Spatiotemporal transmission of SARS-CoV-2 lineages during 2020-2021 in Pernambuco-Brazil.

SARS-CoV-2 virus emerged as a new threat to humans and spread around the world, leaving a large death toll. As of January 2023, Brazil is among the countries with the highest number of registered deaths. Nonpharmacological and pharmacological interventions have been heterogeneously implemented in the country, which, associated with large socioeconomic differences between the country regions, has led to distinct virus spread dynamics. Here, we investigate the spatiotemporal dispersion of SARS-CoV-2 lineages in the Pernambuco state (Northeast Brazil) throughout the distinct epidemiological scenarios that unfolded in the first 2 years of the pandemic. We generated a total of 1,389 new SARS-CoV-2 genomes from June 2020 to August 2021. This sampling captured the arrival, communitary transmission, and the circulation of the B1.1, B.1.1.28, and B.1.1.33 lineages; the emergence of the former variant of interest P.2; and the emergence and fast replacement of all previous variants by the more transmissible variant of concern P.1 (Gamma). Based on the incidence and lineage spread pattern, we observed an East-to-West to inner state pattern of transmission, which is in agreement with the transmission of more populous metropolitan areas to medium- and small-size country-side cities in the state. Such transmission patterns may be partially explained by the main routes of traffic across municipalities in the state. Our results highlight that the fine-grained intrastate analysis of lineages and incidence spread can provide actionable insights for planning future nonpharmacological intervention for air-borne transmissible human pathogens.IMPORTANCEDuring the COVID-19 pandemic, Brazil was one of the most affected countries, mainly due its continental-size, socioeconomic differences among regions, and heterogeneous implementation of intervention methods. In order to investigate SARS-CoV-2 dynamics in the state of Pernambuco, we conducted a spatiotemporal dispersion study, covering the period from June 2020 to August 2021, to comprehend the dynamics of viral transmission during the first 2 years of the pandemic. Throughout this study, we were able to track three significant epidemiological waves of transmission caused by B1.1, B.1.1.28, B.1.1.33, P.2, and P.1 lineages. These analyses provided valuable insights into the evolution of the epidemiological landscape, contributing to a deeper understanding of the dynamics of virus transmission during the early years of the pandemic in the state of Pernambuco.

Assessment of clinical characteristics and viral load in individuals infected by Delta and Omicron variants of SARS-CoV-2.

In late 2021, a new variant of SARS-CoV-2 called Omicron emerged, replacing Delta worldwide. Although it has been associated with a lower risk of hospitalization and severe forms of COVID-19, there is little evidence of its relationship with specific symptoms and viral load. The aim of this study was to verify the relationship between Delta and Omicron variants of concern, viral load, and the occurrence of symptoms in individuals with COVID-19. Nasopharyngeal swab samples were collected and sequenced from patients with COVID-19 from the Northeast Region of Brazil between August 2021 and March 2022. The results showed a gradual replacement of the Delta variant by the Omicron variant during the study period. A total of 316 samples (157 Delta and 159 Omicron) were included. There was a higher prevalence of symptoms in Delta-infected individuals, such as coryza, olfactory and taste disturbances, headache, and myalgia. There was no association between viral load and the variants analyzed. The results reported here contribute to the understanding of the symptoms associated with the Delta and Omicron variants in individuals affected by COVID-19.

SARS-CoV-2 intra-host diversity, antibody response, and disease severity after reinfection by the variant of concern Gamma in Brazil.

The rapid spread of the SARS-CoV-2 Variant of Concern (VOC) Gamma in Amazonas during early 2021 fueled a second large COVID-19 epidemic wave and raised concern about the potential role of reinfections. Very few cases of reinfection associated with the VOC Gamma have been reported to date, and their potential impact on clinical, immunological, and virological parameters remains largely unexplored. Here we describe 25 cases of SARS-CoV-2 reinfection in Brazil. SARS-CoV-2 genomic analysis confirmed that individuals were primo-infected with distinct viral lineages between March and December 2020 (B.1.1, B.1.1.28, B.1.1.33, B.1.195, and P.2) and reinfected with the VOC Gamma between 3 to 12 months after primo-infection. We found a similar mean cycle threshold (Ct) value and limited intra-host viral diversity in both primo-infection and reinfection samples. Sera of 14 patients tested 10-75 days after reinfection displayed detectable neutralizing antibodies (NAb) titers against SARS-CoV-2 variants that circulated before (B.1.*), during (Gamma), and after (Delta and Omicron) the second epidemic wave in Brazil. All individuals had milder or no symptoms after reinfection, and none required hospitalization. These findings demonstrate that individuals reinfected with the VOC Gamma may display relatively high RNA viral loads at the upper respiratory tract after reinfection, thus contributing to onward viral transmissions. Despite this, our study points to a low overall risk of severe Gamma reinfections, supporting that the abrupt increase in hospital admissions and deaths observed in Amazonas and other Brazilian states during the Gamma wave was mostly driven by primary infections. Our findings also indicate that most individuals analyzed developed a high anti-SARS-CoV-2 NAb response after reinfection that may provide some protection against reinfection or disease by different SARS-CoV-2 variants.

Ancient origin of Jingchuvirales derived glycoproteins integrated in arthropod genomes.

Endogenous virus elements (EVEs) are viral-derived sequences integrated into their host genomes. EVEs of the Jingchuvirales order were detected in a wide range of insect genomes covering several distantly related families. Moreover, Jingchuvirales-derived glycoproteins were recently associated by our group with the origin of a putative new retrovirus based on a glycoprotein captured by a mosquito retrotransposon. But, except for mosquitoes, there is a lack of a more detailed understanding of the endogenization mechanism, timing, and frequency per Jingchuvirales viral lineages. Here we screened Jingchuvirales glycoprotein-derived EVEs (Jg-EVEs) in eukaryotic genomes. We found six distinct endogenization events of Jg-EVEs, that belong to two out of five known Jingchuvirales families (Chuviridae and Natareviridae). For seven arthropod families bearing Jg-EVEs there is no register of bona fide circulating chuvirus infection. Hence, our results show that Jingchuvirales viruses infected or still infect these host families. Although we found abundant evidence of LTR-Gypsy retrotransposons fragments associated with the glycoprotein in Hymenoptera and other insect orders, our results show that the widespread distribution of Jingchuvirales glycoproteins in extant Arhtropods is a result of multiple ancient endogenization events and that these virus fossils are being vertically inherited in Arthropods genomes for millions of years.

Real-life evaluation of a rapid antigen test (DPP SARS-CoV-2 Antigen) for COVID-19 diagnosis of primary healthcare patients, in the context of the Omicron-dominant wave in Brazil.

OBJECTIVES: We aimed to investigate the real-life performance of the rapid antigen test in the context of a primary healthcare setting, including symptomatic and asymptomatic individuals that sought diagnosis during an Omicron infection wave. METHODS: We prospectively accessed the performance of the DPP SARS-CoV-2 Antigen test in the context of an Omicron-dominant real-life setting. We evaluated 347 unselected individuals (all-comers) from a public testing centre in Brazil, performing the rapid antigen test diagnosis at point-of-care with fresh samples. The combinatory result from two distinct real-time quantitative PCR (RT-qPCR) methods was employed as a reference and 13 samples with discordant PCR results were excluded. RESULTS: The assessment of the rapid test in 67 PCR-positive and 265 negative samples revealed an overall sensitivity of 80.5% (CI 95% = 69.1%-89.2%), specificity of 99.2% (CI 95% = 97.3%-99.1%) and positive/negative predictive values higher than 95%. However, we observed that the sensitivity was dependent on the viral load (sensitivity in Ct < 31 = 93.7%, CI = 82.8%-98.7%; Ct > 31 = 47.4%, CI = 24.4%-71.1%). The positive samples evaluated in the study were Omicron (BA.1/BA.1.1) by whole-genome sequencing (n = 40) and multiplex RT-qPCR (n = 17). CONCLUSIONS: Altogether, the data obtained from a real-life prospective cohort supports that the rapid antigen test sensitivity for Omicron remains high and underscores the reliability of the test for COVID-19 diagnosis in settings with high disease prevalence and limited PCR testing capability.

Phylogenetic-based inference reveals distinct transmission dynamics of SARS-CoV-2 lineages Gamma and P.2 in Brazil.

The COVID-19 epidemic in Brazil experienced two major lineage replacements until mid-2021. The first was driven by lineage P.2, in late 2020, and the second by lineage Gamma, in early 2021. To understand how these SARS-CoV-2 lineages spread in Brazil, we analyzed 11,724 genomes collected throughout the country between September 2020 and April 2021. Our findings indicate that lineage P.2 probably emerged in July 2020 in the Rio de Janeiro state and Gamma in November 2020 in the Amazonas state. Both states were the main hubs of viral disseminations to other Brazilian locations. We estimate that Gamma was 1.56-3.06 times more transmissible than P.2 in Rio de Janeiro and that the median effective reproductive number (Re) of Gamma varied according to the geographic context (Re = 1.59-3.55). In summary, our findings support that lineage Gamma was more transmissible and spread faster than P.2 in Brazil.

Unusual SARS-CoV-2 intrahost diversity reveals lineage superinfection.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide by July 2021 and the pandemic has been characterized by infection waves of viral lineages showing distinct fitness profiles. The simultaneous infection of a single individual by two distinct SARS-CoV-2 lineages may impact COVID-19 disease progression and provides a window of opportunity for viral recombination and the emergence of new lineages with differential phenotype. Several hundred SARS-CoV-2 lineages are currently well phylogenetically defined, but two main factors have precluded major coinfection/codetection and recombination analysis thus far: (i) the low diversity of SARS-CoV-2 lineages during the first year of the pandemic, which limited the identification of lineage defining mutations necessary to distinguish coinfecting/recombining viral lineages; and the (ii) limited availability of raw sequencing data where abundance and distribution of intrasample/intrahost variability can be accessed. Here, we assembled a large sequencing dataset from Brazilian samples covering a period of 18 May 2020 to 30 April 2021 and probed it for unexpected patterns of high intrasample/intrahost variability. This approach enabled us to detect nine cases of SARS-CoV-2 coinfection with well characterized lineage-defining mutations, representing 0.61 % of all samples investigated. In addition, we matched these SARS-CoV-2 coinfections with spatio-temporal epidemiological data confirming its plausibility with the cocirculating lineages at the timeframe investigated. Our data suggests that coinfection with distinct SARS-CoV-2 lineages is a rare phenomenon, although it is certainly a lower bound estimate considering the difficulty to detect coinfections with very similar SARS-CoV-2 lineages and the low number of samples sequenced from the total number of infections.

ViralFlow: A Versatile Automated Workflow for SARS-CoV-2 Genome Assembly, Lineage Assignment, Mutations and Intrahost Variant Detection.

The COVID-19 pandemic is driven by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) that emerged in 2019 and quickly spread worldwide. Genomic surveillance has become the gold standard methodology used to monitor and study this fast-spreading virus and its constantly emerging lineages. The current deluge of SARS-CoV-2 genomic data generated worldwide has put additional pressure on the urgent need for streamlined bioinformatics workflows. Here, we describe a workflow developed by our group to process and analyze large-scale SARS-CoV-2 Illumina amplicon sequencing data. This workflow automates all steps of SARS-CoV-2 reference-based genomic analysis: data processing, genome assembly, PANGO lineage assignment, mutation analysis and the screening of intrahost variants. The pipeline is capable of processing a batch of around 100 samples in less than half an hour on a personal laptop or in less than five minutes on a server with 50 threads. The workflow presented here is available through Docker or Singularity images, allowing for implementation on laptops for small-scale analyses or on high processing capacity servers or clusters. Moreover, the low requirements for memory and CPU cores and the standardized results provided by ViralFlow highlight it as a versatile tool for SARS-CoV-2 genomic analysis.

The ongoing evolution of variants of concern and interest of SARS-CoV-2 in Brazil revealed by convergent indels in the amino (N)-terminal domain of the spike protein.

Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike (S) glycoprotein can alter its antigenicity and promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. Importantly, we detected the community transmission of different P.1 lineages bearing NTD indels ∆69-70 (which can impact several SARS-CoV-2 diagnostic protocols), ∆144 and ins214ANRN, and a new VOI N.10 derived from the B.1.1.33 lineage carrying three NTD deletions (∆141-144, ∆211, and ∆256-258). These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil generates new viral lineages that might be more resistant to antibody neutralization than parental variants of concern.

Lying in wait: the resurgence of dengue virus after the Zika epidemic in Brazil.

After the Zika virus (ZIKV) epidemic in the Americas in 2016, both Zika and dengue incidence declined to record lows in many countries in 2017-2018, but in 2019 dengue resurged in Brazil, causing ~2.1 million cases. In this study we use epidemiological, climatological and genomic data to investigate dengue dynamics in recent years in Brazil. First, we estimate dengue virus force of infection (FOI) and model mosquito-borne transmission suitability since the early 2000s. Our estimates reveal that DENV transmission was low in 2017-2018, despite conditions being suitable for viral spread. Our study also shows a marked decline in dengue susceptibility between 2002 and 2019, which could explain the synchronous decline of dengue in the country, partially as a result of protective immunity from prior ZIKV and/or DENV infections. Furthermore, we performed phylogeographic analyses using 69 newly sequenced genomes of dengue virus serotype 1 and 2 from Brazil, and found that the outbreaks in 2018-2019 were caused by local DENV lineages that persisted for 5-10 years, circulating cryptically before and after the Zika epidemic. We hypothesize that DENV lineages may circulate at low transmission levels for many years, until local conditions are suitable for higher transmission, when they cause major outbreaks.

COVID-19 in Amazonas, Brazil, was driven by the persistence of endemic lineages and P.1 emergence.

The northern state of Amazonas is among the regions in Brazil most heavily affected by the COVID-19 epidemic and has experienced two exponentially growing waves, in early and late 2020. Through a genomic epidemiology study based on 250 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from different Amazonas municipalities sampled between March 2020 and January 2021, we reveal that the first exponential growth phase was driven mostly by the dissemination of lineage B.1.195, which was gradually replaced by lineage B.1.1.28 between May and June 2020. The second wave coincides with the emergence of the variant of concern (VOC) P.1, which evolved from a local B.1.1.28 clade in late November 2020 and replaced the parental lineage in <2 months. Our findings support the conclusion that successive lineage replacements in Amazonas were driven by a complex combination of variable levels of social distancing measures and the emergence of a more transmissible VOC P.1 virus. These data provide insights to understanding the mechanisms underlying the COVID-19 epidemic waves and the risk of dissemination of SARS-CoV-2 VOC P.1 in Brazil and, potentially, worldwide.

Metatranscriptomic analysis identifies different viral-like sequences in two neotropical Mansoniini mosquito species.

Mosquitoes interact with a wide range of viruses including both arboviruses and insect-specific viruses. This study aimed to characterize the RNA viruses that are interacting with Mansonia wilsoni and Coquillettidia hermanoi mosquito species. The total RNA extracted from mosquito pools were sequenced on a Ion torrent platform. Viral contigs were identified against viral databases and their evolutionary relationship were reconstructed. We identified a total of 107 viral sequences, 11 of which were assigned as endogenous viral elements, and at least six known viral families were identified. Phylogenetic reconstructions were performed for 4 viral families. All Mansoniini viruses investigated through phylogenetic analysis are closely related to insect-specific viruses found in other mosquito species although with considerable divergence at the amino acid level, suggesting that we have detected new viral lineages. This study enhanced our understanding about the virome of two sylvatic Mansoniini mosquitoes.

Systematic Review of Wolbachia Symbiont Detection in Mosquitoes: An Entangled Topic about Methodological Power and True Symbiosis.

Wolbachia is an endosymbiotic bacterium that naturally infects several arthropods and nematode species. Wolbachia gained particular attention due to its impact on their host fitness and the capacity of specific Wolbachia strains in reducing pathogen vector and agricultural pest populations and pathogens transmission. Despite the success of mosquito/pathogen control programs using Wolbachia-infected mosquito release, little is known about the abundance and distribution of Wolbachia in most mosquito species, a crucial knowledge for planning and deployment of mosquito control programs and that can further improve our basic biology understanding of Wolbachia and host relationships. In this systematic review, Wolbachia was detected in only 30% of the mosquito species investigated. Fourteen percent of the species were considered positive by some studies and negative by others in different geographical regions, suggesting a variable infection rate and/or limitations of the Wolbachia detection methods employed. Eighty-three percent of the studies screened Wolbachia with only one technique. Our findings highlight that the assessment of Wolbachia using a single approach limited the inference of true Wolbachia infection in most of the studied species and that researchers should carefully choose complementary methodologies and consider different Wolbachia-mosquito population dynamics that may be a source of bias to ascertain the correct infectious status of the host species.

Identification of a novel SARS-CoV-2 P.1 sub-lineage in Brazil provides new insights about the mechanisms of emergence of variants of concern.

One of the most remarkable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) features is the significant number of mutations they acquired. However, the specific factors that drove the emergence of such variants since the second half of 2020 are not fully resolved. In this study, we describe a new SARS-CoV-2 P.1 sub-lineage circulating in Brazil, denoted here as Gamma-like-II, that as well as the previously described lineage Gamma-like-I shares several lineage-defining mutations with the VOC Gamma. Reconstructions of ancestor sequences support that most lineage-defining mutations of the Spike (S) protein, including those at the receptor-binding domain (RBD), accumulated at the first P.1 ancestor. In contrast, mutations outside the S protein were mostly fixed at subsequent steps. Our evolutionary analyses estimate that P.1-ancestral strains carrying RBD mutations of concern probably circulated cryptically in the Amazonas for several months before the emergence of the VOC Gamma. Unlike the VOC Gamma, the other P.1 sub-lineages displayed a much more restricted dissemination and accounted for a low fraction (<2 per cent) of SARS-CoV-2 infections in Brazil in 2021. The stepwise diversification of lineage P.1 through multiple inter-host transmissions is consistent with the hypothesis that partial immunity acquired from natural SARS-CoV-2 infections in heavily affected regions might have been a major driving force behind the natural selection of some VOCs. The lag time between the emergence of the P.1 ancestor and the expansion of the VOC Gamma and the divergent epidemic trajectories of P.1 sub-lineages support a complex interplay between the emergence of mutations of concern and viral spread in Brazil.

Multiple Introductions Followed by Ongoing Community Spread of SARS-CoV-2 at One of the Largest Metropolitan Areas of Northeast Brazil.

Multiple epicenters of the SARS-CoV-2 pandemic have emerged since the first pneumonia cases in Wuhan, China, such as Italy, USA, and Brazil. Brazil is the third-most affected country worldwide, but genomic sequences of SARS-CoV-2 strains are mostly restricted to states from the Southeast region. Pernambuco state, located in the Northeast region, is the sixth most affected Brazilian state, but very few genomic sequences from the strains circulating in this region are available. We sequenced 101 strains of SARS-CoV-2 from patients presenting Covid-19 symptoms that reside in Pernambuco. Phylogenetic reconstructions revealed that all genomes belong to the B lineage and most of the samples (88%) were classified as lineage B.1.1. We detected multiple viral introductions from abroad (likely from Europe) as well as six local B.1.1 clades composed by Pernambuco only strains. Local clades comprise sequences from the capital city (Recife) and other country-side cities, corroborating the community spread between different municipalities of the state. These findings demonstrate that different from Southeastern Brazilian states where the epidemics were majorly driven by one dominant lineage (B.1.1.28 or B.1.1.33), the early epidemic phase at the Pernambuco state was driven by multiple B.1.1 lineages seeded through both national and international traveling.

In and Outs of Chuviridae Endogenous Viral Elements: Origin of a Potentially New Retrovirus and Signature of Ancient and Ongoing Arms Race in Mosquito Genomes.

BACKGROUND: Endogenous viral elements (EVEs) are sequences of viral origin integrated into the host genome. EVEs have been characterized in various insect genomes, including mosquitoes. A large EVE content has been found in Aedes aegypti and Aedes albopictus genomes among which a recently described Chuviridae viral family is of particular interest, owing to the abundance of EVEs derived from it, the discrepancy among the chuvirus endogenized gene regions and the frequent association with retrotransposons from the BEL-Pao superfamily. In order to better understand the endogenization process of chuviruses and the association between chuvirus glycoproteins and BEL-Pao retrotransposons, we performed a comparative genomics and evolutionary analysis of chuvirus-derived EVEs found in 37 mosquito genomes. RESULTS: We identified 428 EVEs belonging to the Chuviridae family confirming the wide discrepancy among the chuvirus genomic regions endogenized: 409 glycoproteins, 18 RNA-dependent RNA polymerases and one nucleoprotein region. Most of the glycoproteins (263 out of 409) are associated specifically with retroelements from the Pao family. Focusing only on well-assembled Pao retroelement copies, we estimated that 263 out of 379 Pao elements are associated with chuvirus-derived glycoproteins. Seventy-three potentially active Pao copies were found to contain glycoproteins into their LTR boundaries. Thirteen out of these were classified as complete and likely autonomous copies, with a full LTR structure and protein domains. We also found 116 Pao copies with no trace of glycoproteins and 37 solo glycoproteins. All potential autonomous Pao copies, contained highly similar LTRs, suggesting a recent/current activity of these elements in the mosquito genomes. CONCLUSION: Evolutionary analysis revealed that most of the glycoproteins found are likely derived from a single or few glycoprotein endogenization events associated with a recombination event with a Pao ancestral element. A potential functional Pao-chuvirus hybrid (named Anakin) emerged and the glycoprotein was further replicated through retrotransposition. However, a number of solo glycoproteins, not associated with Pao elements, can be found in some mosquito genomes suggesting that these glycoproteins were likely domesticated by the host genome and may participate in an antiviral defense mechanism against both chuvirus and Anakin retrovirus.

Drosophila parasitoid wasps bears a distinct DNA transposon profile.

BACKGROUND: The majority of Eukaryotic genomes are composed of a small portion of stable (non-mobile) genes and a large fraction of parasitic mobile elements such as transposable elements and endogenous viruses: the Mobilome. Such important component of many genomes are normally underscored in genomic analysis and detailed characterized mobilomes only exists for model species. In this study, we used a combination of de novo and homology approaches to characterize the Mobilome of two non-model parasitoid wasp species. RESULTS: The different methodologies employed for TE characterization recovered TEs with different features as TE consensus number and size. Moreover, some TEs were detected only by one or few methodologies. RepeatExplorer and dnaPipeTE estimated a low TE content of 5.86 and 4.57% for Braconidae wasp and 5.22% and 7.42% for L. boulardi species, respectively. Both mobilomes are composed by a miscellaneous of ancient and recent elements. Braconidae wasps presented a large diversity of Maverick/Polintons Class II TEs while other TE superfamilies were more equally diverse in both species. Phylogenetic analysis of reconstructed elements showed that vertical transfer is the main mode of transmission. CONCLUSION: Different methodologies should be used complementarity in order to achieve better mobilome characterization. Both wasps genomes have one of the lower mobilome estimates among all Hymenoptera genomes studied so far and presented a higher proportion of Class II than Class I TEs. The large majority of superfamilies analyzed phylogenetically showed that the elements are being inherited by vertical transfer. Overall, we achieved a deep characterization of the mobilome in two non-model parasitoid wasps improving our understanding of their evolution.

HTT-DB: new features and updates.

Horizontal Transfer (HT) of genetic material between species is a common phenomenon among Bacteria and Archaea species and several databases are available for information retrieval and data mining. However, little attention has been given to this phenomenon among eukaryotic species mainly due to the lower proportion of these events. In the last years, a vertiginous amount of new HT events involving eukaryotic species was reported in the literature, highlighting the need of a common repository to keep the scientific community up to date and describe overall trends. Recently, we published the first HT database focused on HT of transposable elements among eukaryotes: the Horizontal Transposon Transfer DataBase (http://lpa.saogabriel.unipampa.edu.br: 8080/httdatabase/). Here, we present new features and updates of this unique database: (i) its expansion to include virus-host exchange of genetic material, which we called Horizontal Virus Transfer (HVT) and (ii) the availability of a web server for HT detection, where we implemented the online version of vertical and horizontal inheritance consistence analysis (VHICA), an R package developed for HT detection. These improvements will help researchers to navigate through known HVT cases, take data-informed decision and export figures based on keywords searches. Moreover, the availability of the VHICA as an online tool will make this software easily reachable even for researchers with no or little computation knowledge as well as foster our capability to detect new HT events in a wide variety of taxa. (Database URL: http://lpa.saogabriel.unipampa.edu.br:8080/httdatabase/).