Tick-borne encephalitis virus (TBEV) is the most important tick-borne zoonotic virus in Europe. In Belgium, antibodies to TBEV have already been detected in wildlife and domestic animals, but up-to-date prevalence data for TBEV are lacking, and no studies have assessed its seroprevalence in sheep. Serum samples of 480 sheep from all over Belgium and 831 wild boar hunted in Flanders (northern Belgium) were therefore screened for TBEV antibodies by ELISA and plaque reduction neutralization test (PRNT), respectively. The specificity of positive samples was assessed by PRNTs for TBEV and the Louping Ill, West Nile, and Usutu viruses. TBEV seroprevalence was 0.42% (2/480, CI 95%: 0.11-1.51) in sheep and 9.27% (77/831, CI 95%: 7.48-11.43) in wild boar. TBEV seroprevalence in wild boar from the province of Flemish Brabant was significantly higher (22.38%, 15/67) compared to Limburg (7.74%, 34/439) and Antwerp (8.61%, 28/325). Oud-Heverlee was the hunting area harboring the highest TBEV seroprevalence (33.33%, 11/33). In an attempt to obtain a Belgian TBEV isolate, 1983 ticks collected in areas showing the highest TBEV seroprevalence in wild boars were tested by real-time qPCR. No TBEV-RNA-positive tick was detected. The results of this study suggest an increase in TBEV prevalence over the last decade and highlight the need for One-Health surveillance in Belgium.

Respiratory Syncytial Virus (RSV) is a very important viral pathogen in children, immunocompromised and cardiopulmonary diseased patients and the elderly. Most of the published research with RSV was performed on RSV Long and RSV A2, isolated in 1956 and 1961, yet recent RSV isolates differ from these prototype strains. Additionally, these viruses have been serially passaged in cell culture, which may result in adaptations that affect virus-host interactions. We have isolated RSV from mucosal secretions of 12 patients in the winters 2016-2017 and 2017-2018, of which eight RSV-A subtypes and four RSV-B subtypes. Passage 3 of the isolates was assessed for viral replication kinetics and infectious virus production in HEp-2, A549 and BEAS-2B cells, thermal stability at 37 °C, 32 °C and 4 °C, syncytia formation, neutralization by palivizumab and mucin mRNA expression in infected A549 cells. We observed that viruses isolated in one RSV season show differences on the tested assays. Furthermore, comparison with RSV A2 and RSV B1 reveals for some RSV isolates differences in viral replication kinetics, thermal stability and fusion capacity. Major differences are, however, not observed and differences between the recent isolates and reference strains is, overall, similar to the observed variation in between the recent isolates. One clinical isolate (BE/ANT-A11/17) replicated very efficiently in all cell lines, and remarkably, even better than RSV A2 in the HEp-2 cell line.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca2+ and Mg2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6-9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days.

Little cherry disease, caused by little cherry virus 1 (LChV-1) and little cherry virus 2 (LChV-2), which are both members of the family Closteroviridae, severely affects sweet (Prunus avium L.) and sour cherry (P. cerasus L.) orchards lifelong production worldwide. An intensive survey was conducted across different geographic regions of Belgium to study the disease presence on these perennial woody plants and related species. Symptomatic as well as non-symptomatic Prunus spp. trees tested positive via RT-PCR for LChV-1 and -2 in single or mixed infections, with a slightly higher incidence for LChV-1. Both viruses were widespread and highly prevalent in nearly all Prunus production areas as well as in private gardens and urban lane trees. The genetic diversity of Belgian LChV-1 and -2 isolates was assessed by Sanger sequencing of partial genomic regions. A total RNA High-Throughput Sequencing (HTS) approach confirmed the presence of both viruses, and revealed the occurrence of other Prunus-associated viruses, namely cherry virus A (CVA), prune dwarf virus (PDV) and prunus virus F (PrVF). The phylogenetic inference from full-length genomes revealed well-defined evolutionary phylogroups with high genetic variability and diversity for LChV-1 and LChV-2 Belgian isolates, yet with little or no correlation with planting area or cultivated varieties. The global diversity and the prevalence in horticultural areas of LChV-1 and -2 variants, in association with other recently described fruit tree viruses, are of particular concern. Future epidemiological implications as well as new investigation avenues are exhaustively discussed.

To mitigate the massive COVID-19 burden caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccination campaigns were initiated. We performed a single-center observational trial to monitor the mid- (3 months) and long-term (10 months) adaptive immune response and to document breakthrough infections (BTI) in healthcare workers (n = 84) upon BNT162b2 vaccination in a real-world setting. Firstly, serology was determined through immunoassays. Secondly, antibody functionality was analyzed via in vitro binding inhibition and pseudovirus neutralization and circulating receptor-binding domain (RBD)-specific B cells were assessed. Moreover, the induction of SARS-CoV-2-specific T cells was investigated by an interferon-γ release assay combined with flowcytometric profiling of activated CD4+ and CD8+ T cells. Within individuals that did not experience BTI (n = 62), vaccine-induced humoral and cellular immune responses were not correlated. Interestingly, waning over time was more pronounced within humoral compared to cellular immunity. In particular, 45 of these 62 subjects no longer displayed functional neutralization against the delta variant of concern (VoC) at long-term follow-up. Noteworthily, we reported a high incidence of symptomatic BTI cases (17.11%) caused by alpha and delta VoCs, although vaccine-induced immunity was only slightly reduced compared to subjects without BTI at mid-term follow-up.

The National Reference Center for Hantavirus in Belgium is currently using the Hantavirus IgM/IgG ELISA Progen kit (Heidelberg, Germany) for the detection of the most prevalent Hantavirus in Western Europe, Puumala virus (PUUV). Two commercially available PUUV kits were compared: Progen and RIDASCREEN® Hantavirus Puumala IgM/IgG ELISA assay (Darmstadt, Germany).

Since time immemorial, phages-the viral parasites of bacteria-have been protecting Earth's biosphere against bacterial overgrowth. Today, phages could help address the antibiotic resistance crisis that affects all of society. The greatest hurdle to the introduction of phage therapy in Western medicine is the lack of an appropriate legal and regulatory framework. Belgium is now implementing a pragmatic phage therapy framework that centers on the magistral preparation (compounding pharmacy in the US) of tailor-made phage medicines.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compromises the ability of military forces to fulfill missions. At the beginning of May 2020, 22 out of 70 Belgian soldiers deployed to a military education and training center in Maradi, Niger, developed mild COVID-19 compatible symptoms. Immediately upon their return to Belgium, and two weeks later, all seventy soldiers were tested for SARS-CoV-2 RNA (RT-qPCR) and antibodies (two immunoassays). Nine soldiers had at least one positive COVID-19 diagnostic test result. Five of them exhibited COVID-19 symptoms (mainly anosmia, ageusia, and fever), while four were asymptomatic. In four soldiers, SARS-CoV-2 viral load was detected and the genomes were sequenced. Conventional and genomic epidemiological data suggest that these genomes have an African most recent common ancestor and that the Belgian military service men were infected through contact with locals. The medical military command implemented testing of all Belgian soldiers for SARS-CoV-2 viral load and antibodies, two to three days before their departure on a mission abroad or on the high seas, and for specific missions immediately upon their return in Belgium. Some military operational settings (e.g., training camps in austere environments and ships) were also equipped with mobile infectious disease (COVID-19) testing capacity.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee (Apis mellifera) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites (Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>107 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

More than a year after the first identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of the 2019 coronavirus disease (COVID-19) in China, the emergence and spread of genomic variants of this virus through travel raise concerns regarding the introduction of lineages in previously unaffected regions, requiring adequate containment strategies. Concomitantly, such introductions fuel worries about a possible increase in transmissibility and disease severity, as well as a possible decrease in vaccine efficacy. Military personnel are frequently deployed on missions around the world. As part of a COVID-19 risk mitigation strategy, Belgian Armed Forces that engaged in missions and operations abroad were screened (7683 RT-qPCR tests), pre- and post-mission, for the presence of SARS-CoV-2, including the identification of viral lineages. Nine distinct viral genotypes were identified in soldiers returning from operations in Niger, the Democratic Republic of the Congo, Afghanistan, and Mali. The SARS-CoV-2 variants belonged to major clades 19B, 20A, and 20B (Nextstrain nomenclature), and included "variant of interest" B.1.525, "variant under monitoring" A.27, as well as lineages B.1.214, B.1, B.1.1.254, and A (pangolin nomenclature), some of which are internationally monitored due to the specific mutations they harbor. Through contact tracing and phylogenetic analysis, we show that isolation and testing policies implemented by the Belgian military command appear to have been successful in containing the influx and transmission of these distinct SARS-CoV-2 variants into military and civilian populations.

HIV-1 pol sequences obtained through baseline drug resistance testing of patients newly diagnosed between 2013 and 2017 were analyzed for genetic similarity. For 927 patients the information on genetic similarity was combined with demographic data and with information on the recency of infection. Overall, 48.3% of the patients were genetically linked with 11.4% belonging to a pair and 36.9% involved in a cluster of ≥3 members. The percentage of early diagnosed (≤4 months after infection) was 28.6%. Patients of Belgian origin were more frequently involved in transmission clusters (49.7% compared to 15.3%) and diagnosed earlier (37.4% compared to 12.2%) than patients of Sub-Saharan African origin. Of the infections reported to be locally acquired, 69.5% were linked (14.1% paired and 55.4% in a cluster). Equal parts of early and late diagnosed individuals (59.9% and 52.4%, respectively) were involved in clusters. The identification of a genetically linked individual for the majority of locally infected patients suggests a high rate of diagnosis in this population. Diagnosis however is often delayed for >4 months after infection increasing the opportunities for onward transmission. Prevention of local infection should focus on earlier diagnosis and protection of the still uninfected members of sexual networks with human immunodeficiency virus (HIV)-infected members.

Double-stranded RNA (dsRNA) molecules of viral origin trigger a post-transcriptional gene-silencing mechanism called RNA interference (RNAi). Specifically, virally derived dsRNA is recognized and cleaved by the enzyme Dicer2 into short interfering RNAs (siRNAs), which further direct sequence-specific RNA silencing, ultimately silencing replication of the virus. Notably, RNAi can also be artificially triggered by the delivery of gene-specific dsRNA, thereby leading to endogenous gene silencing. This is a widely used technology that holds great potential to contribute to novel pest control strategies. In this regard, research efforts have been set to find methods to efficiently trigger RNAi in the field. In this article, we demonstrate the generation of dsRNA- and/or virus-derived siRNAs-the main RNAi effectors-in six insect species belonging to five economically important orders (Lepidoptera, Orthoptera, Hymenoptera, Coleoptera, and Diptera). In addition, we describe that the siRNA length distribution is species-dependent. Taken together, our results reveal interspecies variability in the (antiviral) RNAi mechanism in insects and show promise to contribute to future research on (viral-based) RNAi-triggering mechanisms in this class of animals.

In times where only a few novel antibiotics are to be expected, antimicrobial resistance remains an expanding global health threat. In case of chronic infections caused by therapy-resistant pathogens, physicians have limited therapeutic options, which are often associated with detrimental consequences for the patient. This has resulted in a renewed interest in alternative strategies, such as bacteriophage (phage) therapy. However, there are still important hurdles that currently impede the more widespread implementation of phage therapy in clinical practice. First, the limited number of good-quality case series and clinical trials have failed to show the optimal application protocol in terms of route of administration, frequency of administration, treatment duration and phage titer. Second, there is limited information on the systemic effects of phage therapy. Finally, in the past, phage therapy has been applied intuitively in terms of the selection of phages and their combination as parts of phage cocktails. This has led to an enormous heterogeneity in previously published studies, resulting in a lack of reliable safety and efficacy data for phage therapy. We hereby present a study protocol that addresses these scientific hurdles using a multidisciplinary approach, bringing together the experience of clinical, pharmaceutical and molecular microbiology experts.

Genetic analyses play a central role in infectious disease research. Massively parallelized "mechanical cloning" and sequencing technologies were quickly adopted by HIV researchers in order to broaden the understanding of the clinical importance of minor drug-resistant variants. These efforts have, however, remained largely limited to small genomic regions. The growing need to monitor multiple genome regions for drug resistance testing, as well as the obvious benefit for studying evolutionary and epidemic processes makes complete genome sequencing an important goal in viral research. In addition, a major drawback for NGS applications to RNA viruses is the need for large quantities of input DNA. Here, we use a generic overlapping amplicon-based near full-genome amplification protocol to compare low-input enzymatic fragmentation (Nextera™) with conventional mechanical shearing for Roche 454 sequencing. We find that the fragmentation method has only a modest impact on the characterization of the population composition and that for reliable results, the variation introduced at all steps of the procedure--from nucleic acid extraction to sequencing--should be taken into account, a finding that is also relevant for NGS technologies that are now more commonly used. Furthermore, by applying our protocol to deep sequence a number of pre-therapy plasma and PBMC samples, we illustrate the potential benefits of a near complete genome sequencing approach in routine genotyping.

Countries rely on good diagnostic tests and appropriate testing schemes to fight against economically important small ruminant lentivirus (SRLV) infections. We undertook an extensive comparative analysis of seven commercially available serological tests and one in-house real-time PCR (qPCR) detecting genotype A and B strains using a large panel of representative Belgian field samples and samples from experimentally infected sheep and goats. ELISAs generally performed well and detected seroconversion within three weeks post experimental infection. Two enzyme-linked immunosorbent assays (ELISAs) (Elitest and IDscreen® kits) showed the highest sensitivities (>96%) and specificities (>95%) in both species, and their combined use allowed to correctly identify the infection status of all animals. Individual agar gel immunodiffusion (AGIDs) kits lacked sensitivity, but interestingly, the combined use of both kits had a sensitivity and specificity of 100%. qPCRs detected SRLV infection before seroconversion at two weeks post infection and showed a specificity of 100%. Sensitivity however remained suboptimal at 85%. These results allow to propose a faster and cheaper diagnostic testing strategy for Belgium by combining a first ELISA screening, followed by confirmation of positive samples in AGID and/or a second ELISA. Since genotypes A and B strains are predominant in many countries, these results are interesting for other countries implementing SRLV control programs.

Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.

Bacteriophage therapy has recently attracted increased interest, particularly in difficult-to-treat infections. Although it is not a novel concept, standardized treatment guidelines are currently lacking. We present the first steps towards the establishment of a "multidisciplinary phage task force" (MPTF) and a standardized treatment pathway, based on our experience of four patients with severe musculoskeletal infections. After review of their medical history and current clinical status, a multidisciplinary team found four patients with musculoskeletal infections eligible for bacteriophage therapy within the scope of Article 37 of the Declaration of Helsinki. Treatment protocols were set up in collaboration with phage scientists and specialists. Based on the isolated pathogens, phage cocktails were selected and applied intraoperatively. A draining system allowed postoperative administration for a maximum of 10 days, 3 times per day. All patients received concomitant antibiotics and their clinical status was followed daily during phage therapy. No severe side-effects related to the phage application protocol were noted. After a single course of phage therapy with concomitant antibiotics, no recurrence of infection with the causative strains occurred, with follow-up periods ranging from 8 to 16 months. This study presents the successful outcome of bacteriophage therapy using a standardized treatment pathway for patients with severe musculoskeletal infection. A multidisciplinary team approach in the form of an MPTF is paramount in this process.

Chikungunya and yellow fever virus cause vector-borne viral diseases in humans. There is currently no specific antiviral drug for either of these diseases. Banana plants are used in traditional medicine for treating viral diseases such as measles and chickenpox. Therefore, we tested selected banana cultivars for their antiviral but also cytotoxic properties. Different parts such as leaf, pseudostem and corm, collected separately and extracted with four different solvents (hexane, acetone, ethanol, and water), were tested for in vitro antiviral activity against Chikungunya virus (CHIKV), enterovirus 71 (EV71), and yellow fever virus (YFV). Extracts prepared with acetone and ethanol from leaf parts of several cultivars exhibited strong (EC50 around 10 μg/mL) anti-CHIKV activity. Interestingly, none of the banana plant extracts (concentration 1-100 µg/mL) were active against EV71. Activity against YFV was restricted to two cultivars: Namwa Khom-Pseudostem-Ethanol (5.9 ± 5.4), Namwa Khom-Corm-Ethanol (0.79 ± 0.1) and Fougamou-Corm-Acetone (2.5 ± 1.5). In most cases, the cytotoxic activity of the extracts was generally 5- to 10-fold lower than the antiviral activity, suggesting a reasonable therapeutic window.

Metagenomics studies have accelerated the discovery of novel or divergent viruses of the honey bee. However, most of these studies predominantly focused on RNA viruses, and many suffer from the relatively low abundance of viral nucleic acids in the samples (i.e., compared to that of the host). Here, we explored the virome of the Ethiopian honey bee, Apis mellifera simensis, using an unbiased metagenomic approach in which the next-generation sequencing step was preceded by an enrichment protocol for viral particles. Our study revealed five well-known bee viruses and 25 atypical virus species, most of which have never been found in A. mellifera before. The viruses belong to Iflaviridae, Dicistroviridae, Secoviridae, Partitiviridae, Parvoviridae, Potyviridae, and taxonomically unclassified families. Fifteen of these atypical viruses were most likely plant-specific, and the remaining ten were presumed to be insect-specific. Apis mellifera filamentous virus (AmFV) was found in one sampling site out of 10. Two samples contained high read counts of a virus similar to Diatraea saccharales densovirus (DsDNV), which is a virus that causes high mortality in the sugarcane borer. AmFV and the DsDNV-like virus were the only DNA viruses found. Three viruses that primarily infect Drosophila spp. were also discovered: La Jolla virus (LJV), Kilifi virus (KiV), and Thika virus. Our study suggests that phoretic varroa mites are involved in the transmission of LJV and KiV and that both viruses replicate in mites and adult bees. We also found an overwhelming dominance of the deformed wing virus type B variant, which fits well with the apparently harmless infestation by Varroa destructor. It was suggested that Ethiopian bees have developed tolerance against virus infections as the result of natural selection.