Closing the Rift: Discovery of a novel virus receptor.

In this issue of Cell, Ganaie et al. reports the identification of LRP1 as a receptor of the highly pathogenic Rift Valley fever virus. By using genome-wide CRISPR-Cas9 screening and functional studies, Ganaie et al. identified LRP1 and several co-factors as essential elements for virus infection.

A trans-complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence.

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.

Loss of furin cleavage site attenuates SARS-CoV-2 pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

Nucleocapsid mutations in SARS-CoV-2 augment replication and pathogenesis.

While SARS-CoV-2 continues to adapt for human infection and transmission, genetic variation outside of the spike gene remains largely unexplored. This study investigates a highly variable region at residues 203-205 in the SARS-CoV-2 nucleocapsid protein. Recreating a mutation found in the alpha and omicron variants in an early pandemic (WA-1) background, we find that the R203K+G204R mutation is sufficient to enhance replication, fitness, and pathogenesis of SARS-CoV-2. The R203K+G204R mutant corresponds with increased viral RNA and protein both in vitro and in vivo. Importantly, the R203K+G204R mutation increases nucleocapsid phosphorylation and confers resistance to inhibition of the GSK-3 kinase, providing a molecular basis for increased virus replication. Notably, analogous alanine substitutions at positions 203+204 also increase SARS-CoV-2 replication and augment phosphorylation, suggesting that infection is enhanced through ablation of the ancestral 'RG' motif. Overall, these results demonstrate that variant mutations outside spike are key components in SARS-CoV-2's continued adaptation to human infection.

Can we screen for heart disease in children at public health centres? A multicentre observational study of screening for heart disease with a risk of sudden death in children.

Sudden cardiac death in children is a rare event, but of great social significance. Generally, it is related to heart disease with a risk of sudden cardiac death (SCD), which may occur with cardiovascular symptoms and/or electrocardiographic markers; thus, a primary care paediatrician (PCP) could detect them. Therefore, we proposed a study that assesses how to put into practice and conduct a cardiovascular assessment within the routine healthy-child check-ups at six and twelve years of age; that reflects cardiovascular signs and symptoms, as well as the electrocardiographic alterations that children with a risk of SCD in the selected population present; and that assesses the PCP's skill at electrocardiogram (ECG) interpretation. In collaboration with PCPs, primary care nurses, and paediatric cardiologists, an observational, descriptive, multicentre, cross-sectional study was carried out in the Balearic Islands (Spain), from April 2021 to January 2022, inclusive. The PCPs gathered patient data through forms (medical record, electrocardiogram, and physical examination) and sent them to the investigator, together with the informed consent document and electrocardiogram. The investigator passed the electrocardiogram on to the paediatric cardiologists for reading, in an identical form to those the paediatricians had filled in. The variables were collected, and a descriptive analysis performed. Three paediatric cardiologists, twelve PCPs, and nine nurses from seven public health centres took part. They collected the data from 641 patients, but 233 patients did not participate (in 81.11% due to the PCP's workload). Therefore, the study coverage was around 64%, representing the quotient of the total number of patients who participated, divided by the total number of patients who were eligible for the study. We detected 30 patients with electrocardiographic alterations compatible with SCD risk. Nine of these had been examined by a paediatric cardiologist at some time (functional murmur in 8/9), five had reported shortness of breath with exercise, and four had reported a family history of sudden death. The physical examination of all the patients whose ECG was compatible with a risk of SCD was normal. Upon analysing to what extent the ECG results of the PCP and the paediatric cardiologist agreed, the percentage of agreement in the final interpretation (normal/altered) was 91.9%, while Cohen's kappa coefficient was 31.2% (CI 95%: 13.8-48.6%). The sensitivity of the ECG interpretation by the PCP to detect an ECG compatible with a risk of SCD was 29% and the positive predictive value 45%.     Conclusions: This study lays the foundations for future SCD risk screening in children, performed by PCPs. However, previously, it would be important to optimise their training in reading and interpreting paediatric ECGs. What is Known: • In Spain at present, there is a programme in place to detect heart disease with a risk of sudden death [1], but it targets only children who are starting on or are doing a physical activity as a federated sport. Implementing such screening programmes has proven effective in several countries [2]. However, several studies showed that the incidence of sudden cardiac death is no higher in children competing in sport activities than in those who do not do any sport [3]. This poses an ethical conflict, because at present, children who do not do any federated sport are excluded from screening. According to the revised literature, so far, only in two studies did they screen the child population at schools, and in both, they successfully detected patients with heart disease associated to the risk of sudden death [4, 5]. We have found no studies where the screening of these features was included within the routine healthy-child check-ups by primary care paediatricians. What is New: • We did not know whether-in our setting, at present-the primary care paediatrician could perform a screening method within the routine healthy-child check-ups, in order to detect presumably healthy children at risk of sudden cardiac death, as they present one of the SCD risks. In this regard, we proposed our project: to assess how to put into practice and conduct a cardiovascular assessment via SCD risk screening in the healthy child population by primary care paediatricians and appraise primary care paediatricians' skills in identifying the electrocardiographic alterations associated with SCD risk. The ultimate intention of this pilot study was to make it possible, in the future, to design and justify a study aimed at universalising cardiovascular screening and achieving a long-term decrease in sudden cardiac death events in children.

A multi-centre randomized controlled trial investigating Consolidation Chemotherapy with and without oxaliplatin in distal rectal cancer and Watch & Wait.

BACKGROUND: Neoadjuvant chemoradiation(nCRT) has been considered the preferred initial treatment strategy for distal rectal cancer. Advantages of this approach include improved local control after radical surgery but also the opportunity for organ preserving strategies (Watch and Wait-WW). Consolidation chemotherapy(cCT) regimens using fluoropyrimidine-based with or without oxalipatin following nCRT have demonstrated to increase complete response and organ preservation rates among these patients. However, the benefit of adding oxaliplatin to cCT compared to fluoropirimidine alone regimens in terms of primary tumor response remains unclear. Since oxalipatin-treatment may be associated with considerable toxicity, it becomes imperative to understand the benefit of its incorporation into standard cCT regimens in terms of primary tumor response. The aim of the present trial is to compare the outcomes of 2 different cCT regimens following nCRT (fluoropyrimidine-alone versus fluoropyrimidine + oxaliplatin) for patients with distal rectal cancer. METHODS: In this multi-centre study, patients with magnetic resonance-defined distal rectal tumors will be randomized on a 1:1 ratio to receive long-course chemoradiation (54 Gy) followed by cCT with fluoropyrimidine alone versus fluoropyrimidine + oxaliplatin. Magnetic resonance(MR) will be analyzed centrally prior to patient inclusion and randomization. mrT2-3N0-1 tumor located no more than 1 cm above the anorectal ring determined by sagittal views on MR will be eligible for the study. Tumor response will be assessed after 12 weeks from radiotherapy(RT) completion. Patients with clinical complete response (clinical, endoscopic and radiological) may be enrolled in an organ-preservation program(WW). The primary endpoint of this trial is decision to organ-preservation surveillance (WW) at 18 weeks from RT completion. Secondary endpoints are 3-year surgery-free survival, TME-free survival, distant metastases-free survival, local regrowth-free survival and colostomy-free survival. DISCUSSION: Long-course nCRT with cCT is associated with improved complete response rates and may be a very attractive alternative to increase the chances for organ-preservation strategies. Fluoropyrimidine-based cCT with or without oxaliplatin has never been investigated in the setting of a randomized trial to compare clinical response rates and the possibility of organ-preservation. The outcomes of this study may significantly impact clinical practice of patients with distal rectal cancer interested in organ-preservation. TRIAL REGISTRATION: www. CLINICALTRIALS: gov NCT05000697; registered on August 11th, 2021.

Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 in Aerosol Suspensions.

We aerosolized severe acute respiratory syndrome coronavirus 2 and determined that its dynamic aerosol efficiency surpassed those of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome. Although we performed experiment only once across several laboratories, our findings suggest retained infectivity and virion integrity for up to 16 hours in respirable-sized aerosols.

Separation of influenza virus-like particles from baculovirus by polymer-grafted anion exchanger.

The baculovirus expression vector system is a very powerful tool to produce virus-like particles and gene-therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for wider industrial use. We used chimeric human immunodeficiency virus-1 (HIV-1) gag influenza-hemagglutin virus-like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus-like particles. A fast and simple purification method for these virus-like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer-grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus-like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography-mass spectrometry. When considering a vaccination dose of 109 particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double-stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus-like particles.

Organ Preservation in cT2N0 Rectal Cancer After Neoadjuvant Chemoradiation Therapy: The Impact of Radiation Therapy Dose-escalation and Consolidation Chemotherapy.

OBJECTIVE: To demonstrate the difference in organ-preservation rates and avoidance of definitive surgery among cT2N0 rectal cancer patients undergoing 2 different chemoradiation (CRT) regimens. BACKGROUND: Patients with cT2N0 rectal cancer are more likely to develop complete response to neoadjuvant CRT. Organ preservation has been considered an alternative treatment strategy for selected patients. Radiation dose-escalation and consolidation chemotherapy have been associated with increased rates of response and may improve chances of organ preservation among these patients. METHODS: Patients with distal and nonmetastatic cT2N0 rectal cancer managed by neoadjuvant CRT were retrospectively reviewed. Patients undergoing standard CRT (50.4 Gy and 2 cycles of 5-FU-based chemotherapy) were compared with those undergoing extended CRT (54 Gy and 6 cycles of 5-FU-based chemotherapy). Patients were assessed for tumor response at 8 to 10 weeks. Patients with complete clinical response (cCR) underwent organ-preservation strategy ("Watch and Wait"). Patients were referred to salvage surgery in the event of local recurrence during follow-up. RESULTS: Thirty-five patients underwent standard and 46 patients extended CRT. Patients undergoing extended CRT were more likely to undergo organ preservation and avoid definitive surgical resection at 5years (67% vs 30%; P = 0.001). After development of a cCR, surgery-free survival is similar between extended and standard CRT groups at 5 years (78% vs 56%; P = 0.12). CONCLUSIONS: Dose-escalation and consolidation chemotherapy leads to increased long-term organ-preservation rates among cT2N0 rectal cancer. After achievement of a cCR, the risk for local recurrence and need for salvage surgery is similar, irrespective of the CRT regimen.

At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification.

At-line static light scattering and fluorescence monitoring allows direct in-process tracking of fluorescent virus-like particles. We have demonstrated this by coupling at-line multi-angle light scattering and fluorescence detectors to the downstream processing of enveloped virus-like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus-like particles containing the Human Immunodeficiency Virus-1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single-column anion-exchange chromatography method was used for direct capture and purification. The majority of host-cell protein impurities passed through the column without binding. Virus-like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi-angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus-like particles were concentrated 17-fold to final a concentration of 4.45 × 1010 particles/mL. Simple buffers and operation make this process suitable for large scale purposes.

Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus.

UNLABELLED: Severe fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genus Phlebovirus in the family Bunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12 to 30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the beta interferon (IFN-ß) promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus-infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens. IMPORTANCE: SFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We sought to determine whether these "infectious" extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point toward a novel mechanism for virus replication among arthropod-borne viruses.

Itaya virus, a Novel Orthobunyavirus Associated with Human Febrile Illness, Peru.

Our genetic analyses of uncharacterized bunyaviruses isolated in Peru identified a possible reassortant virus containing small and large gene segment sequences closely related to the Caraparu virus and a medium gene segment sequence potentially derived from an unidentified group C orthobunyavirus. Neutralization tests confirmed serologic distinction among the newly identified virus and the prototype and Caraparu strains. This virus, named Itaya, was isolated in 1999 and 2006 from febrile patients in the cities of Iquitos and Yurimaguas in Peru. The geographic distance between the 2 cases suggests that the Itaya virus could be widely distributed throughout the Amazon basin in northeastern Peru. Identification of a new Orthobunyavirus species that causes febrile disease in humans reinforces the need to expand viral disease surveillance in tropical regions of South America.

Hijacking of RIG-I signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type I interferon responses.

UNLABELLED: Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family results in the activation of type I interferon (IFN) responses. To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural protein NSs of the newly described severe fever with thrombocytopenia syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the beta interferon (IFN-ß) promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of the Atg7 gene, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures colocalize with Rab5 but not with Golgi apparatus or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and point toward a novel mechanism for the suppression of IFN responses. IMPORTANCE: The mechanism by which the newly described SFTSV inhibits host antiviral responses has not yet been fully characterized. In this study, we describe the redistribution of RIG-I signaling components into virus-induced cytoplasmic structures in cells infected with SFTSV. This redistribution correlates with the inhibition of host antiviral responses. Further characterization of the interplay between the viral protein and components of the IFN responses could potentially provide targets for the rational development of therapeutic interventions.

Arboretum and Puerto Almendras viruses: two novel rhabdoviruses isolated from mosquitoes in Peru.

Arboretum virus (ABTV) and Puerto Almendras virus (PTAMV) are two mosquito-associated rhabdoviruses isolated from pools of Psorophora albigenu and Ochlerotattus fulvus mosquitoes, respectively, collected in the Department of Loreto, Peru, in 2009. Initial tests suggested that both viruses were novel rhabdoviruses and this was confirmed by complete genome sequencing. Analysis of their 11 482 nt (ABTV) and 11 876 (PTAMV) genomes indicates that they encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with an additional gene (U1) encoding a small hydrophobic protein. Evolutionary analysis of the L protein indicates that ABTV and PTAMV are novel and phylogenetically distinct rhabdoviruses that cannot be classified as members of any of the eight currently recognized genera within the family Rhabdoviridae, highlighting the vast diversity of this virus family.

Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors.

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth decade of life.

Robust and resource-efficient production process suitable for large-scale production of baculovirus through high cell density seed train and optimized infection strategy.

The aim of this study was the development of a scalable production process for high titer (108 pfu/mL and above) recombinant baculovirus stocks with low cell line-derived impurities for the production of virus-like particles (VLP). To achieve this, we developed a high cell density (HCD) culture for low footprint cell proliferation, compared different infection strategies at multiplicity of infection (MOI) 0.05 and 0.005, different infection strategies and validated generally applicable harvest criteria of cell viability ≤ 80%. We also investigated online measurable parameters to observe the baculovirus production. The infection strategy employing a very low virus inoculum of MOI 0.005 and a 1:2 dilution with fresh medium one day after infection proved to be the most resource efficient. There, we achieved higher cell-specific titers and lower host cell protein concentrations at harvest than other tested infection strategies with the same MOI, while saving half of the virus stock for infecting the culture compared to other tested infection strategies. HCD culture by daily medium exchange was confirmed as suitable for seed train propagation, infection, and baculovirus production, equally efficient as the conventionally propagated seed train. Online measurable parameters for cell concentration and average cell diameter were found to be effective in monitoring the production process. The study concluded that a more efficient VLP production process in large scale can be achieved using this virus stock production strategy, which could also be extended to produce other proteins or extracellular vesicles with the baculovirus expression system.

Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells.

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

Virome analyses of Amblyomma cajennense and Rhipicephalus microplus ticks collected in Colombia.

Tick-borne viruses (TBV) have gained public health relevance in recent years due to the recognition of human-associated fatal cases and the increase in tick-borne disease and transmission. However, many tick species have not been studied for their potential to transmit pathogenic viruses, especially those found in Latin America. To gain better understanding of the tick virome, we conducted targeted amplification using broadly-reactive consensus-degenerate pan-viral targeting viruses from the genera Flavivirus, Bandavirus, Uukuvirus, and Orthonairovirus genus. Additionally, we conducted unbiased metagenomic analyses to investigate the presence of viral RNA sequences in Amblyomma cajennense, A. patinoi and Rhipicephalus microplus ticks collected from a horse slaughter plant in Medellín, Colombia. While no viral products were detected by PCR, results of the metagenomic analyses revealed the presence of viral genomes belonging to the genera Phlebovirus, Bandavirus, and Uukuvirus, including Lihan Tick Virus (LTV), which was previously reported in Rhipicephalus microplus from Colombia. Overall, the results emphasized the enormous utility of the next-generation sequencing in identifying virus genetic diversity presents in ticks and other species of vectors and reservoirs.