibility test of the proposed technology was performed in a Biosafety Level 4 classified laboratory using the Wuhan SARS-CoV-2 strain. The laboratory scale device actually captured 120,000 virus particles from the culture media circulation proving the feasibility of the suggested technology. This performance has an estimated capture ability of 15 million virus particles by using the therapeutic size column design, representing three times over-engineering with the assumption of 5 million genomic virus copies in an average viremic patient. Our results suggested that this new therapeutic virus capture device could significantly lower virus load thus preventing the development of more severe COVID-19 cases and consequently reducing mortality rate.

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.

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.

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.

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.

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.

Analysis of COVID-19-Related RT-qPCR Test Results in Hungary: Epidemiology, Diagnostics, and Clinical Outcome.

Background: Effective testing is an essential tool for controlling COVID-19. We aimed to analyse the data from first-wave PCR test results in Hungary's Southern Transdanubian region to improve testing strategies. Methods: We performed a retrospective analysis of all suspected COVID-19 cases between 17 March and 8 May 2020, collecting epidemiological, demographic, clinical and outcome data (ICU admission and mortality) with RT-qPCR test results. Descriptive and comparative statistical analyses were conducted. Results: Eighty-six infections were confirmed among 3,657 tested patients. There was no difference between the positive and negative cases in age and sex distribution; however, ICU admission (8.1 vs. 3.1%, p = 0.006) and in-hospital mortality (4.7 vs. 1.6%, p = 0.062) were more frequent among positive cases. Importantly, none of the initially asymptomatic patients (n = 20) required ICU admission, and all survived. In almost all cases, if the first test was negative, second and third tests were performed with a 48-h delay for careful monitoring of disease development. However, the positive hit rate decreased dramatically with the second and third tests compared to the first (0.3 vs. 2.1%, OR = 0.155 [0.053-0.350]). Higher E-gene copy numbers were associated with a longer period of PCR positivity. Conclusion: In our immunologically naïve suspected COVID-19 population, coronavirus infection increased the need for intensive care and mortality by 3-4 times. In the event of the exponential phase of the pandemic involving a bottleneck in testing capacity, a second or third test should be reconsidered to diagnose more coronavirus infections.

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.

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.