Novel members of the order Picornavirales identified in freshwater from Guarapiranga reservoir in São Paulo.

The global challenge of water resource availability is exacerbated by anthropogenic influences that promote the emergence of pollutants. Among these pollutants are microbiological agents, including viruses, which are ubiquitous in the biosphere and play a pivotal role in both ecological balance and the occurrence of diseases in animals and plants. Consequently, monitoring viruses in water sources becomes indispensable for the establishment of effective prevention, promotion, and control strategies. Within this context, the study focuses on the identification of novel viruses belonging to the Picornavirales order in freshwater from the Guarapiranga Reservoir in the state of São Paulo, Brazil. The samples were subjected to viral metagenomics. Our analysis led to the characterization of four distinct sequences (GinkV-05, AquaV_10, MarV_14, and MarV_64), which exhibited significant divergence compared to other members of the Picornavirales order. This remarkable diversity prompted the identification of a potential new genus within the Marnaviridae family, tentatively named Ginkgonavirus. Additionally, we characterized four sequences in a very distinct clade and propose the recognition of a novel family (named Aquaviridae) within the Picornavirales order. Our findings contribute valuable insights into the previously uncharted diversity of Picornavirales present in water sources, shedding light on an important facet of viral ecology and evolution in aquatic environments.

Interfering factors in the diagnosis of Senecavirus A.

BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

Use of FTA card for the detection of two RNA (CSFV and SV-A) and two DNA viruses (ASFVand SuHV-1) of importance in veterinary medicine.

The FTA card has emerged as a promising alternative for nucleic acid extraction. The FTA card is a filter paper impregnated with chemicals that preserve and stabilize the genetic material present in the sample, allowing for its storage and transport at room temperature. The aim of this study was to test the card for the detection of RNA and DNA nucleic acids. Two RNA viruses (Senecavirus A and classical swine fever virus) and two DNA viruses (African swine fever virus and suid alphaherpesvirus 1) were tested, and in all cases, there was a decrease in sensitivity. The methods exhibited good repeatability and demonstrated a rapid and practical use for sample transport and nucleic acid extraction.

Four novel picornaviruses detected in Magellanic Penguins (Spheniscus magellanicus) in Chile.

Members of the Picornaviridae family comprise a significant burden on the poultry industry, causing diseases such as gastroenteritis and hepatitis. However, with the advent of metagenomics, a number of picornaviruses have now been revealed in apparently healthy wild birds. In this study, we identified four novel viruses belonging to the family Picornaviridae in healthy Magellanic penguins, a near threatened species. All samples were subsequently screened by RT-PCR for these new viruses, and approximately 20% of the penguins were infected with at least one of these viruses. The viruses were distantly related to members of the genera Hepatovirus, Tremovirus, Gruhelivirus and Crahelvirus. Further, they had more than 60% amino acid divergence from other picornaviruses, and therefore likely constitute novel genera. Our results demonstrate the vast undersampling of wild birds for viruses, and we expect the discovery of numerous avian viruses that are related to hepatoviruses and tremoviruses in the future.

Comparison of historical and contemporary isolates of Senecavirus A.

Senecavirus A (SVA) was discovered as a cell culture contaminant in 2002, and multiple attempts to experimentally reproduce disease were unsuccessful. Field reports of porcine idiopathic vesicular disease (PIVD) cases testing PCR positive for SVA in addition to outbreaks of PIVD in Brazil and the United States in 2015 suggested SVA was a causative agent, which has now been consistently demonstrated experimentally. Ease of experimental reproduction of disease with contemporary strains of SVA raised questions concerning the difficulty of reproducing vesicular disease with historical isolates. The following study was conducted to compare the pathogenicity of SVA between historical and contemporary isolates in growing pigs. Six groups of pigs (n = 8) were intranasally inoculated with the following SVA isolates: SVV001/2002, CAN/2011, HI/2012, IA/2015, NC/2015, SD/2015. All isolates induced vesicular disease in at least half of the inoculated pigs from each group. All pigs replicated virus as demonstrated by serum and/or swab samples positive for SVA by quantitative PCR. Pig sera tested by virus neutralization assay demonstrated cross-neutralizing antibodies against all viruses utilized in the study. Cross-neutralizing antibodies from pigs inoculated with historical isolates were lower than those pigs that were inoculated with contemporary isolates. Phylogenetic analysis revealed two clades with SVV001/2002 being in a separate clade compared to the other five isolates. Although differences in the infection kinetics and sequences of these six isolates were found, clinical presentation of vesicular disease was similar between both historical and contemporary isolates.

Persistent Detection of Cosavirus and Saffold Cardiovirus in Riachuelo River, Argentina.

Cosaviruses (CoSV) and Saffold cardiovirus (SAFV) are novel members of the Picornaviridae family. The Matanza-Riachuelo river basin covers a total area of 2200 km2 with approximately 60 km long. Its last section is called Riachuelo River. The aim of this study was to describe the circulation of both picornaviruses and their relationship with the environmental situation of the Riachuelo River using 274 samples collected from 2005 to 2015. CoSV and SAFV were investigated in samples available by two periods: 2005-2006 and 2014-2015 (103 and 101, respectively). Physicochemical and bacteriological parameters confirmed very high levels of human fecal contamination during the 11 years evaluated. CoSV was detected in 85.7% (66/77) and 65.4% (17/26) of the samples collected in 2005-2006 and 2014-2015 periods, respectively. Species A and D were identified, the first one being widely predominant: 74.1% (20/27) and 75.0% (3/4) in both periods. SAFV virus was detected in 47.1% (32/68) and 52.6% (10/19) in periods 2005-2006 and 2014-2015, respectively. SAFV-6 was the most identified genotype in the entire study, while SAFV-3 was predominant in 2005-2006. The contribution of genotypes 1, 2, 4 and 8 was minor. The high prevalence of CoSV and SAFV suggests that both viruses have been circulating in Argentina at least since 2005. Our results show that a watercourse with high rates of human fecal contamination can become a persistent source of new viruses which capacity to produce human diseases is unknown.

Near-complete genome of cosavirus A from a child hospitalized with acute gastroenteritis, Brazil.

Acute gastroenteritis (AG) is responsible for 525,000 deaths worldwide in children under-5-years and is caused by the Human Cosavirus (HCoSV; family Picornaviridae, Genus Cosavirus). Although its health importance, a significant percentage of diarrhea cases (≈ 40 %) still of unknown etiology. In Brazil, few studies have reported HCoSV-A sequences analyzing partial 5' UTR. This study characterized the first near-complete genome of a Cosavirus A (strain AM326) from a child hospitalized with AG in Amazonas state, Northern Brazil. High throughput sequencing (HTS) was performed using the HiSeq™ 2500 platform (Illumina) in one fecal specimen collected from the Surveillance of Rotavirus Network of the Evandro Chagas Institute collected in 2017. Sequence reads were assembled by the De Novo approach using three distinct algorithmic (IDBA-UD, Spades, and MegaHit). The final contig was recovered from the HCoSV-AM326 sample revealing 7,735 nt in length (SRA number SRR12535029; GenBank MT023104) and the genetic characterization, as well as phylogenetic analysis demonstrated a new variant strain from Brazil, highlighting the association of HCoSV-A as a possible causative agent of AG. This finding demonstrates the importance of the metagenomic approach to elucidate cases of diarrhea without a defined etiology, as well as providing a better understanding about the virus genetics, evolution and epidemiology.

Molecular Characterization of Hovenia Dulcis-Associated Virus 1 (HDaV1) and 2 (HDaV2): New Tentative Species within the Order Picornavirales.

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5' ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

Análise do desempenho do sistema de vigilância sanitária animal no surto de doença vesicular em suínos no estado de Santa Catarina - Brasil / Analysis of the performance of the animal health surveillance system in the outbreak of swine vesicular disease in the state of Santa Catarina - Brazil

Background: The occurrence of vesicular disease associated with Senecavirus A in a pig-producing region of Santa Catarina increased in 2015, reflected by the number of syndromic notifications to the official animal health service. In view of the recurrence of this event in 2018, the objectives of this study were to analyze the official data related to cases of suspected vesicular disease in pigs and to evaluate whether the experience in conducting the investigations of 2015 was incorporated into the years subsequent to 2015. We addressed this goal by analysis of the performance parameters of the state animal health surveillance system. Materials, Methods & Results: Descriptive analyses of data from official investigations of suspected vesicular disease in swine in different regions were carried out, and statistical models were used to: i) test the effect of the year on the age of the investigated injuries; ii) assess whether there was an association between the year and the type of outcome of the official investigation (discarded case or a probable case of vesicular disease, which resulted in the collection of samples for laboratory diagnosis and interdiction of the affected properties); iii) evaluate whether there was an association between the year and the detection of Senecavirus A RNA among the molecular analyses carried out after a case was classified as probable vesicular disease. From 05/22/2015 to 03/28/2019, there were 2093 notifications of suspected vesicular disease in pigs to the official service of Santa Catarina, with 1538 (73.5%) occurring in 2015 and 555 (26.5%) in subsequent years. After 2015, when compared to the base year, the chances of detecting late vesicular lesions (>3 days) were similar (increased 1.11 times, but there was no statistically significant association), in view of a panorama in which 55.29% of cases had the lesions classified as late throughout the analyzed period. The...

Human virome in nasopharynx and tracheal secretion samples.

BACKGROUND: In Brazil the implementation of the Sentinel Surveillance System of Influenza began in 2000. Central public health laboratories use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for diagnosis of respiratory viruses, but this protocol identifies only specific targets, resulted in inconclusive diagnosis for many samples. Thus, high-throughput sequencing (HTS) would be complementary method in the identification of pathogens in inconclusive samples for RT-qPCR or other specific detection protocols. OBJECTIVES: This study aimed to detect unidentified viruses using HTS approach in negative samples of nasopharynx/tracheal secretions by the standard RT-qPCR collected in the Federal District, Brazil. METHODS: Nucleic acids were extracted from samples collected in winter period of 2016 and subjected to HTS. The results were confirmed by the multiplex PR21 RT-qPCR, which identifies 21 respiratory pathogens. FINDINGS: The main viruses identified by HTS were of families Herpesviridae, Coronaviridae, Parvoviridae and Picornaviridae, with the emphasis on rhinoviruses. The presence of respiratory viruses in the samples was confirmed by the PR21 multiplex RT-qPCR. Coronavirus, enterovirus, bocavirus and rhinovirus were found by multiplex RT-qPCR as well as by HTS analyses. MAIN CONCLUSIONS: Wide virus diversity was found by different methodologies and high frequency of rhinovirus occurrence was confirmed in population in winter, showing its relevance for public health.

Seneca Valley virus RNA detection in pig feed and feed ingredients in Brazil.

We investigated Seneca Valley virus (SVV) contamination in pig feed and feed ingredients. Twenty-seven samples were collected from two Brazilian feed mills and subjected to conventional RT-nested-PCR and qRT-PCR assays. Seven samples were SVV-positive with viral loads of 3.94-4.33 log10 genomic copies/g of feed. The study reveals SVV feed and feed ingredient contamination under natural conditions in Brazil.

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples.

Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot-and-mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT-ddPCR) using one-step and two-step approaches. Analytical sensitivity and specificity were done in parallel with real-time PCR, RT-qPCR (one-step and two-step) for comparison of sensitivity and specificity of both methods. In the standardization of RT-ddPCR, the double-quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one-step techniques showed superior performance than two-step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT-ddPCR and RT-qPCR using the one-step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT-PCR had a better performance than RT-PCR when swine serum pools were tested. According to the results, the one-step RT-ddPCR and RT-qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT-ddPCR), being a useful tool for vesicular diseases control programs.

Longitudinal survey of Teschovirus A, Sapelovirus A, and Enterovirus G fecal excretion in suckling and weaned pigs.

Fecal samples from 27 pigs were longitudinally analyzed for Teschovirus A (TV-A), Sapelovirus A (SV-A), and Enterovirus G (EV-G) RNA presence. Suckling piglet fecal samples were negative for the three enteric picornaviruses. However, these picornaviruses were detected in 22/27 weaned pig fecal samples. This study provides new data on TV-A, SV-A, and EV-G infection dynamics.

Human virome in nasopharynx and tracheal secretion samples

BACKGROUND In Brazil the implementation of the Sentinel Surveillance System of Influenza began in 2000. Central public health laboratories use reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for diagnosis of respiratory viruses, but this protocol identifies only specific targets, resulted in inconclusive diagnosis for many samples. Thus, high-throughput sequencing (HTS) would be complementary method in the identification of pathogens in inconclusive samples for RT-qPCR or other specific detection protocols. OBJECTIVES This study aimed to detect unidentified viruses using HTS approach in negative samples of nasopharynx/tracheal secretions by the standard RT-qPCR collected in the Federal District, Brazil. METHODS Nucleic acids were extracted from samples collected in winter period of 2016 and subjected to HTS. The results were confirmed by the multiplex PR21 RT-qPCR, which identifies 21 respiratory pathogens. FINDINGS The main viruses identified by HTS were of families Herpesviridae, Coronaviridae, Parvoviridae and Picornaviridae, with the emphasis on rhinoviruses. The presence of respiratory viruses in the samples was confirmed by the PR21 multiplex RT-qPCR. Coronavirus, enterovirus, bocavirus and rhinovirus were found by multiplex RT-qPCR as well as by HTS analyses. MAIN CONCLUSIONS Wide virus diversity was found by different methodologies and high frequency of rhinovirus occurrence was confirmed in population in winter, showing its relevance for public health.

Development and evaluation of a nested-PCR assay for Senecavirus A diagnosis.

Senecavirus A (SVA) has been associated with vesicular disease in weaned and adult pigs and with high mortality of newborn piglets. This study aimed to establish a nested-PCR assay for the routine diagnosis of SVA infection. Tissue samples (n = 177) were collected from 37 piglets of 18 pig farms located in four different Brazilian states. For the nested-PCR, a primer set was defined to amplify an internal VP1 fragment of 316 bp of SVA genome. Of the 37 piglets, 15 (40.5%) and 23 (62.2%) were positive for the SVA in the RT-PCR and nested-PCR assays, respectively. The SVA RNA was detected in 61/177 (34.5%) samples with the RT-PCR, while the nested-PCR assay showed 84/177 (47.5%) samples with the virus (p < 0.05). According to the herds, 11 (61.1%) and 16 (88.9%) of the 18 pig herds were positive for the SVA in the RT-PCR and nested-PCR assays, respectively. Nucleotide sequencing analysis revealed similarities of 98.7-100% among SVA Brazilian strains and of 86.6-98% with SVA strains from other countries. The nested-PCR assay in this study was suitable to recover the SVA RNA in biological specimens, piglets, and/or herds that were considered as negative in the RT-PCR assay, and is proposed for the routine investigation of the SVA infection in piglets, especially when other techniques are not available or when a great number of samples has to be examined.

Quantitative analysis of senecavirus A in tissue samples from naturally infected newborn piglets.

In this study, we determined the distribution of senecavirus A (SVA) and viral RNA load in different organs and tissues of naturally infected piglets. A TaqMan-based qRT-PCR assay was performed using RNA extracted from brainstem, cerebellum, cerebrum, heart, kidney, liver, lungs, small intestine, spleen, urinary bladder, and tonsils of seven newborn piglets. SVA was detected in 57 out of 70 tissue samples (81.4%). Viral loads ranged from 4.07 to 10.38 log10 genomic copies per g of tissue. The results show that SVA has tropism for various organs in naturally infected newborn piglets, especially for tonsils, spleen, lungs, and liver. Lymphoid organs had the highest viral loads and may be important sites for SVA replication.

Emergence and whole-genome sequence of Senecavirus A in Colombia.

In 2015 and 2016, Senecavirus A (SVA) emerged as an infectious disease in Brazil, China and the United States (US). In a Colombian commercial swine farm, vesicles on the snout and coronary bands were reported and tested negative for foot-and-mouth disease virus (FMDv), but positive for SVA. The whole-genome phylogenetic analysis indicates the Colombian strain clusters with the strains from the United States, not with the recent SVA strains from Brazil.

Genomic characterization and phylogenetic analysis of Chinese sacbrood virus isolated from Loess Plateau, China.

The complete genomic RNA of the Chinese sacbrood virus (CSBV) strain, which infects the honeybees in the Loess plateau, was sequenced and analyzed. The CSBV-SX strain contains 8705 nucleotides, which includes a single large open reading frame (99-8681 nucleotides) encoding 2860 amino acids. A novel efficient identification method was used to investigate the samples infected by CSBV. The putative amino acid sequence alignment analysis showed that, except for some normal well characterized domains such as RNA helicase, RNA protease, and RNA-dependent RNA polymerase domains, a calicivirus coat protein domain was identified at amino acids 493-564. Phylogenetic analysis indicated that CSBV-SX was closely related to CSBV-BJ, and this result was supported by nucleotide multiple sequence alignment and protein multiple sequence alignment analysis results. These differences in the CSBV-SX strain may be related to virus adaptation to the xerothermic, low relative humidity, and strong ultraviolet radiation conditions in the Loess Plateau.

Clinical Manifestations of Senecavirus A Infection in Neonatal Pigs, Brazil, 2015.

We identified new clinical manifestations associated with Senecavirus A infection in neonatal piglets in Brazil in 2015. Immunohistochemical and molecular findings confirmed the association of Senecavirus A with these unusual clinical signs and more deaths. Other possible disease agents investigated were not associated with these illnesses.

Invasive ants carry novel viruses in their new range and form reservoirs for a honeybee pathogen.

When exotic animal species invade new environments they also bring an often unknown microbial diversity, including pathogens. We describe a novel and widely distributed virus in one of the most globally widespread, abundant and damaging invasive ants (Argentine ants, Linepithema humile). The Linepithema humile virus 1 is a dicistrovirus, a viral family including species known to cause widespread arthropod disease. It was detected in samples from Argentina, Australia and New Zealand. Argentine ants in New Zealand were also infected with a strain of Deformed wing virus common to local hymenopteran species, which is a major pathogen widely associated with honeybee mortality. Evidence for active replication of viral RNA was apparent for both viruses. Our results suggest co-introduction and exchange of pathogens within local hymenopteran communities. These viral species may contribute to the collapse of Argentine ant populations and offer new options for the control of a globally widespread invader.