Biology subjects

Vasilakis, N.

Publications and source records attributed to Vasilakis, N..

16 recordsLinked to original sources

bioRxiv2026

Systematic genome analysis enables long-overdue taxonomic reorganization of genus Orthoflavivirus; potential assignment of over 50 new species

Members of the Orthoflavivirus genus in the family Flaviviridae are primarily vector-transmitted RNA viruses infecting humans, other mammals and birds, including major pathogens such as tick borne encephalitis, West Nile, Zika and dengue viruses. Vector-borne flaviviruses are assigned to the Euflavivirus subgenus of orthoflaviviruses, with more divergent largely unclassified insect-specific flaviviruses, including cell fusing agent virus assigned to the second subgenus, Fusivirus. The currently classified 57 orthoflavivirus species have traditionally been assigned descriptively on the basis of their genome and antigenic relatedness, ecology and host and vector tropisms, although many more orthoflavi-like viruses remain unassigned. To investigate whether species might be demarcated using a fixed genetic divergence threshold, we examined distributions of pairwise distances in NS5 and E genes (which encode the RNA-dependent RNA polymerase and envelope protein, respectively) of classified orthoflaviviruses. Demarcation thresholds of 0.1 in the two regions would be most parsimonious with current euflavivirus classification. Exceptions include species in the tick-borne encephalitis virus complex with divergences below the threshold, and dengue virus types currently assigned to a single species that are substantially above. Application of the proposed thresholds would provide the means to establish 25 new euflavivirus and 33 new fusivirus species and reconcile several anomalies and inconsistencies in the current species taxonomy. The use of a species demarcation threshold brings consistency with the classification of viruses in other families. A robust and evolutionarily consistent framework furthermore enables taxonomic assignments of the numerous currently unassigned orthoflaviviruses that lack the phenotypic descriptions required for the current species definition.

microbiology↗

bioRxiv2026

An Integrated Computational Antigen Discovery Pipeline with Hierarchical Filtering for Emerging Viral Variants

Emerging and evolving viral diseases, such as SARS-CoV-2, continue to pose significant global health challenges, underscoring the urgent need for rapid and scalable antigen discovery pipelines. This work presents a computational pipeline that integrates diverse computational tools and machine learning models to accelerate the identification and optimization of antigen candidates. The pipeline employs efficient filtering and consensus-based strategies to highlight epitopes with high therapeutic potential. We demonstrate its utility by significantly narrowing the antigen search space for Rift Valley fever virus (RVFV) and Mayaro virus (MAYV), and by effectively identifying conserved neutralizing epitopes in SARS-CoV-2. Our proposed computational antigen pipeline offers a powerful framework for expediting the development of future vaccines and therapeutics in response to emerging pathogens.

bioinformatics↗

bioRxiv2025

Inside-Out: Modeling the link between Zika virus viral dynamics within hosts and transmission to vectors across host species and virus strains

Epidemiological models of mosquito-borne virus transmission often lack accurate estimates of host-to-vector transmission probability. Here, we estimated this probability for two strains of Zika virus (ZIKV)--one sylvatic and one human-endemic--from two monkey species to Aedes albopictus mosquitoes using experimental infection data. Viral dynamics did not differ between monkey species, although one (cynomolgus macaque) is a native ZIKV host and the other (squirrel monkey) a novel host, but differed between strains, with viremia for the human-endemic strain peaking later and lower than the sylvatic strain. Only the sylvatic strain was transmitted to mosquitoes. In mosquitoes, anatomical barriers influence viral progression to salivary glands, complicating host infectiousness estimation. We quantified the probability of viral dissemination to the legs in Ae. albopictus, which increased with host viral load and was higher after feeding on squirrel monkeys than on cynomolgus macaques. We also found a positive relationship between virus titer in mosquito legs and virus detection in saliva after a 14-day extrinsic incubation period. Combining these factors, we found that squirrel monkeys were on average 1.5 times more infectious to Ae. albopictus than cynomolgus macaques. These estimates will help assess ZIKVs potential to establish an enzootic, sylvatic cycle in the Americas.

systems biology↗

bioRxiv2025

Human herpesvirus 1 associated with epizootics in Belo Horizonte, Minas Gerais, Brazil

Human activity in sylvatic environments and resulting contact with wildlife, such as non-human primates (NHP), can lead to pathogen spillover or spillback. Both NHPs and humans host a variety of herpesviruses. While these viruses typically cause asymptomatic infections in their natural hosts, they can lead to severe disease or even death when they move into novel hosts. In early 2024, deaths of Callithrix penicillata, the black-tufted marmoset, were reported in an urban park in Belo Horizonte, Minas Gerais, Brazil. The epizootic was investigated in collaboration with CETAS/IBAMA and the Zoonoses Department of Belo Horizonte. Nine marmoset carcasses, and 4 sick marmosets were found in the park; the latter exhibited severe neurological symptoms and systemic illness before succumbing within 48 hours. Carcasses were tested for rabies virus and were all negative, and necropsy findings revealed widespread organ damage. In addition, the samples were tested for yellow fever virus, with negative results. Finally, molecular testing, viral isolation and phylogenetic analysis demonstrated human herpesvirus 1 (HHV-1) as the causative agent. The likely source of infection was human-to-marmoset transmission, facilitated by close interactions such as feeding and handling. This study highlights the risks of pathogen spillover between humans and nonhuman primates, emphasizing the need for enhanced surveillance and public awareness to mitigate future epizootics.

microbiology↗

bioRxiv2025

Integrated analysis of protein sequence and structureredefines viral diversity and the taxonomy of the Flaviviridae

The Flaviviridae are a family of non-segmented positive-sense enveloped RNA viruses containing significant pathogens including hepatitis C virus and yellow fever virus. Recent large-scale metagenomic surveys have identified many diverse RNA viruses related to classical orthoflaviviruses and pestiviruses but quite different genome lengths and configurations, and with a hugely expanded host range that spans multiple animal phyla, including molluscs, cnidarians and stramenopiles,, and plants. Grouping of RNA-directed RNA polymerase (RdRP) hallmark gene sequences of flavivirus and flavi-like viruses into four divergent clades and multiple lineages within them was congruent with helicase gene phylogeny, PPHMM profile comparisons, and comparison of RdRP protein structure predicted by AlphFold2. These results support their classification into the established order, Amarillovirales, in three families (Flaviviridae, Pestiviridae, and Hepaciviridae), and 14 genera. This taxonomic framework informed by RdRP hallmark gene evolutionary relationships provides a stable reference from which major genome re-organisational events can be understood.

microbiology↗

bioRxiv2024

Detection of neutralizing antibodies against arboviruses from liver homogenates

Yellow fever virus (YFV) circulates in a sylvatic cycle between non-human primates (NHPs) and arboreal mosquitoes in Brazil. Passive monitoring of ill or deceased NHPs is a key component of the Brazilian yellow fever (YF) surveillance program. Samples from NHPs carcasses are usually suitable for molecular tests but not for serological assays. As an alternative to the conventional plaque reduction neutralization test (PRNT) based on sera, we tested the utility of liver homogenates from experimentally infected (YFV, Mayaro virus [MAYV], chikungunya virus [CHIKV], or mock) mice to quantify PRNTs. Although homogenates from mock-infected mice showed a low level of nonspecific virus neutralization against YFV, MAYV or CHIKV, homogenates from YFV-, MAYV- and CHIKV-infected mice demonstrated significantly higher levels of virus neutralization compared to controls. Receiver operating characteristic (ROC) curves analyses were performed using the median neutralization values of three technical replicates for each infected group separately or collectively. Results showed scores [≥]0.97 (95% CI [≥] 0.89-1.0) for the area under the curve at dilutions 1:20 to 1:80, suggesting that median virus neutralization values effectively differentiated YFV-, MAYV-, or CHIKV-infected groups from controls. Liver homogenates obtained from 25 NHPs carcasses (collected during the 2017 YF outbreak in Brazil) were also tested using the adapted PRNT as well as rapid anti-YFV IgM immunochromatographic tests. Neutralization activity was detected in six NHPs samples that were also positive by PCR and anti-YFV IgM tests and one sample that tested negative by PCR and IgM test. Our results demonstrate the feasibility of using liver homogenates as an alternative approach for serological investigation in viral epidemiologic surveillance.

microbiology↗

bioRxiv2024

Forest edge landscape context affects mosquito community composition and risk of pathogen emergence

Forest edges, where humans, mosquitoes, and wildlife interact, may serve as a nexus for zoonotic arbovirus exchange. Although often treated as uniform interfaces, the landscape context of edge habitats can greatly impact ecological interactions. Here, we investigated how the landscape context of forest edges shapes mosquito community structure in an Amazon rainforest reserve near the city of Manaus, Brazil, using hand-nets to sample mosquitoes at three distinct forest edge types. Sampling sites were situated at edges bordering urban land cover, rural land cover, and natural treefall gaps, while sites in continuous forest served as controls. Community composition differed substantially among edge types, with rural edges supporting the highest species diversity. Rural edges also provided suitable habitat for forest specialists, including key sylvatic vectors, of which Haemagogus janthinomys was the most abundant species sampled overall. Our findings emphasize the importance of landscape context in assessing pathogen emergence risk at forest edges.

ecology↗

bioRxiv2024

Yellow Fever Emergence: Role of Heterologous Flavivirus Immunity in Preventing Urban Transmission

During major, recent yellow fever (YF) epidemics in Brazil, human cases were attributed only to spillover infections from sylvatic transmission with no evidence of human amplification. Furthermore, the historic absence of YF in Asia, despite abundant peridomestic Aedes aegypti and naive human populations, represents a longstanding enigma. We tested the hypothesis that immunity from dengue (DENV) and Zika (ZIKV) flaviviruses limits YF virus (YFV) viremia and transmission by Ae. aegypti. Prior DENV and ZIKV immunity consistently suppressed YFV viremia in experimentally infected macaques, leading to reductions in Ae. aegypti infection when mosquitoes were fed on infected animals. These results indicate that, in DENV- and ZIKV-endemic regions such as South America and Asia, flavivirus immunity suppresses YFV human amplification potential, reducing the risk of urban outbreaks. One-Sentence SummaryImmunity from dengue and Zika viruses suppresses yellow fever viremia, preventing infection of mosquitoes and reducing the risk of epidemics.

microbiology↗

bioRxiv2024

Aedes albopictus is not an arbovirus aficionado - Impacts of sylvatic flavivirus infection in vectors and hosts on mosquito engorgement on non-human primates

The contact structure between vertebrate hosts and arthropod vectors plays a key role in the spread of arthropod-borne viruses (arboviruses); thus, it is important to determine whether arbovirus infection of either host or vector alters vector feeding behavior. Here we leveraged a study of the replication dynamics of two arboviruses isolated from their ancestral cycles in paleotropical forests, sylvatic dengue-2 (DENV-2) and Zika (ZIKV), in one non-human primate (NHP) species from the paleotropics (cynomolgus macaques, Macaca fascicularis) and one from the neotropics (squirrel monkeys, Saimiri boliviensis) to test the effect of both vector and host infection with each virus on completion of blood feeding (engorgement) of the mosquito Aedes albopictus. Although mosquitoes were starved and given no choice of hosts, engorgement rates varied dramatically, from 0% to 100%. While neither vector nor host infection systematically affected engorgement, NHP species and body temperature at the time of feeding did. We also interrogated the effect of repeated mosquito bites on cytokine expression and found that epidermal growth factor (EGF) and macrophage migration inhibitory factor (MIF) concentrations were dynamically associated with exposure to mosquito bites. This study highlights the importance of incorporating individual-level heterogeneity of vector biting in arbovirus transmission models.

systems biology↗

bioRxiv2023

Potential of Ilheus virus to emerge

Ilheus virus (ILHV)(Flaviviridae: Orthoflavivirus) is an arthropod-borne virus (arbovirus) endemic to Central and South America and the Caribbean. First isolated in 1944, most of our knowledge derives from surveillance and seroprevalence studies. These efforts have detected ILHV in a broad range of mosquito and vertebrate species, including humans, but laboratory investigations of pathogenesis and vector competence have been lacking. Here, we develop several immune intact murine models that closely recapitulate human neuroinvasive disease with strain- and age-specific virulence, as well as a uniformly lethal immunocompromised model. Replication kinetics in several vertebrate and invertebrate cell lines demonstrate that ILHV is capable of replicating to high titers in a wide variety of potential host and vector species. Lastly, vector competence studies provide strong evidence for efficient infection of and potential transmission by Aedes species mosquitoes, despite ILHVs phylogenetically clustering with Culex vectored flaviviruses, suggesting ILHV is poised for emergence in the neotropics. TeaserMurine models of ILHV mimic human disease, and Aedes species of mosquitoes are highly susceptible to infection and dissemination.

microbiology↗

bioRxiv2023

Madariaga and Venezuelan equine encephalitis virus seroprevalence in rodent enzootic hosts in Eastern and Western Panama

While rodents are primary reservoirs of Venezuelan equine encephalitis virus (VEEV), their role in Madariaga virus (MADV) transmission remains uncertain, particularly given their overlapping geographic distribution. This study explores the interplay of alphavirus prevalence, rodent diversity, and land use within Darien and Western Panama provinces. A total of three locations were selected for rodent sampling in Darien province: Los Pavitos, El Real de Santa Maria and Santa Librada. Two sites were selected in Western Panama province: El Cacao and Ciri Grande. We used plaque reduction neutralization tests to assess MADV and VEEV seroprevalences in 599 rodents of 16 species across five study sites. MADV seroprevalence was observed at higher rates in Los Pavitos (Darien province), 9.0%, 95% CI: 3.6-17.6, while VEEV seroprevalence was elevated in El Cacao (Western Panama province), 27.3%, 95% CI: 16.1-40.9, and El Real de Santa Maria (Darien province), 20.4%, 95% CI: 12.6-29.7. Species like Oryzomys coesi, 23.1%, 95% CI: 5.0-53.8, and Transandinomys bolivaris, 20.0%, 95% CI: 0.5-71.6 displayed higher MADV seroprevalences than other species, whereas Transandinomys bolivaris, 80.0%, 95% CI: 28.3-99.4, and Proechimys semispinosus, 27.3%, 95% CI: 17.0-39.6, exhibited higher VEEV seroprevalences. Our findings provide support to the notion that rodents are vertebrate reservoirs of MADV and reveal spatial variations in alphavirus seropositivity among rodent species, with different provinces exhibiting distinct rates for MADV and VEEV. Moreover, specific rodent species are linked to unique seroprevalence patterns for these viruses, suggesting that rodent diversity and environmental conditions might play a significant role in shaping alphavirus distribution.

microbiology↗

bioRxiv2023

Immunologically mediated trade-offs shaping transmission of sylvatic dengue and Zika viruses in native and novel non-human primate hosts

Mosquito-borne dengue (DENV) and Zika (ZIKV) viruses originated in Old World sylvatic cycles involving monkey hosts, spilled over into human transmission, and were translocated to the Americas, creating potential for spillback into neotropical sylvatic cycles. Studies of the trade-offs that shape within-host dynamics and transmission of these viruses are lacking, hampering efforts to predict spillover and spillback. We exposed native (cynomolgus macaque) or novel (squirrel monkey) hosts to mosquitoes infected with either sylvatic DENV or ZIKV and monitored viremia, natural killer cells, transmission to mosquitoes, cytokines, and neutralizing antibody titers. Unexpectedly, DENV transmission from both host species occurred only when serum viremia was undetectable or near the limit of detection. ZIKV replicated in squirrel monkeys to much higher titers than DENV and was transmitted more efficiently but stimulated lower neutralizing antibody titers. Increasing ZIKV viremia led to greater instantaneous transmission and shorter duration of infection, consistent with a replication-clearance trade-off.

microbiology↗

bioRxiv2022

Ecological drivers of sustained enzootic yellow fever virus transmission in Brazil, 2017-2021

Beginning December 2016, sylvatic yellow fever (YF) outbreaks spread into southeastern Brazil, and Minas Gerais state experienced two sylvatic YF waves (2017 and 2018). Following these massive YF waves, we screened 187 free-living non-human primate (NHPs) carcasses collected throughout the state between January 2019 and June 2021 for YF virus (YFV) using qPCR. One sample belonging to a Callithrix, collected in June 2020, was positive for YFV. The viral strain belonged to the same lineage associated with 2017-2018 outbreaks, showing the continued enzootic circulation of YFV in the state. Next, using data from 781 NHPs carcasses collected in 2017-18, we used generalized additive mixed models (GAMMs) to identify the spatiotemporal and host-level drivers of YFV infection and intensity (an estimation of genomic viral load in the liver of infected NHP). Our GAMMs explained 65% and 68% of variation in virus infection and intensity, respectively, and uncovered strong temporal and spatial patterns for YFV infection and intensity. NHP infection was higher in the eastern part of Minas Gerais state, where 2017-2018 outbreaks affecting humans and NHPs were concentrated. The odds of YFV infection were significantly lower in NHPs from urban areas than from urban-rural or rural areas, while infection intensity was significantly lower in NHPs from urban areas or the urban-rural interface relative to rural areas. Both YFV infection and intensity were higher during the warm/rainy season compared to the cold/dry season. The higher YFV intensity in NHPs in warm/rainy periods could be a result of higher exposure to vectors and/or higher virus titers in vectors during this time resulting in the delivery of a higher virus dose and higher viral replication levels within NHPs. Further studies are needed to better test this hypothesis and further compare the dynamics of YFV enzootic cycles between different seasons. Author SummaryIn 2017 and 2018 massive sylvatic yellow fever (YF) outbreaks took place in Minas Gerais Brazil. To investigate yellow fever virus (YFV) circulation following these massive outbreaks, we investigated samples from 187 free-living non-human primate (NHPs) collected between January 2019 and June 2021. One sample belonging to a Callithrix, collected in June 2020 was positive for YFV. This virus was closely related to YFV from previous outbreaks (2017-2018) showing the continued enzootic circulation of YFV in the state. Next, we investigated the drivers of YFV infection and intensity (an estimation of viral load in each infected NHP) during the 2017-18 outbreaks. The odds of YFV infection in NHPs were lower in urban areas compared to rural ones as expected in sylvatic cycles. There were strong temporal and spatial patterns were observed for YFV infection and intensity, especially in the eastern part of Minas Gerais state. The higher YFV infection and intensity observed during the warm/rainy season (as opposed to the cold/dry one) could be related to higher exposure to vectors and/or higher virus titers in vectors during this time. The possible delivery of a higher virus dose in NHPs could lead to higher viral replication levels within NHPs explaining the higher intensity of infection during warm/rainy season. Further studies are needed to better test this hypothesis and further compare the dynamics of YFV enzootic cycles between different seasons.

microbiology↗

bioRxiv2020

Emergence of Zika virus: Role of founder effects, drift and mutational reversions in spread to the Americas

Summary ParagraphMosquito-borne viruses have recently spread globally, with major impacts on human health. Zika virus (ZIKV) emerged from obscurity in 2013 to spread from Asia to the South Pacific and the Americas, where millions of people were infected. For the first time, severe clinical manifestations, including Guillain Barre syndrome and defects to the fetuses of pregnant women, were detected. Phylogenetic studies have shown that ZIKV evolved in Africa and later spread to Asia, and that the Asian lineage is responsible for the recent epidemics. However, the reasons for the sudden emergence of ZIKV remain incompletely understood. Accumulating evidence on other arboviruses like chikungunya and West Nile suggest the likelihood that viral mutations could be determinants of change in ZIKV transmission efficiency responsible for efficient spread. Using evolutionary analyses, we determined that four mutations, which occurred just before ZIKV introduction to the Americas, represent direct reversions of previous mutations that accompanied spread many decades ago from ZIKVs native Africa to Asia and early circulation there. Experimental infections of mosquitoes, human cells, and mice using ZIKV strains with and without these mutations demonstrated that the original mutations reduced fitness for urban transmission, while the reversions restored fitness, increasing epidemic risk. Overall, our findings include the three newly identified, transmission-adaptive ZIKV mutations, and demonstration that these and one identified previously restored fitness for epidemic transmission soon before introduction into the America. The initial mutations may have followed founder effects and/or drift when the virus was introduced into Asia, or could be related to changes on host or vector utilization within Asia.

microbiology↗

bioRxiv2020

Evolution of resistance to fluoroquinolones by dengue virus serotype 4 provides insight into mechanism of action and consequences for viral fitness

Drugs against flaviviruses such as dengue (DENV) and Zika (ZIKV) virus are urgently needed. We previously demonstrated that three fluoroquinolones, ciprofloxacin, enoxacin, and difloxacin, suppress replication of six flaviviruses. To investigate the barrier to resistance and mechanism(s) of action of these drugs, DENV-4 was passaged in triplicate in HEK-293 cells in the presence or absence of each drug. Resistance to ciprofloxacin was detected by the seventh passage and to difloxacin by the tenth, whereas resistance to enoxacin did not occur within ten passages. Two putative resistance-conferring mutations were detected in the envelope gene of ciprofloxacin and difloxacin-resistant DENV-4. In the absence of ciprofloxacin, ciprofloxacin-resistant viruses sustained a significantly higher viral titer than control viruses in HEK-293 and HuH-7 cells and resistant viruses were more stable than control viruses at 37{degrees}C. These results suggest that the mechanism of action of ciprofloxacin and difloxacin involves interference with virus binding or entry.

evolutionary biology↗

bioRxiv2018

Novel modular pressure and flow rate balanced microfluidic serial dilution networks on printed circuit boards: Designs, Simulations and Fabrication

Fast, efficient and more importantly accurate serial dilution is a requirement for many chemical and biological microfluidic-based applications. Over the last decade, a large number of microfluidic devices has been proposed, each demonstrating either a different type of dilution technique or complex system architectures based on various flow source combinations. In this work, a novel serial dilution architecture is demonstrated, implemented on a commercially fabricated printed circuit board (PCB). The proposed single layer, stepwise serial diluter comprises an optimised microfluidic network, where identical dilution ratio per stage can be ensured, either by applying equal pressure or equal flow rates at both inlets. The advantages of the proposed serial diluter are twofold. Firstly, it is structured as a modular unit cell, simplifying the required fluid driving mechanism to a single source for both sample and buffer solution. Thus, this unit cell can be seen as a fundamental microfluidic building block, which can form multistage serial dilution cascades, once combined appropriately with itself or other similar unit cells. Secondly, the serial diluter has been fabricated entirely using commercial PCB technologies, allowing the device to be interfaced with standard electronic components, if more complex miniature point-of-care (PoC) systems are desired, where the small footprint and accuracy of the device is of paramount importance.

bioengineering↗