Phylogenetic analysis of papillomaviruses in dogs from southern Brazil: molecular epidemiology and investigation of mixed infections and spillover events.

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.

Novel high-coverage primers for detection of canine morbillivirus by end-point and real-time RT-PCR assays.

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.

Reverse Genetics of Dengue Virus.

Dengue virus (DENV) is one of the most important and widespread arthropod-borne viruses, causing millions of infections over the years. Considering its epidemiological importance, efforts have been directed towards understanding various aspects of DENV biology, which have been facilitated by the development of different molecular strategies for engineering viral genomes, such as reverse genetics approaches. Reverse genetic systems are a powerful tool for investigating virus-host interaction, for vaccine development, and for high-throughput screening of antiviral compounds. However, stable manipulation of DENV genomes is a major molecular challenge, especially when using conventional cloning systems. To circumvent this issue, we describe a simple and efficient yeast-based reverse genetics system to recover infectious DENV clones.

Novel genomic targets for proper subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2.

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.

Emergence of SARS-CoV-2 serotype(s): Is it a matter of time?

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.

An end-point multiplex PCR/reverse transcription-PCR for detection of five agents of bovine neonatal diarrhea.

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.

Synergism between chikungunya virus infection and rheumatoid arthritis on cytokine levels: Clinical implications?

Post-chikungunya virus (CHIKV) chronic arthritis shares several immunopathogenic mechanisms with rheumatoid arthritis (RA), which has led to discussions about the probable relationship between the two diseases. Indeed, some studies have suggested a role for CHIKV infection in RA development. However, to the best of our knowledge, the influence of CHIKV on previous RA has not yet been demonstrated. Herein, we analyzed the potential synergism between CHIKV infection and RA on cytokine and chemokine levels. For this, we compared the IL-1ß, IL-6, IL-10, IL-17A, CCL2, CXCL8, CXCL9 and CXCL10 levels, in addition to rheumatoid factor (RF) and C-reactive protein (CRP), in patients with post-CHIKV chronic arthritis (named CHIKV group), patients with RA (RA group), and patients with previous RA who were later infected by CHIKV (RA-CHIKV). History of CHIKV infection was confirmed by serology (IgG, ELISA). Cytokines/chemokines were quantified by flow cytometry. RF, CRP, age and sex data were obtained from medical records. IL-1ß, IL-6, IL-10 and IL-17A levels were significantly higher in RA-CHIKV compared to the other groups. CXCL8 levels were higher in the CHIKV group than in RA. CXCL9 was higher in CHIKV than in the RA-CHIKV group. CXCL10 was higher in CHIKV than in the other groups. FR levels were higher in RA than in the CHIKV group, and in RA-CHIKV than in CHIKV. No significant difference was observed in CCL2 and CRP, as well as in age and sex. Finally, our findings suggest an interplay between CHIKV infection and RA, which must be analyzed for its possible clinical impact.

Epidemiological profile of arboviruses in two different scenarios: dengue circulation vs. dengue, chikungunya and Zika co-circulation.

BACKGROUND: The severity and distribution of dengue virus (DENV) infections have been attributed to a complex interaction among viral, host and environmental factors. Herein, we investigated the influence of chikungunya (CHIKV) and Zika (ZIKV) viruses on the epidemiological profile of dengue cases, using Recife, Pernambuco state, Brazil, as a study model. In addition, we described and compared the epidemiological profile related to each arbovirus (DENV vs. CHIKV vs. ZIKV). METHODS: All cases of dengue, chikungunya and Zika reported to the Pernambuco Health Department in 2011-2013 (DENV circulation) and 2016-2018 (DENV, CHIKV and ZIKV co-circulation) were included in our study. The cases were classified by sex, age and race/color and their distribution was analyzed by the χ2 test. Furthermore, the data were also analyzed for co-infections. Temperature, humidity and rainfall data were analyzed using one-way ANOVA and paired t-test. RESULTS: During 2011-2013, 15,315 dengue cases were diagnosed, most of them female, brown and 20-29 age group. Between 2016 and 2018, 15,870 dengue cases were described, which presented the same profile described above. In the two triennia, the female/male dengue ratio fluctuated significantly, ranging from 1.07 to 1.52. Regarding chikungunya, 7076 cases were reported, most of them female and brown. The female/male ratio also fluctuated significantly, ranging from 1.62 to 2.1. Two main age groups were observed in chikungunya: ≤ 19 years (minority of diagnoses) and ≥ 20 years (majority of diagnoses). In the same triennium, 266 Zika cases were reported to the Pernambuco Health Department, mainly in females and in the 0-9 and 20-39 age groups. In general, 119 co-infections were identified: 117 DENV-CHIKV, 1 CHIKV-ZIKV and 1 DENV-CHIKV-ZIKV. Concerning climate data, only the humidity in 2011 was significantly different from the other years. CONCLUSION: The epidemiological profile of dengue cases did not change after the introduction of CHIKV and ZIKV. Females were the most diagnosed with dengue, chikungunya or Zika, however we found important differences in the age profile of these arboviruses, which should be considered by public health policies, as well as investigated in future studies of virus-host interaction.

An appraisal of gene targets for phylogenetic classification of canine distemper virus: Is the hemagglutinin the best candidate?

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.

Phylogeny and amino acid analysis in single and mixed bovine papillomavirus infections in Southern Brazil, 2016-2020.

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.

Analysis of seroprevalence and risk factors for hepatitis E virus (HEV) in donation candidates and blood donors in Northeast Brazil.

Oral transmission is the main route of hepatitis E virus (HEV) infection; however, genotypes 3 and 4 may also be transmitted by blood transfusion. Individuals who need blood products are often immunosuppressed, which increase the risk of severe disease and death by HEV. Despite this, blood banks in Brazil do not screen for HEV and epidemiological studies in this population are rare; this is an important issue as HEV-3 is frequently identified in the country. Herein, we analyzed the seroprevalence and risk factors for HEV seropositivity in donor candidates/blood donors from Northeast Brazil. Nine hundred and ninety-six donor candidates/blood donors from Foundation of Hematology and Hemotherapy of Pernambuco (HEMOPE) were interviewed regarding socioeconomic, sociodemographic, and behavioral data and analyzed for anti-HEV IgG. Anti-HEV IgG was detected using the HEV IgG (EUROIMMUN) kit. Associations between seropositivity and potential risk factors were analyzed by the χ2 test and Fisher's exact test. Seroprevalence was 0.9% (9/996), 77.77% (7/9) and 22.22% (2/9) in blood donors and donor candidates, respectively. HEV seropositivity was associated with male (OR: 11.65; CI: 0.6755-200.9; p = 0.0163), income higher than BRL 20,000/month (p = 0.0002), and lake bathing (OR: 4.553; CI: 1.391-15.25; p = 0.0258). Importantly, about 43% (3/7) of anti-HEV positive donors made their first donation more than 20 years ago, which must be taken as a warning sign, given the possibility that these individuals may have been infected after registration as donors. Finally, the report of HEV seropositivity, especially in regular blood donors, as well as the identification of potential risk factors, reinforces the need for viral screening in Brazilian blood banks.

A new (old) bovine viral diarrhea virus 2 subtype: BVDV-2e.

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.

Retrospective observational study on the epidemiological profile of people living with HIV/AIDS in Pernambuco state, Brazil.

INTRODUCTION: HIV/AIDS is a major global public health concern. In Pernambuco state, Brazil, the number of people living with HIV/AIDS (PLWHA) is among the highest in the country. Herein, a cross-sectional retrospective observational study was carried out with 811 PLWHA followed up at the Clinical Hospital, Pernambuco, Brazil, between 2013 and 2017. METHODOLOGY: The patients' sociodemographic and behavioral data were obtained by interview. Information about HIV load and CD4 T lymphocyte count were obtained from patients' records. Data were analyzed for both the total number of PLWHA and gender. RESULTS: Recife municipality had the highest number of PLWHA. Most PLWHA were 40-44 years old, male, brown ethnicity, heterosexual, single, with elementary education, used condoms regularly, shared sharp objects, had surgery, had no non-HIV sexual infection, did not receive transfusions, did not use injectable drugs, and had no tattoo. The median of first and last CD4 T lymphocyte counts were 241 and 549.5 cells/mm³, respectively. The first HIV load had a median of 14,882 copies/mL (IQR = 613-109,750 copies/mL). Regarding the last viral load, 63.74% had an undetectable load. All patients were using antiretroviral therapy, mean time of 5.9 (± 5.5) years. This epidemiological and medical profile was maintained when PLWHA were analyzed according to gender, except for the report of another sexually transmitted infection, in which 51.4% of men (268/521) reported having/or having had it. CONCLUSIONS: The epidemiological profile of PLWHA in Pernambuco, Brazil, was described. This regional characterization is useful for directing public health policies, contributing to population-directed decision making.

Prevalence and risk factor analysis for HIV/HTLV 1/2 coinfection in Paraíba state, Brazil.

INTRODUCTION: Human T-lymphotropic virus (HTLV) 1 and 2 infections can lead to neurological diseases, mainly in HIV/HTLV 1 coinfected. Furthermore, HTLV 1 infection in HIV/AIDS patients has also been associated with AIDS progression. Despite this, HTLV 1/2 infections are not of mandatory notification in Brazil. Here, we describe the prevalence of HTLV 1/2 in HIV/AIDS patients from Paraíba state, Brazil, as well as the sociodemographic characteristics of the coinfected individuals. METHODOLOGY: Information about HIV viral load and TCD4 lymphocyte count were obtained from patients' records. Data on the patients' sociodemographic characteristics were obtained by interview conducted after signing the informed consent form. The serological diagnosis for HTLV 1/2 was performed by Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB). RESULTS: A total of 401 HIV/AIDS patients participated in the study, of whom about 1.5% (6/401) were positive for antibodies against HTLV, specifically for HTLV 1, evaluated by both ELISA and WB. No risk factors were found associated with HIV/HTLV 1/2 coinfection. CONCLUSIONS: We report a 1.5% prevalence of HTLV 1 infection in HIV/AIDS patients from Paraíba state. Although we have not identified risk factors associated with HTLV 1, we describe the most observed sociodemographic characteristics in HIV/HTLV 1 coinfection.

Subtyping bovine viral diarrhea virus (BVDV): Which viral gene to choose?

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.

Sequence analysis of the DA domain of glycoprotein E2 of pestiviruses isolated from beef cattle in Southern Brazil.

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.

Evaluation of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ cytokines in HIV/HHV-8 coinfection.

Imbalance in the immune response is one of the main pathogenic mechanisms of diseases related with human immunodeficiency virus (HIV)/human gammaherpesvirus 8 (HHV-8) coinfection, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD) and the Kaposi's sarcoma-associated herpesvirus inflammatory cytokine syndrome (KICS). However, significant changes in pro- and anti-inflammatory cytokine levels may be observed in HIV/HHV-8 individuals who are negative for KS, PEL, MCD, and/or KICS. In this study, serum levels of interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor nucrosis factor α (TNF-α) and interferon γ (IFN-γ) were assessed in 69 HIV and 48 HIV/HHV-8 individuals, all negatives for HHV-8-related diseases. The cytokines were measured by flow cytometry and analyzed by the Mann-Whitney test. The p < .05 and 95% confidence interval were considered in all analyzes. IL-4 (p = .0155), IL-6 (p = .0036), and IL-10 (p = .0036) levels were significantly higher in HIV/HHV-8 patients than in the HIV group. On the other hand, IL-2 (p = .2295), TNF-α (p = .1216) and IFN-γ (p = .1178) did not differ between the groups analyzed. To our knowledge, to date, this is the first report on significant differences in the levels of IL-4 and IL-6 in HIV versus HIV/HHV-8 individuals. Finally, these early findings are important as a prognostic tool and contribute to clarifying the HHV-8-host interaction.

End-point RT-PCR: A potential alternative for diagnosing coronavirus disease 2019 (COVID-19).

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.