RESUMO
The multiplex molecular diagnostic assays described for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A (IAV) and B (IBV) viruses have been mainly based on real-time reaction, which limits their access to many laboratories or diagnostic institutions. To contribute to available strategies and expand access to differential diagnosis, we describe an end-point multiplex RT-PCR targeting SARS-CoV-2, IAV and IBV with simultaneous endogenous control amplification. Initially, we looked for well-established primers sets for SARS-CoV-2, IAV, IBV and RNAse P whose amplicons could be distinguished on agarose gel. The multiplex assay was then standardized by optimizing the reaction mix and cycle conditions. The limit of detection (LoD) was determined using titrated viruses (for SARS-CoV-2 and IAV) and by dilution from a pool of IBV-positive samples. The diagnostic performance of the multiplex was evaluated by testing samples with different RNAse P and viral loads, previously identified as positive or negative for the target viruses. The amplicons of IAV (146 bp), SARS-CoV-2 (113 bp), IBV (103 bp) and RNAse P (65 bp) were adequately distinguished in our multiplex. The LoD for SARS-CoV-2, IAV and IBV was 0.02 TCID50/ml, 0.07 TCID50/ml and 10-3 from a pool of positive samples, respectively. All samples positive for SARS-CoV-2 (n=70, Ct 17.2-36.9), IAV (n=53, Ct 14-34.9) and IBV (n=12, Ct 23.9-31.9) remained positive in our multiplex assay. RNAse P from negative samples (n=40, Ct 25.2-30.2) was also amplified in the multiplex. Overall, our assay is a timely and alternative tool for detecting SARS-CoV-2 and influenza viruses in laboratories with limited access to supplies/equipment.
Assuntos
COVID-19 , Vírus da Influenza A , Vírus da Influenza B , Reação em Cadeia da Polimerase Multiplex , Ribonuclease P , SARS-CoV-2 , Humanos , Ribonuclease P/genética , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/genética , Vírus da Influenza B/isolamento & purificação , Vírus da Influenza B/genética , COVID-19/diagnóstico , COVID-19/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Diagnóstico Diferencial , Influenza Humana/diagnóstico , Influenza Humana/virologia , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Limite de Detecção , RNA Viral/genética , RNA Viral/análiseRESUMO
Papillomaviruses (PVs) have been identified in several animal species, including dogs (canine papillomaviruses, CPVs) and cattle (bovine papillomaviruses, BPVs). Although some BPVs may occasionally infect species other than cattle, to the best of our knowledge, BPVs have not been reported in dogs to date. Herein, we carried out a retrospective phylogenetic study of PVs circulating in dogs from southern Brazil between 2017 and 2022, also investigating possible mixed infections and spillover events. For this, we screened 32 canine papilloma samples by PCR using the degenerate primers FAP59/64 and/or MY09/11, which amplify different regions of the L1 gene; the genomic target often used for PV classification/typing. Out these, 23 PV DNA samples were successfully amplified and sequenced. All PVs amplified by FAP59/64 (n = 22) were classified as CPV-1. On the other hand, PVs amplified by MY09/11 (n = 4) were classified as putative BPV-1. Among these, three samples showed mixed infection by CPV-1 and putative BPV-1. One of the putative BPV-1 detected in co-infected samples had the L1 gene full-sequenced, confirming the gene identity. Furthermore, the phylogenetic classifications from the FAP59/64 and/or MY09/11 amplicons were supported by a careful in silico analysis, which demonstrated that the analysis based on them matches to the classification from the complete L1 gene. Overall, we described CPV-1 circulation in southern Brazil over the years and the potencial BPV infection in dogs (potential spillover event), as well as possible CPV/1/BPV-1 co-infections. Finally, we suggest the analysis of the complete genome of the putative BPVs detected in dogs in order to deepen the knowledge about the PV-host interactions.
Assuntos
Coinfecção , Doenças do Cão , Epidemiologia Molecular , Papillomaviridae , Infecções por Papillomavirus , Filogenia , Animais , Cães , Brasil/epidemiologia , Doenças do Cão/virologia , Doenças do Cão/epidemiologia , Infecções por Papillomavirus/veterinária , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/epidemiologia , Papillomaviridae/genética , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Estudos Retrospectivos , Coinfecção/virologia , Coinfecção/veterinária , Coinfecção/epidemiologia , DNA Viral/genéticaRESUMO
Canine distemper virus (CDV) is a major threat to domestic dogs and wildlife worldwide. Molecular assays are the most sensitive and specific tests to diagnose the disease, however, the high CDV genetic variability may compromise laboratory diagnosis. Herein, we designed a high-coverage primer set for end-point (RT-PCR) and real-time (RT-qPCR) for CDV detection. Initially, we collected 194 complete/near-complete CDV genomes (GenBank) and analyzed them for highly conserved regions for primer design. We then assessed the in silico coverage, analytical sensitivity, specificity and diagnostic performance of RT-PCR/RT-qPCR reactions based on our primers. Furthermore, the coverage of our primers, as well as their analytical sensitivity and diagnostic performance, were compared to a commonly used primer set for CDV detection (named PP-I). Our forward (F) and reverse (R) primers fully matched 100 % (194/194) and 99 % (192/194) of the analyzed sequences, whereas the PP-I F and R primers fully matched 15 % (29/194) and 9 % (18/194) sequences, respectively. The detection limit of our RT-PCR and RT-qPCR was equivalent to that of PP-I primers (0.001 TCID50/mL). Out of 70 clinical samples tested, 38 were positive by our RT-PCR/RT-qPCR assays, whereas reactions with primers PP-I failed to detect 9/28 (32 %) positive samples selected for comparison purposes. In addition, our assays did not amplify other canine viruses associated with respiratory and neurological diseases: canine adenovirus 2, canine parainfluenza virus 2, canine herpesvirus 1 and rabies virus. Overall, we describe a high-coverage primer set for CDV detection, which represents an attractive tool for laboratory diagnosis of canine distemper.
Assuntos
Vírus da Cinomose Canina , Cinomose , Animais , Cães , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus da Cinomose Canina/genética , Sensibilidade e Especificidade , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Cinomose/diagnósticoRESUMO
The mpox outbreaks reported in several countries from May 2022 have shown an epidemiological profile different from that observed in previous years, raising a global public health alert. This issue is particularly important for Brazil, the second country with the highest number of mpox cases. Herein, we performed a retrospective cross-sectional study on mpox cases notified in Pernambuco state, northeastern Brazil, between July 2022 and March 2023. Confirmed mpox cases were analyzed in a space-time series and their social and clinical characteristics were compared with those of suspect-negative cases, including a multivariate logistic regression to identify predictors associated with a positive diagnosis. A total of 1493 suspected mpox cases were reported, of which 362 cases (24.2%) were confirmed and distributed in 33 municipalities. Most mpox cases occurred between epidemiological weeks (EW) 33 and 39 of 2022, with the highest moving average in EW 34 and 35 (36 and 31.5, respectively). The most frequent clinical signs and symptoms were rash (87.3%), fever (60.2%), headache (45.3%), and genital/perianal lesions (40.3%). In the multivariate analysis, three variables showed considerable performance in predicting a positive mpox diagnosis (area under the ROC curve = 0.87; 95% CI: 0.84-0.90): sexual orientation (nonheterosexual; OR: 23.08; 95% CI: 13.97-38.15), male sex (OR: 2.05; 95% CI: 1.10-3.85), and multiple partnerships (OR: 1.95; 95% CI: 1.15-3.32). Overall, in addition to the detailed spatiotemporal description of mpox cases, which may contribute to appropriate public health measures, our study brings insights into mpox epidemiology by describing predictors associated with a positive diagnosis.
Assuntos
Mpox , Feminino , Humanos , Masculino , Brasil/epidemiologia , Estudos Transversais , Estudos Retrospectivos , Análise Espaço-TemporalRESUMO
Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped based on analysis of single genomic regions, mainly the 5' untranslated region (UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classification. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for BVDV subtyping. Initially, genomic regions previously described as the most suitable targets for BVDV subtyping were analyzed for design of high-coverage primers. The putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 BVDV-1 and 88 BVDV-2 complete/near-complete genomes (CNCGs) (GenBank). This analysis was also performed considering the region amplifiable by primers HCV90-368, 324-326 and BP189-389 (5'UTR), which have been used for BVDV diagnosis and/or classification. After confirming the agreement between the analyses of our primers' amplicon versus the CNCGs, we optimized the RT-PCRs and evaluated their performance for amplification of BVDV isolates/strains (n = 35 for BVDV-1; n = 33 for BVDV-2). Among the potential targets for BVDV subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526 bp amplicon) and NS5B (BVDV-2) (728 bp). The classification based on these regions fully reproduced the subtyping of all CNCGs. On the other hand, subtyping based on the putative amplicons from primers HCV90-368, 324-326 and BP189-389 showed disagreements in relation the CNCG analysis. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains tested. Finally, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDVs.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Vírus da Diarreia Viral Bovina Tipo 1 , Vírus da Diarreia Viral Bovina Tipo 2 , Vírus da Diarreia Viral Bovina , Animais , Bovinos , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Filogenia , Genômica , Regiões 5' não Traduzidas/genética , Vírus da Diarreia Viral Bovina/genéticaRESUMO
Since its identification in late 2019, SARS-CoV-2 has undergone numerous mutations, resulting in the emergence of several viral variants, which may differ in transmissibility, virulence and/or evasion from host immunity. Particularly, immunity-related changes have been well documented in the Omicron variant, including reports of escaping neutralizing antibodies induced by infection/vaccination with heterologous SARS-CoV-2 or used in serological therapy. These findings may encourage some discussions about the possibility that Omicron is a distinct SARS-CoV-2 serotype. To contribute to this issue, we combined concepts from immunology, virology and evolution and performed an interesting brainstorm on the hypothesis that Omicron is a distinct SARS-CoV-2 serotype. Furthermore, we also discussed the likelihood of emergence of SARS-CoV-2 serotypes over time, which may not necessarily be related to Omicron. Finally, insights into this topic may have direct implications for vaccine formulations, immunodiagnostic platforms and serological therapies, contributing to better management of future outbreaks or waves.
Assuntos
COVID-19 , Humanos , SARS-CoV-2/genética , Sorogrupo , Anticorpos Neutralizantes , Anticorpos Antivirais , Glicoproteína da Espícula de CoronavírusRESUMO
Neonatal calf diarrhea (NCD) is frequently associated with single or mixed viral, bacterial and/or protozoal infections. Consequently, laboratory diagnostic of NCD usually requires specific tests for each potential agent; a time-consuming, laborious and expensive process. Herein, we describe an end-point multiplex PCR/reverse transcription-PCR (RT-PCR) for detection of five major NCD agents: bovine rotavirus (BRV), bovine coronavirus (BCoV), Escherichia coli K99 (E. coli K99), Salmonella enterica (S. enterica) and Cryptosporidium parvum (C. parvum). Initially, we selected and/or designed high-coverage primers. Subsequently, we optimized multiplex PCR/RT-PCR conditions. Next, we evaluated the analytical sensitivity of the assay and assessed the performance of the reaction by testing 95 samples of diarrheic calf feces. The analytical specificity was evaluated against bovine viral diarrhea virus (BVDV), E. coli heat-stable enterotoxin (STa) and Eimeria spp. The detection limit of our assay was about 10 infectious units of BRV, 10-2 dilution of a BCoV positive sample pool, about 5 × 10-4 CFU for S. enterica, 5 × 10-6 CFU for E. coli K99 and 50 oocysts for C. parvum. No non-specific amplification of other bovine diarrhea agents was detected. Out of 95 samples analyzed, 50 were positive for at least one target, being 35 single and 15 mixed infections. BRV was the most frequent agent detected in single infections (16/35), followed by Cryptosporidium spp. (11/35), which was the most frequent in mixed infections (11/15). Positive and negative multiplex results were confirmed in individual reactions. In conclusion, we described an end-point multiplex PCR/RT-PCR for faster and easier NCD diagnosis, which may be useful for routine diagnosis and surveillance studies.
Assuntos
Coinfecção , Criptosporidiose , Cryptosporidium parvum , Cryptosporidium , Doenças não Transmissíveis , Recém-Nascido , Humanos , Reação em Cadeia da Polimerase Multiplex , Escherichia coli , Criptosporidiose/diagnóstico , Transcrição Reversa , Diarreia/diagnóstico , Diarreia/veterinária , Cryptosporidium parvum/genéticaRESUMO
Bovine papillomaviruses (BPVs) exhibit a high degree of genetic variability, and several viral types have been identified based on analysis of the L1 gene. The L1 is the main capsid protein and the main target for neutralizing antibodies. We performed a retrospective study on BPVs circulating in Rio Grande do Sul state, Southern Brazil, in 2016-2020. DNA from 43 bovine papilloma samples were amplified using two degenerate primer sets - FAP59/64 and MY09/11 - targeting the L1 region, and analyzed for phylogeny, mixed BPV infections (coinfections) and amino acid (aa) sequences. We also performed an in silico analysis with 114 BPV L1 sequences from the GenBank database to assess the agreement between the phylogeny obtained based on complete L1 sequences versus that based on the region amplified using the FAP59/64 and MY09/11 primer sets. Considering single and coinfections, we identified 31 BPV-1 (31/43; 72.1%), 27 BPV-2 (27/43; 62.8%) and 4 BPV-6 (4/43; 9.3%). Coinfections with BPV-1 and BPV-2 were observed in 61.3% of the samples. Our results are supported by in silico analyses that demonstrate that the classification using FAP59/64 or MY09/11 matches the complete L1 results, except for BPV-17 and -18, which may be mistakenly classified depending on the primers used. Furthermore, we found unique or rare amino acids in at least one L1 sequence of each BPV type identified in our study, some of which have been identified previously in papillomavirus epitopes, suggesting immune-mediated selection. Finally, our study provides an overview of BPVs circulating in Southern Brazil over the last five years and point to the combined use of primers FAP59/64 and MY09/11 for analysis of BPV coinfections and putative epitopes.
Assuntos
Papillomavirus Bovino 1 , Doenças dos Bovinos , Coinfecção , Infecções por Papillomavirus , Animais , Bovinos , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/veterinária , Filogenia , Brasil/epidemiologia , Aminoácidos/genética , Estudos Retrospectivos , DNA Viral/genética , Papillomaviridae/genética , Doenças dos Bovinos/epidemiologiaRESUMO
Sequence analysis of the canine distemper virus (CDV) hemagglutinin (H) gene may provide important insights on virus-host interactions and has also been frequently used for CDV phylogenetic classification. Herein, we performed an in silico analysis of CDV complete genomes (CGs) available in GenBank in order to investigate the suitability of H for CDV classification into lineages/genotypes. In addition, we analyzed the other viral genes for their potential use in CDV classification. Initially, we collected 116 CDV CGs from GenBank and compared their phylogenetic classification with that of their respective H nucleotide (nt) and amino acid (aa) sequences. Subsequently, we calculated the geodesic distance between the CG and H phylogenetic trees. These analyses were later performed with other CDV genes. All CDV CGs were also evaluated for possible recombination events. Nucleotide and aa analyses of H misclassified some Vaccine/America 1/Asia 3 lineage sequences compared to CG analysis, finding supported by both Maximum Likelihood (ML) and Bayesian Markov Chain Monte Carlo (B-MCMC) methods. Moreover, aa-based H analysis showed additional disagreements with the classification obtained by CG. The geodesic distance between the H and CG trees was 0.0680. Strong recombination signals were identified in the H gene, including Vaccine/America 1/Asia 3 lineage sequences. In contrast, C and P were the only genes that fully reproduced the CG classification (by ML and/or B-MCMC) and that did not show strong recombination signals. Furthermore, the P phylogenetic tree showed the lowest geodesic distance from the CG tree (0.0369). These findings suggest C and P as potential targets for CDV phylogenetic classification, especially when full genome sequencing is not possible. Finally, since our results were obtained considering the CDV CGs available to date, future analyses performed as more CDV sequences become available will be useful to assess probable issues of H-based phylogeny and to consolidate the suitability of the C and P genes for CDV classification.
Assuntos
Vírus da Cinomose Canina , Cinomose , Animais , Cães , Filogenia , Vírus da Cinomose Canina/genética , Hemaglutininas , Teorema de Bayes , NucleotídeosRESUMO
Bovine pestiviruses are members of the species Pestivirus A (bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (BVDV-2) or Pestivirus H (HoBiPeV). To date, BVDV-2 isolates/strains have been classified into three subtypes (a-c) by phylogenetic analysis, and an additional subtype (d) has been proposed based on 5' untranslated region (UTR) secondary structures. In a previous study, we identified some BVDV-2 sequences in the GenBank database that could not be classified as subtype a, b or c by phylogenetic analysis of their genomes, UTRs or individual genes. Here, we performed a detailed study of these sequences and assessed whether they might represent a distinct BVDV-2 subtype. Initially, we collected 85 BVDV-2 complete/near-complete genomes (CNCGs) from GenBank and performed a "proof of equivalence" between phylogenetic analyses based on CNCGs and open reading frames (ORFs), which showed that ORFs may be reliably used as a reference target for BVDV-2 phylogeny, allowing us to increase our dataset to 139 sequences. Among these, we found seven sequences that could not be classified as BVDV-2a-c. The same was observed in the phylogenetic analysis of CNCGs and viral genes. In addition, the seven non-BVDV-2a-c sequences formed a distinct cluster in all phylogenetic trees, which we propose to term BVDV-2e. BVDV-2e also showed 44 amino acid changes compared to BVDV-2a-c, 20 of which are in well-defined positions. Importantly, an additional phylogenetic analysis including BVDV-2d and a pairwise comparison of BVDV-2e and BVDV-2d sequences also supported the difference between these subtypes. Finally, we propose the recognition of BVDV-2e as a distinct BVDV-2 subtype and encourage its inclusion in future phylogenetic analyses to understand its distribution and evolution.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina , Vírus da Diarreia Viral Bovina Tipo 1 , Vírus da Diarreia Viral Bovina Tipo 2 , Vírus da Diarreia Viral Bovina , Pestivirus , Animais , Bovinos , Vírus da Diarreia Viral Bovina Tipo 2/genética , Filogenia , Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina/genética , Pestivirus/genética , Regiões 5' não Traduzidas/genéticaRESUMO
We report the nearly complete genome sequence of a Brazilian bovine enterovirus (genus Enterovirus, family Picornavirus). This enterovirus was isolated from an enteric and respiratory disease outbreak in a beef cattle herd in southern Brazil. Phylogeny indicates that this isolate belongs to the species Enterovirus E.
RESUMO
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease characterized by reproductive impairment or failure in breeding animals, and a respiratory disease in pigs of any age. Brazil is the fourth largest pork producer and exporter globally, and PRRS virus (PRRSV) infection has never been reported in the country. This study aimed to investigate the status of porcine biological samples from commercial swine herds, quarantined imported boars, wild boars and feral pigs to update PRRS information in Brazil. A total of 14,382 samples were collected from 2008 to 2020, including sera (n = 12,841), plasma (n = 1,000) and oral fluids (n = 541), comprehending 137 herds and free-living pigs in eight Brazilian states. One out of 1,000 (0.1%) plasma and 15 out of 12,841 (0.11%) serum samples tested positive for PRRSV antibodies through ELISA. Upon ELISA retesting, only the plasma sample, from one 8-day-old piglet remained positive. All sixteen previously PRRSV antibody-positive samples were tested through RT-PCR and found to be negative. The presence of false-positive or singleton reactors are quite expected. Thus, the use of different/alternative diagnostic tests is indicated for an efficient PRRSV detection. Taken together, our findings demonstrated no conclusive evidence of PRRSV infection in the tested pigs, highlighting the importance to reinforce the surveillance program to prevent the introduction and eventual dissemination of PRRSV in Brazil.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Doenças dos Suínos , Animais , Anticorpos Antivirais , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Estudos Retrospectivos , SuínosRESUMO
Bovine viral diarrhea virus-1 (BVDV-1, Pestivirus A) and BVDV-2 (Pestivirus B) have been clustered into 21 and 4 subtypes, respectively. This genetic diversity, in addition to the lack of consensus on which genomic region to use for BVDV subtyping, has resulted in conflicting classifications depending on the target analyzed. Here, we investigated which genes or UTRs would reproduce the phylogeny obtained by complete genome (CG) analyses. The study was carried out with 91 (BVDV-1) and 85 (BVDV-2) CG available on GenBank database. The viruses were subtyped by analyzing their CG, as well as their individual genes and UTRs (complete 3' and 5'UTRs, and partial 5'UTR); and the phylogeny results were compared to each other. The sequences were aligned using the ClustalW multiple method (BioEdit Alignment Editor software, v.7.0.5.3) and the phylogenetic analyses were performed by the Maximum Likelihood method (MEGA-X software, v.10.2.4), with 1000 bootstrap replicates. The best analysis model for each gene/UTR was defined using the jModelTest software. The geodesic distance between the CG (reference) and individual genes/UTRs trees was also calculated (TreeCmp software, v.2.0). In general, 3'UTR-based analyses, followed by 5'UTR, presented the least reliable subtyping results. Regarding BVDV-1, phylogeny based on C, Erns, E1, E2, p7, NS2, NS3, NS4B, NS5A and NS5B was consistent with that of CG. In contrast, analyses performed with individual BVDV-2 genes showed at least one different clustering from the phylogeny based on the CG. After analyzing the geodesic distance between the CG and genes/UTRs trees, we observed that NS4B (for BVDV-1) and NS5A (BVDV-2) presented the closest topology and edge length to the CG analyses. Finally, comparing the phylogeny performed with the CG and the genes/UTRs, as well as the geodesic distance between them, we understand that NS4B and NS5A represent the most suitable targets for BVDV-1 and -2 subtyping, respectively, and may be considered in future phylogenetic studies.
Assuntos
Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Genes Virais , Filogenia , Vírus da Diarreia Viral Bovina Tipo 1/classificação , Vírus da Diarreia Viral Bovina Tipo 2/classificação , Sequenciamento Completo do GenomaRESUMO
The parapoxvirus orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host innate and pro-inflammatory responses to infection. Using the ORFV IA82 strain as the parental virus, recombinant viruses with individual deletions in the genes encoding the IMPs chemokine binding protein (CBP; ORFV112), inhibitor of granulocyte-monocyte colony-stimulating factor and IL-2 (GIF, ORFV117) and interleukin 10 homologue (vIL-10; ORFV127) were generated and characterized in vitro and in vivo. The replication properties of the individual gene deletion viruses in cell culture was not affected comparing with the parental virus. To investigate the effect of the individual gene deletions in ORFV infection and pathogenesis, groups of four lambs were inoculated with each virus and were monitored thereafter. Lambs inoculated with either recombinant or with the parental ORFV developed characteristic lesions of contagious ecthyma. The onset, nature and severity of the lesions in the oral commissure were similar in all inoculated groups from the onset (3 days post-inoculation [pi]) to the peak of clinical lesions (days 11-13 pi). Nonetheless, from days 11-13 pi onwards, the oral lesions in lambs inoculated with the recombinant viruses regressed faster than the lesions produced by the parental virus. Similarly, the amount of virus shed in the lesions were equivalent among lambs of all groups up to day 15 pi, yet they were significantly higher in the parental virus group from day 16-21 pi. In conclusion, individual deletion of these IMP genes from the ORFV genome resulted in slight reduction in virulence in vivo, as evidenced by a reduction in the duration of the clinical disease and virus shedding.
Assuntos
Genes Virais/imunologia , Vírus do Orf/genética , Vírus do Orf/patogenicidade , Doenças dos Ovinos/virologia , Fatores Etários , Animais , Citocinas/genética , Citocinas/imunologia , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Genoma Viral , Mutação , Vírus do Orf/imunologia , Ovinos/virologia , Doenças dos Ovinos/imunologia , Transdução de Sinais , Virulência , Replicação Viral/genética , Eliminação de Partículas Virais , Sequenciamento Completo do GenomaRESUMO
The envelope glycoprotein E2 of pestiviruses is a major target for neutralizing antibodies. In this study, we analyzed the E2 DA domain of 43 pestiviruses from Southern Brazil. The isolates were identified as Bovine viral diarrhea virus (BVDV) subtypes 1a and 1b or BVDV-2b. Compared to reference strains, the BVDV-1 and -2 isolates had four and two mutations in the DA domain, respectively. All BVDV-2 isolates had a deletion of residues 724 and 725. All mutated amino acids in the BVDV isolates had the same aa substitution, and all were in previously identified antibody binding sites. It is possible that an immunity-mediated selection is acting on the pestiviruses circulating in Southern Brazil.
Assuntos
Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/isolamento & purificação , Proteínas do Envelope Viral/genética , Animais , Antígenos Virais/genética , Sítios de Ligação de Anticorpos/genética , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Brasil/epidemiologia , Bovinos , Vírus da Diarreia Viral Bovina/classificação , Vírus da Diarreia Viral Bovina/imunologia , Mutação , Filogenia , RNA Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Real-time reverse transcription-polymerase chain reaction (RT-qPCR) is considered the "gold standard" for the direct diagnosis of SARS-CoV-2 infections. However, routine diagnosis by RT-qPCR is a limitation for many laboratories, mainly due to the infrastructure and/or disproportionate relationship between demand and supply of inputs. In this context, and to increase the diagnostic coverage of SARS-CoV-2 infections, we describe an alternative, sensitive and specific one-step end-point RT-PCR for the detection of the SARS-CoV-2 E gene. The performance of the RT-PCR was evaluated in 43 clinical samples, of which 10 and 33 were previously identified as negative and positive, respectively, by RT-qPCR. Among the positive samples, 15 and 18 were from asymptomatic and symptomatic individuals, respectively. Here, 32/33 of the positive samples in the RT-qPCR, including from asymptomatic individuals, were found positive in the RT-PCR (Ct 15.94-34.92). The analytical sensitivity of the assay was about 7.15-9 copies of vRNA/µL, and nonspecific amplifications were not observed in SARS-CoV-2 negative samples. Importantly, the RT-PCR reactions were performed in a 10 µL final volume. Finally, considering specificity, analytical sensitivity and cost reduction, we believe that the RT-PCR platform described here may be a viable option for the diagnostic of SARS-CoV-2 infections in laboratories in which RT-qPCR is not available.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , COVID-19/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Humanos , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
HoBi-like pestiviruses (HoBiPeV) constitute a novel group of bovine pestiviruses, genetically and antigenically related to bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2. Recent data shows that HoBiPeV are endemic among Brazilian cattle, yet bovine reproductive/respiratory vaccines contain only BVDV-1 and BVDV-2 strains. The present study investigated the neutralizing antibody response against these pestiviruses induced by two commercial vaccines (VA = attenuated, VI = inactivated) and by three experimental, replicative, vaccine formulations (VAC1 = monovalent, BVDV-1; VAC2 = bivalent, BVDV-1 + BVDV-2; VAC3 = trivalent, BVDV-1 + BVDV-2 and HoBiPeV). Seronegative beef calves were immunized once (replicative vaccines) or twice (inactivated vaccine) and serum samples were tested by virus-neutralization (VN) 30 days after vaccination (dpv) (replicative vaccines) or 30 days after the second dose (VI). We considered a threshold VN titer of ≥60 indicative of protection against clinical disease. At 30 dpv, VA induced protective titers against BVDV-2 in 7/7 animals (GMT=289.8) and against BVDV-1 and HoBiPeV in 5/7 animals (GMTs=97.5 and 80, respectively). VI induced protective titers against BVDV-1 in 1/7 animal (GMT=16.4), 2/7 animals against BVDV-2 (GMT=53.8) and in none of the calves against HoBiPeV (GMT=12.2). When a pool of sera of each vaccine group was tested against individual Brazilian isolates, VA induced protective titers against 3/7 BVDV-1 isolates, to 9/10 (BVDV-2) and 1/8 (HoBiPeV); VI induced protective titers against 1/7 (BVDV-1), 1/10 (BVDV-2) and none (0/8) HoBiPeV isolates. The experimental vaccine VAC1 induced protective titers against BVDV-1 in 9/9 animals (GMT=320) but in no animal against BVDV-2 or HoBiPeV (GMT<10). VAC2 induced protective titers to BVDV-1 and BVDV-2 in 9/9 animals (GMTs=160 and 640, respectively), and against HoBiPeV in 7/9 animals (GMT=108.5). Finally, VAC3 induced protective titers in all animals against BVDV-1 (GMT=234.3), BVDV-2 (294.9) and HoBiPeV (201.1). Testing the pool of sera against pestivirus isolates, VAC1 induced titers ≥ 60 against 4/7 BVDV-1 but to none BVDV-2/HoBiPeV isolate; VAC2 induced protective titers against 4/7 BVDV-1; 10/10 BVDV-2 and 2/8 HoBiPeV; VAC3 induced protective titers against all BVDV-1, BVDV-2 and HoBiPeV isolates. These results indicate that vaccines composed by BVDV-1+BVDV-2, especially those containing inactivated virus, may not induce serological response against a variety of HoBiPeV isolates. Thus, the need of inclusion of HoBiPeV in vaccine formulations should be considered.(AU)
Os pestivírus HoBi-like (HoBiPeV) compõe um grupo novo de pestivírus de bovinos, genética e antigenicamente relacionados com os vírus da diarreia viral bovina 1 e 2 (BVDV-1, BVDV2). Dados recentes indicam que os HoBiPeV são endêmicos na população bovina do Brasil, mas as vacinas respiratórias e reprodutivas bovinas contêm apenas cepas de BVDV-1 e BVDV-2. O presente estudo investigou a atividade neutralizante contra estes pestivírus induzidas por duas vacinas comerciais (VA = atenuada, VI = inativada) e por três vacinas experimentais replicativas (VAC1 = monovalente, BVDV-1; VAC2 = bivalente, BVDV-1 + BVDV-2; VAC3 = trivalente, BVDV-1 + BVDV-2 e HoBiPeV). Bezerros soronegativos foram imunizados uma vez (vacinas replicativas) ou duas (vacina inativada) e amostras de soro foram testadas por vírus-neutralização (VN) 30 dias após a vacinação (dpv) (vacinas replicativas) ou 30 dias após a segunda dose (VI). Títulos neutralizantes ≥60 foram considerados indicativos de proteção contra doença clínica. Nesta data, a VA induziu títulos protetivos contra o BVDV-2 em 7/7 animais (GMT=289,8) e contra BVDV-1 e HoBiPeV em 5/7 animals (GMTs=97,5 e 80, respectivamente). VI induziu títulos protetores contra BVDV-1 em 1/7 animal (GMT=16,4), em 2/7 animais contra BVDV-2 (GMT=53,8) e em nenhum contra HoBiPeV (GMT=12,2). Quando um pool de soro de cada grupo vacinal foi testado frente a isolados Brasileiros, a VA induziu títulos protetores contra 3/7 isolados de BVDV-1, 9/10 (BVDV-2) e 1/8 (HoBiPeV); VI induziu títulos protetores em 1/7 contra BVDV-1, 1/10 (BVDV-2) e em nenhum (0/8) contra isolados de HoBiPeV. A VAC1 induziu títulos protetores contra BVDV-1 em 9/9 animais (GMT=320) mas em nenhum animal contra BVDV-2 ou HoBiPeV (GMT<10). VAC2 induziu títulos protetores contra BVDV-1e BVDV-2 em 9/9 animais (GMTs=160 e 640, respectivamente),e contra HoBiPeV em 7/9 animais (GMT=108,5). Finalmente, VAC3 induziu títulos protetores em todos os animais contra BVDV-1 (GMT=234,3), BVDV-2 (294,9) e HoBiPeV (201,1). No teste de pool de soro contra isolados de pestivírus, VAC1 induziu títulos ≥60 contra 4/7 BVDV-1 mas contra nenhum isolado de BVDV-2/HoBiPeV; VAC2 induziu títulos protetores contra 4/7 BVDV-1; 10/10 BVDV-2 e 2/8 HoBiPeV; VAC3 induziu títulos protetores contra todos BVDV-1, BVDV-2 e HoBiPeV. Esses resultados indicam que vacinas contendo apenas BVDV-1 BVDV-2, especialmente aquelas inativadas, podem não conferir resposta sorológica protetora contra vários isolados de HoBiPeV. Portanto, a necessidade de se incluir cepas de HoBiPeV nas vacinas deve ser considerada.(AU)
Assuntos
Animais , Bovinos , Bovinos/virologia , Vacinas Virais/administração & dosagem , Pestivirus/química , Variação AntigênicaRESUMO
Porphyrins are photoactive compounds that can absorb the energy of light and transfer it to oxygen molecules, producing reactive oxygen species (ROS). Once produced, ROS may alter biological molecules and cellular mechanisms, leading to cell apoptosis or inactivation of microorganisms, such as bacteria, fungi, and viruses. Therefore, the objective of this study was to evaluate the in vitro virucidal activity of six tetra-cationic porphyrins against two bovine viruses (Bovine alphaherpesvirus 1, BoHV-1, enveloped; and Bovine adenovirus, BAV, non-enveloped). For this, viral suspensions were incubated with each porphyrin (H2TMeP, ZnTMeP, and CuTMeP at 4.0 µM, NiTMeP at 5.0 µM, and CoClTMeP and MnClTMeP at 1.0 µM) and exposed to white-light irradiation for 0, 60, 120, and 180 min (BAV) or 0, 30, 60, 90, and 120 min (BoHV-1). Then, the remaining viral titers were determined by limiting dilution and compared with the control (virus + porphyrins without light exposition). The porphyrins H2TMeP and ZnTMeP showed the highest virucidal activity against both viruses. ZnTMeP inactivated BoHV-1 after 30 min of photoactivation and H2TMeP after 60 min. The BAV was photo-inactivated by both porphyrins at 180 min of white-light exposition. CuTMeP, NiTMeP, and CoClTMeP porphyrins had weak virucidal activity against BoHV-1 and MnClTMeP showed no virucidal activity against both viruses. These results indicated that free-base H2TMeP and ZnTMeP porphyrins present virucidal activity against non-enveloped and enveloped viruses, opening the possibility for their use to inactivate viruses on surfaces, biological substrates, and solutions.