Identification and characterization of novel alphacoronaviruses in Tadarida brasiliensis (Chiroptera, Molossidae) from Argentina: insights into recombination as a mechanism favoring bat coronavirus cross-species transmission.

Bats are reservoirs of various coronaviruses that can jump between bat species or other mammalian hosts, including humans. This article explores coronavirus infection in three bat species (Tadarida brasiliensis, Eumops bonariensis, and Molossus molossus) of the family Molossidae from Argentina using whole viral metagenome analysis. Fecal samples of 47 bats from three semiurban or highly urbanized areas of the province of Santa Fe were investigated. After viral particle enrichment, total RNA was sequenced using the Illumina NextSeq 550 instrument; the reads were assembled into contigs and taxonomically and phylogenetically analyzed. Three novel complete Alphacoronavirus (AlphaCoV) genomes (Tb1-3) and two partial sequences were identified in T. brasiliensis (Tb4-5), and an additional four partial sequences were identified in M. molossus (Mm1-4). Phylogenomic analysis showed that the novel AlphaCoV clustered in two different lineages distinct from the 15 officially recognized AlphaCoV subgenera. Tb2 and Tb3 isolates appeared to be variants of the same virus, probably involved in a persistent infectious cycle within the T. brasiliensis colony. Using recombination analysis, we detected a statistically significant event in Spike gene, which was reinforced by phylogenetic tree incongruence analysis, involving novel Tb1 and AlphaCoVs identified in Eptesicus fuscus (family Vespertilionidae) from the U.S. The putative recombinant region is in the S1 subdomain of the Spike gene, encompassing the potential receptor-binding domain of AlphaCoVs. This study reports the first AlphaCoV genomes in molossids from the Americas and provides new insights into recombination as an important mode of evolution of coronaviruses involved in cross-species transmission. IMPORTANCE This study generated three novel complete AlphaCoV genomes (Tb1, Tb2, and Tb3 isolates) identified in individuals of Tadarida brasiliensis from Argentina, which showed two different evolutionary patterns and are the first to be reported in the family Molossidae in the Americas. The novel Tb1 isolate was found to be involved in a putative recombination event with alphacoronaviruses identified in bats of the genus Eptesicus from the U.S., whereas isolates Tb2 and Tb3 were found in different collection seasons and might be involved in persistent viral infections in the bat colony. These findings contribute to our knowledge of the global diversity of bat coronaviruses in poorly studied species and highlight the different evolutionary aspects of AlphaCoVs circulating in bat populations in Argentina.

The Lambda Variant in Argentina: Analyzing the Evolution and Spread of SARS-CoV-2 Lineage C.37.

The second wave of COVID-19 occurred in South America in early 2021 and was mainly driven by Gamma and Lambda variants. In this study, we aimed to describe the emergence and local genomic diversity of the SARS-CoV-2 Lambda variant in Argentina, from its initial entry into the country until its detection ceased. Molecular surveillance was conducted on 9356 samples from Argentina between October 2020 and April 2022, and sequencing, phylogenetic, and phylogeographic analyses were performed. Our findings revealed that the Lambda variant was first detected in Argentina in January 2021 and steadily increased in frequency until it peaked in April 2021, with continued detection throughout the year. Phylodynamic analyses showed that at least 18 introductions of the Lambda variant into the country occurred, with nine of them having evidence of onward local transmission. The spatial--temporal reconstruction showed that Argentine clades were associated with Lambda sequences from Latin America and suggested an initial diversification in the Metropolitan Area of Buenos Aires before spreading to other regions in Argentina. Genetic analyses of genome sequences allowed us to describe the mutational patterns of the Argentine Lambda sequences and detect the emergence of rare mutations in an immunocompromised patient. Our study highlights the importance of genomic surveillance in identifying the introduction and geographical distribution of the SARS-CoV-2 Lambda variant, as well as in monitoring the emergence of mutations that could be involved in the evolutionary leaps that characterize variants of concern.

Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

Viral Metagenomic Data Analyses of Five New World Bat Species from Argentina: Identification of 35 Novel DNA Viruses.

Bats are natural reservoirs of a variety of zoonotic viruses, many of which cause severe human diseases. Characterizing viruses of bats inhabiting different geographical regions is important for understanding their viral diversity and for detecting viral spillovers between animal species. Herein, the diversity of DNA viruses of five arthropodophagous bat species from Argentina was investigated using metagenomics. Fecal samples of 29 individuals from five species (Tadarida brasiliensis, Molossus molossus, Eumops bonariensis, Eumops patagonicus, and Eptesicus diminutus) living at two different geographical locations, were investigated. Enriched viral DNA was sequenced using Illumina MiSeq, and the reads were trimmed and filtered using several bioinformatic approaches. The resulting nucleotide sequences were subjected to viral taxonomic classification. In total, 4,520,370 read pairs were sequestered by sequencing, and 21.1% of them mapped to viral taxa. Circoviridae and Genomoviridae were the most prevalent among vertebrate viral families in all bat species included in this study. Samples from the T. brasiliensis colony exhibited lower viral diversity than samples from other species of New World bats. We characterized 35 complete genome sequences of novel viruses. These findings provide new insights into the global diversity of bat viruses in poorly studied species, contributing to prevention of emerging zoonotic diseases and to conservation policies for endangered species.

Cost-Effective Method to Perform SARS-CoV-2 Variant Surveillance: Detection of Alpha, Gamma, Lambda, Delta, Epsilon, and Zeta in Argentina.

SARS-CoV-2 variants with concerning characteristics have emerged since the end of 2020. Surveillance of SARS-CoV-2 variants was performed on a total of 4,851 samples from the capital city and 10 provinces of Argentina, during 51 epidemiological weeks (EWs) that covered the end of the first wave and the ongoing second wave of the COVID-19 pandemic in the country (EW 44/2020 to EW 41/2021). The surveillance strategy was mainly based on Sanger sequencing of a Spike coding region that allows the identification of signature mutations associated with variants. In addition, whole-genome sequences were obtained from 637 samples. The main variants found were Gamma and Lambda, and to a lesser extent, Alpha, Zeta, and Epsilon, and more recently, Delta. Whereas, Gamma dominated in different regions of the country, both Gamma and Lambda prevailed in the most populated area, the metropolitan region of Buenos Aires. The lineages that circulated on the first wave were replaced by emergent variants in a term of a few weeks. At the end of the ongoing second wave, Delta began to be detected, replacing Gamma and Lambda. This scenario is consistent with the Latin American variant landscape, so far characterized by a concurrent increase in Delta circulation and a stabilization in the number of cases. The cost-effective surveillance protocol presented here allowed for a rapid response in a resource-limited setting, added information on the expansion of Lambda in South America, and contributed to the implementation of public health measures to control the disease spread in Argentina.

A Preliminary Study of the Virome of the South American Free-Tailed Bats (Tadarida brasiliensis) and Identification of Two Novel Mammalian Viruses.

Bats provide important ecosystem services as pollinators, seed dispersers, and/or insect controllers, but they have also been found harboring different viruses with zoonotic potential. Virome studies in bats distributed in Asia, Africa, Europe, and North America have increased dramatically over the past decade, whereas information on viruses infecting South American species is scarce. We explored the virome of Tadarida brasiliensis, an insectivorous New World bat species inhabiting a maternity colony in Rosario (Argentina), by a metagenomic approach. The analysis of five pooled oral/anal swab samples indicated the presence of 43 different taxonomic viral families infecting a wide range of hosts. By conventional nucleic acid detection techniques and/or bioinformatics approaches, the genomes of two novel viruses were completely covered clustering into the Papillomaviridae (Tadarida brasiliensis papillomavirus type 1, TbraPV1) and Genomoviridae (Tadarida brasiliensis gemykibivirus 1, TbGkyV1) families. TbraPV1 is the first papillomavirus type identified in this host and the prototype of a novel genus. TbGkyV1 is the first genomovirus reported in New World bats and constitutes a new species within the genus Gemykibivirus. Our findings extend the knowledge about oral/anal viromes of a South American bat species and contribute to understand the evolution and genetic diversity of the novel characterized viruses.

Assessing Gammapapillomavirus infections of mucosal epithelia with two broad-spectrum PCR protocols.

BACKGROUND: Human papillomaviruses (HPVs) have been divided into mucosal and cutaneous types according to their primary epithelial tissue tropism. However, recent studies showed the presence of several cutaneous types in mucosal lesions and healthy mucosa from different anatomical sites. METHODS: Here, the HPV prevalence and type-specific distribution were assessed in a variety of mucosal samples from 435 individuals using a combination of two established broad-spectrum primer systems: Gamma-PV PCR and CUT PCR. RESULTS: Overall HPV prevalence in anal canal swabs, cervical cancer biopsies, genital warts and oral swabs was 85, 47, 62 and 4%, respectively. In anal canal swabs, Alpha-PVs were most frequently found (59%), followed by Gamma- (37%) and Beta-PVs (4%). The prevalence and persistence of HPV infection in the anal canal of 226 individuals were further explored. Overall HPV, Gamma-PVs and multiple HPV infections were significantly higher in men vs. women (p = 0.034, p = 0.027 and p = 0.003, respectively); multiple HPV infections were more common in individuals ≤40 years (p = 0.05), and significantly higher prevalence of Gamma-PVs and multiple HPV infections was observed in HIV-1-positive vs. HIV-1-negative individuals (p = 0.003 and p = 0.04, respectively). Out of 21 patients with follow-up anal swabs, only one persistent infection with the same type (HPV58) was detected. CONCLUSIONS: Our findings suggest that Gamma-PVs (except species Gamma-6) are ubiquitous viruses with dual muco-cutaneous tissue tropism. Anal canal Gamma-PV infections may be associated with sexual behavior and the host immune status. This study expands the knowledge on Gamma-PVs' tissue tropism, providing valuable data on the characteristics of HPV infection in the anal canal.

Detection of HEV in naturally infected swine from central Argentina by an optimized HEV/MS2 duplex RT-qPCR.

Hepatitis E virus (HEV) is currently considered as a global health concern due to the recognition of its zoonotic transmission to humans, mainly from swine, and its association with the development of severe cases of hepatitis in human risk populations. The lack of updated data on HEV state of infection in swineherds of Argentina, and the necessity of robust technologies for its detection in complex biological samples, positions HEV as an emerging issue in public health. Here, we have optimized a RT-qPCR with internal control for a more precise and accurate HEV RNA detection in swine stool samples. We implemented this optimized molecular tool to analyse the current epidemiological scenario of HEV infection in swine from the core region of commercial activity of Argentina. A total of 135 stool samples were collected from 16 different farms and tested for HEV presence, resulting in 11 positive cases (8.1%). Phylogenetic analysis demonstrated that all of them correspond to HEV genotype 3 and that different subtypes circulate in the region. Moreover, two of the detected strains presented a high nucleotide similarity with a previously identified isolate from human sewage discharges, suggesting the zoonotic transmission of HEV to humans. Collectively, this work provides a better understanding of HEV epidemiology in Argentina while contributes to the improvement of HEV detection technologies.

High prevalence of Gammapapillomaviruses (Gamma-PVs) in pre-malignant cutaneous lesions of immunocompetent individuals using a new broad-spectrum primer system, and identification of HPV210, a novel Gamma-PV type.

Genus Gammapapillomavirus (Gamma-PV) is the most diverse and largest clade within the Papillomaviridae family. A novel set of degenerate primers targeting the E1 gene was designed and further used in combination with the well-known CUT PCR assay to assess HPV prevalence and genus distribution in a variety of cutaneous samples from 448 immunocompetent individuals. General HPV, Gamma-PV and mixed infections prevalence were significantly higher in actinic keratosis with respect to benign and malignant neoplasms, respectively (p = 0.0047, p = 0.0172, p = 0.00001). Gamma-PVs were significantly more common in actinic keratosis biopsies than Beta- and Alpha-PVs (p = 0.002). The full-length genome sequence of a novel putative Gamma-PV type was amplified by 'hanging droplet' long-range PCR and cloned. The novel virus, designated HPV210, clustered within species Gamma-12. This study provides an additional tool enabling detection of HPV infections in skin and adds new insights about possible early roles of Gamma-PVs in the development of cutaneous malignant lesions.

Detection of novel Betapapillomaviruses and Gammapapillomaviruses in eyebrow hair follicles using a single-tube 'hanging droplet' PCR assay with modified pan-PV CODEHOP primers.

A modified pan-PV consensus-degenerate hybrid oligonucleotide primer (CODEHOP) PCR was developed for generic and sensitive detection of a broad-spectrum of human papillomaviruses (HPVs) infecting the cutaneous epithelium. To test the analytical sensitivity of the assay we examined 149 eyebrow hair follicle specimens from immunocompetent male patients. HPV DNA was detected in 60 % (89/149) of analysed eyebrow samples with a total of 48 different HPV sequences, representing 21 previously described HPVs and 27 putative novel HPV types. Evidence for ten novel HPV subtypes and seven viral variants, clustering to three out of five genera containing cutaneous HPVs, was also obtained. Thus, we have shown that the modified pan-PV CODEHOP PCR assay is able to identify multiple HPV types, even from different genera, in the same clinical sample. Overall, these results demonstrate that the pan-PV CODEHOP PCR is an excellent tool for screening and identification of novel cutaneous HPVs, even in samples with low viral loads.

Natural history of human papillomavirus infection of sun-exposed healthy skin of immunocompetent individuals over three climatic seasons and identification of HPV209, a novel betapapillomavirus.

We present the first longitudinal study reporting the natural history of human papillomavirus (HPV) infection in sun-exposed skin of healthy individuals living in a geographical area in which solar UV radiation is influenced by the ozone content of the atmosphere. During three climatic seasons, skin swab samples were obtained from 78 healthy individuals and the prevalence of cutaneous HPVs was assessed with broad-spectrum FAP and CUT primers and determined at 54, 45 and 47 % in spring, summer and winter, respectively. Frequencies of mixed HPV infections were significantly higher in spring with respect to summer and winter (P=0.02). Seventy-one different HPV types/putative types were identified. While 62 volunteers were HPV-infected in at least one season, 23 had persistent infections. ß-PVs (ß-1) were the most prevalent and persistent. Age was associated with both the infection status (P=0.01) and the type of HPV infection (no infection, indeterminate/transient, persistent P=0.02). The molecular/phylogenetic analysis of the newly identified ß-PV, officially designated as HPV209, showed that the virus has a typical genomic organization of cutaneous HPVs with five early (E6, E7, E1, E2 and E4) and two late genes (L2 and L1), which clusters to the species ß-2. This provides useful data on cutaneous HPV infections in high UV-exposed regions.

Molecular characterization, tissue tropism, and genetic variability of the novel Mupapillomavirus type HPV204 and phylogenetically related types HPV1 and HPV63.

HPV204 is the only newly identified Mupapillomavirus (Mu-PV) type in more than a decade. To comprehensively characterize HPV204, we performed a detailed molecular analysis of the viral genome and evaluated its clinical relevance in comparison to the other Mu-PVs, HPV1 and HPV63. The 7,227-bp long genome of HPV204 exhibits typical genomic organization of Mu-PVs with eight open reading frames (ORFs) (E6, E7, E1, E2, E8, E4, L2, and L1). We developed three type-specific quantitative real-time PCRs and used them to test a representative collection (n = 1,006) of various HPV-associated benign and malignant neoplasms, as well as samples of clinically normal cutaneous, mucosal, and mucocutaneous origins. HPV204, HPV1, and HPV63 were detected in 1.1%, 2.7%, and 1.9% of samples tested, respectively, and were present in skin and mucosa, suggesting dual tissue tropism of all Mu-PVs. To evaluate the etiological role of Mu-PVs in the development of HPV-associated neoplasms, Mu-PV viral loads per single cell were estimated. HPV1 and HPV63 were present in high viral copy numbers in 3/43 and 1/43 cutaneous warts, respectively, and were identified as the most likely causative agents of these warts. HPV204 viral load was extremely low in a single HPV204-positive cutaneous wart (7.4 × 10-7 viral copies/cell). Hence, etiological association between HPV204 and the development of cutaneous warts could not be established. To the best of our knowledge, this is the first study to evaluate the genetic variability of Mu-PVs by sequencing complete LCR genomic regions of HPV204, HPV1, and HPV63. We detected several nucleotide substitutions and deletions within the LCR genomic regions of Mu-PVs and identified two genetic variants of HPV204 and HPV63 and five genetic variants of HPV1.

Characterization of novel human papillomavirus types 157, 158 and 205 from healthy skin and recombination analysis in genus γ-Papillomavirus.

Gammapapillomavirus (γ-PV) is a diverse and rapidly expanding genus, currently consisting of 79 fully characterized human PV (HPV) types. In this study, three novel types, HPV157, HPV158 and HPV205, obtained from healthy sun-exposed skin of two immunocompetent individuals, were amplified by the "Hanging droplet" long PCR technique, cloned, sequenced and characterized. HPV157, HPV158 and HPV205 genomes comprise 7154-bp, 7192-bp and 7298-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2). Phylogenetic analysis of the L1 ORF placed all novel types within the γ-PV genus: HPV157 was classified as a new member of species γ-12 while HPV158 and HPV205 belong to species γ-1. We then explored potential recombination events in genus γ-PV with the RDP4 program in a dataset of 74 viruses (71 HPV types with available full-length genomes and the 3 novel types). Two events, both located in the E1 ORF, met the inclusion criterion (p-values <0.05 with at least four methods) and persisted in different ORF combinations: an inter-species recombination in species γ-8 (major and minor parents: species γ-24 and γ-11, respectively), and an intra-species recombination in species γ-7 (recombinant strain: HPV170; major and minor parents: HPV-109 and HPV-149, respectively). These findings were confirmed by phylogenetic tree incongruence analysis. An additional incongruence was found in members of species γ-9 but it was not detected by the RDP4. This report expands our knowledge of the family Papillomaviridae and provides for the first time in silico evidence of recombination in genus γ-PV.

Genome announcement: complete genome sequence of a novel Mupapillomavirus, HPV204.

Human papillomaviruses (HPVs) are small, non-enveloped viruses with a circular double-stranded DNA genome, etiologically associated with various benign and malignant neoplasms of the skin and mucosa. As of May 30, 2015, 201 different HPV types had been completely sequenced and officially recognized and divided into five PV-genera: Alpha-, Beta-, Gamma-, Mu-, and Nupapillomavirus. The Mupapillomavirus genus currently consists of only two HPV types: HPV1 and HPV63, identified in 1980 and 1993, respectively, both associated with sporadic cases of cutaneous warts. In this preliminary study, we announce the complete genome sequence of a novel HPV type, now officially recognized as HPV204. Based on preliminary data, the genome of HPV204 comprises a total of 7,227 bp and contains five early open reading frames (E1, E2, E4, E6, and E7) and two late ORFs (L1 and L2). No E5 ORF could be identified. Preliminary HPV204 clusters to the Mu-PV genus, species Mu-3.

Evidence for homologous recombination in Chikungunya Virus.

Chikungunya Virus (CHIKV), a mosquito-transmitted alphavirus, causes acute fever and joint pain in humans. Recently, endemic CHIKV infection outbreaks have jeopardized public health in wider geographical regions. Here, we analyze the phylogenetic associations of CHIKV and explore the potential recombination events on 152 genomic isolates deposited in GenBank database. The CHIKV genotypes [West African, Asian, East/Central/South African (ECSA)], and a clear division of ECSA clade into three sub-groups (I-II-III), were defined by Bayesian analysis; similar results were obtained using E1 gene sequences. A nucleotide identity-based approach is provided to facilitate CHIKV classification within ECSA clade. Using seven methods to detect recombination, we found a statistically significant event (p-values range: 1.14×10(-7)-4.45×10(-24)) located within the nsP3 coding region. This finding was further confirmed by phylogenetic networks (PHI Test, p=0.004) and phylogenetic tree incongruence analysis. The recombinant strain, KJ679578/India/2011 (ECSA III), derives from viruses of ECSA III and ECSA I. Our study demonstrates that recombination is an additional mechanism of genetic diversity in CHIKV that might assist in the cross-species transmission process.

Analysis of the genetic diversity and phylogenetic relationships of putative human papillomavirus types.

More than 170 human papillomavirus (HPV) types have been completely sequenced, curated and divided into five genera: Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Mupapillomavirus and Nupapillomavirus. With the application of PCR methods, hundreds of putative novel HPV types have been identified as PCR amplicons in mucosa and skin. However, at present there are no studies reporting a systematic search of the currently known L1 amplicons and their phylogenetic relationships. This survey revealed the existence of at least 202 different putative HPV types that are pending for full-genome characterization: five alphapapillomaviruses, 37 betapapillomaviruses, 159 gammapapillomaviruses and one mupapillomavirus. All potential viruses of the genera Alphapapillomavirus and Betapapillomavirus were grouped in the defined species, while 59 putative gammapapillomaviruses types were segregated in 21 unidentified putative species. These data highlight the need for progress in the identification of additional taxa of the family Papillomaviridae in order to elucidate the diversity, evolution and medical implications of these viruses.

Identification of human papillomavirus type 156, the prototype of a new human gammapapillomavirus species, by a generic and highly sensitive PCR strategy for long DNA fragments.

This study developed a hanging-droplet long PCR, a generic and highly sensitive strategy to facilitate the identification of new human papillomavirus (HPV) genomes. This novel procedure used for the first time the hanging-droplet PCR technique for the amplification of long DNA fragments with generic primers targeting the L1 and E1 regions. It was first applied to the amplification of types belonging to the highly divergent genus Gammapapillovirus (γ-PV). The hanging-droplet long PCR was 100-fold more sensitive than a simple long PCR procedure, detecting as few as ten copies of HPV-4. Nineteen skin samples, potentially containing putative HPV types from the γ-PV genus, were also screened. The method identified four γ-PV genomic halves from new and previously described putative types, and made the full characterization of HPV-156 possible. This novel virus meets the criteria for a new species within the γ-PV genus, with nucleotide identities in the L1 ORF ranging from 58.3 to 67.3 % compared with representative types of the current γ-PV species. HPV-156 showed the highest identity to HPV-60 (67.3 %) from species γ-4, and was consistently closely related to it in both late- and early-gene-derived phylogenies. In conclusion, this report provides a versatile and highly sensitive approach that allowed identification of the prototype of a new species within the γ-PV genus. Its application with primers targeting the different genera in which both human and non-human PVs are distributed may facilitate characterization of the missing members of the family Papillomaviridae.