Mpox in Northeast Brazil: Spatiotemporal analysis and predictors associated with confirmed diagnosis.

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.

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.

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.

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.

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.

Diphtheric aspergillosis tracheitis with gastrointestinal dissemination secondary to viral infections in a dairy calf.

Diphtheric aspergillosis tracheitis is an uncommon syndrome described in human pathology, usually associated with immunosuppression in the affected individuals. Interestingly, no comparative/equivalent cases were found in domestic animals. This report describes the pathological and mycological findings associated with diphtheric aspergillosis tracheitis in an immunocompromised calf. The main pathological findings were diphtheric tracheitis and rhinitis, and necrotizing ruminitis associated with intralesional septate, acute branching fungal hyphae consistent with Aspergillus spp. Mycological culture and isolation confirmed the fungal hyphae as A. fumigatus due to characteristic features. Immunohistochemistry (IHC) assays identified intralesional antigens of bovine viral diarrhea virus (BVDV) and malignant catarrhal fever virus (MCFV) at the trachea and small intestine; IHC detected intralesional antigens of bovine alphaherpesvirus 1 (BoHV-1) only at the trachea. These findings confirmed the simultaneous occurrence of A. fumigatus with concomitant infections due to BVDV, MCFV, and BoHV-1 in this calf. Since ovine gammaherpesvirus-2 (OvHV-2) is the cause of MCF in Brail, it is likely that the intralesional MCFV antigens identified were those of OvHV-2. In this case, disseminated aspergillosis was probably associated with the undeveloped immunological status of the calf that was further impaired due to the combined immunodepressive effects of BVDV and BoHV-1 infections. Although BVDV and BoHV-1 are infectious disease pathogens frequently associated with the development of bovine respiratory disease (BRD) in feedlot and dairy cattle, the identification of intralesional OvHV-2-like antigens in several parts of the lungs suggest that this MCFV also played a role in the BRD-associated lesions identified in this calf.

New variants of canine parvovirus in dogs in southern Brazil.

Carnivore protoparvovirus 1 (canine parvovirus 2, CPV-2) has undergone a rapid evolution through mutations in the capsid protein VP2, giving rise to variants associated with unique clinicopathological and immunological features. VP2 is a major capsid protein involved in key steps of virus biology, including interactions with cellular receptors and with the immune system. This study analyzed the complete VP2 coding sequence of 38 CPV-2 isolates obtained from dogs with clinical parvovirosis in southern Brazil. Amplicons encompassing the whole VP2 coding region were subjected to nucleotide sequencing, and predicted amino acid sequences were analyzed to identify molecular markers of viral variants. Viral variants were classified as CPV-2a, -2b or -2c based on the presence of the amino acid Asn, Asp or Glu, respectively, at VP2 residue 426. Amino acid sequence analysis identified 20 CPV-2c and four CPV-2b isolates. Eleven viruses were identified as New CPV-2a, two as New CPV-2b, and one resembled the original CPV-2 and was designated CPV-2-like. In addition to the mutation at amino acid 426 of VP2, new 2a/2b variants containing a Ser297Ala mutation at residue 297 were identified. CPV-2-like samples contained some mutations that were also present in the original CPV-2 isolate, including as Leu, Thr, Ala and Asp at residues 87, 101, 300 and 305, respectively. The New CPV-2a isolates had three additional mutations (Phe267Tyr, Tyr324Ile and Thr440Ala) associated with selective pressure and development of disease in vaccinated dogs. The resemblance of the CPV-2-like isolate to CPV-2 suggests reemergence of CPV-2 and/or evolution from vaccine strains. Phylogenetic analysis grouped the variants with their respective reference strains, in general, according to amino acid changes. These results demonstrate the high VP2 diversity of CPV circulating in dogs in southern Brazil and indicate the emergence of new viral variants that differ markedly from the current vaccine strains.

Programmed cell death-associated gene transcripts in bovine embryos exposed to bovine Herpesvirus type 5.

In vitro-produced bovine embryos become infected after exposure to bovine Herpesvirus type 5 (BoHV-5), yet no changes in developmental rates, mitochondrial activity and inhibition of apoptosis are detected in comparison to unexposed embryos. Thus, the aim of the present study was to assess the transcription of mitochondria-mediated apoptosis genes using TaqMan real-time polymerase chain reaction. Transcripts of mcl-1, caspase-2, -3, Apaf-1 and Bax genes were measured after exposure to BoHV-5 in vitro. Mitochondrial dehydrogenase activity was evaluated by MTT test and compared between groups of exposed and unexposed embryos, at day 7 of development. The rate of oocyte maturation was assessed by the extrusion of the first polar body. In summary, BoHV-5 exposed embryos retained their viability, mitochondrial dehydrogenase activity and displayed up-regulation of transcription of survival mcl-1 gene and down-regulation of Bax transcription in relation to mitochondria-mediated pathway which might improve embryo viability. These findings demonstrate that BoHV-5 exposed embryos maintain their viability and mitochondrial dehydrogenase activity with no compromise of embryos produced in vitro.

Genetic diversity of 3' region of glycoprotein D gene of bovine herpesvirus 1 and 5.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses of cattle. While BoHV-1 is mainly associated with respiratory/genital disease and rarely associated with neurological disease, BoHV-5 is the primary agent of meningoencephalitis in cattle. The envelope glycoprotein D of alphaherpesviruses (BoHV-1/gD1 and BoHV-5/gD5) is involved in the early steps of virus infection and may influence virus tropism and neuropathogenesis. This study performed a sequence analysis of the 3' region of gD gene (gD3') of BoHV-1 isolates recovered from respiratory/genital disease (n = 6 and reference strain Cooper) or from neurological disease (n = 7); and from seven typical neurological BoHV-5 isolates. After PCR amplification, nucleotide (nt) sequencing, and aminoacid (aa) sequence prediction; gD3' sequences were compared, identity levels were calculated, and selective pressure was analyzed. The phylogenetic reconstruction based on nt and aa sequences allowed for a clear differentiation of BoHV-1 (n = 14) and BoHV-5 (n = 7) clusters. The seven BoHV-1 isolates from neurological disease are grouped within the BoHV-1 branch. A consistent alignment of 346 nt revealed a high similarity within each viral species (gD1 = 98.3 % nt and aa; gD5 = 97.8 % nt and 85.8 % aa) and an expected lower similarity between gD1 and gD5 (73.7 and 64.1 %, nt and aa, respectively). The analysis of molecular evolution revealed an average negative selection at gD3'. Thus, the phylogeny and similarity levels allowed for differentiation of BoHV-1 and BoHV-5 species, but not further division in subspecies. Sequence analysis did not allow for the identification of genetic differences in gD3' potentially associated with the respective clinical/pathological phenotypes, yet revealed a lower level of gD3' conservation than previously reported.

Influence of bovine pestivirus heterogeneity on serological responses to 10 different commercial vaccine formulation.

Bovine Pestivirus typically involves one or more organ systems, with clinical manifestations ranging from mild to severe fatal systemic illness that lead to significant reproductive, productive, and economic losses. Vaccines face the challenge of addressing the significant variability of pestiviruses, which affects the interaction between viral antigens and the immune system's ability to provide protection. This study aimed to evaluate the serological responses against bovine viral diarrhea virus 1 (Pestivirus A) and Pestivirus B induced by 10 commercial vaccines, including one recombinant (vaccine E), two modified live (MLV multivalent, vaccine I, and MLV monovalent, vaccine J), and seven killed vaccines (KLV, vaccines A to H). Additionally, we evaluated the cross-reactivity between Pestivirus A and B from vaccines and HoBi-like pestivirus (Pestivirus H). In Phase 1, guinea pigs were used to screen for non-MLVs. They were divided into nine groups (n=6 each) and received two doses (⅕ of bovine dose) of eight different non-MLV on Days 0 and 21. Serum samples were collected on Days 0 and 30 for serological analyse. In Phase 2, Holstein × Gir heifers (n= 45) were divided into five groups, comprising 6-9 animals. They were vaccinated either once with MLVs or twice with the top non-MLVs screened in Phase 1. Serum samples were harvested on d0 (vaccination day) and d60 (60 days after the first dose) for MLV and non-MLV. Specific antibody titers were assessed virus neutralization (VN) and transformed in log2 for statistical analysis using PROC-MIXED. Significant effects were observed for vaccine groups, time points, and their interactions concerning neutralizing antibodies against Pestivirus A and B in both Guinea pigs and heifers. The Phase 1 study revealed serological responses against Pestivirus A exclusively in non-MLV D (85.33±13.49) and E (72.00±19.26). In the bovine study, the KLD vaccine D (72.00±15.10), recombinant vaccine E (90.66±25.85), and MLV I (170.66±28.22) resulted in an average of neutralizing antibodies against Pestivirus A that exceeded the protective threshold (≥ 60). However,individual analysis of heifers showed a higher frequency of animals presenting titers of Pestivirus A Ab surpassing 32 following vaccination with MLV I and J. None of the vaccine formulations in either study elicited a protective immune response against Pestivirus B or demonstrated cross-reactivity against Pestivirus H.

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.

An end-point multiplex RT-PCR for SARS-CoV-2, Influenza A and B detection, including simultaneous RNAse P amplification: a timely tool for more accessible differential diagnosis.

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.

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.

Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos.

The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.

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.

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.

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.

Detection of Equus caballus papillomavirus-2 in equine penile/preputial papillomas and squamous cell carcinomas in southern Brazil.

For approximately one decade, a novel papillomavirus termed Equus caballus papillomavirus-2 (EcPV-2) has been associated with equine penile/preputial papillomas and squamous cell carcinomas (SCCs). It is currently believed that the virus has a carcinogenic activity, being able to induce such neoplastic lesions. After being first described, EcPV-2 has been detected in many countries from North America, Europe, and Asia; however, to date, it has not been reported in Brazil. The aim of this research was to investigate the presence of EcPV-2 in penile/preputial papillomas and SCCs of Brazilian horses. Forty samples diagnosed as equine penile and/or preputial papillomas, carcinomas in situ (CIS), or SCCs in two veterinary anatomic pathology services from southern Brazil were investigated. Histologic evaluation and immunohistochemistry (IHC) using a BPV-1 antibody were performed. Posteriorly, the samples were submitted to polymerase chain reaction using two broad-spectrum (MY09/11 and FAP) and one EcPV-2-specific primer sets. Positive samples were sequenced. PV antigen expression was detected in one papilloma, one CIS, and two SCCs by IHC. Five SCCs, one papilloma, and one CIS were PV-positive on PCR. Sequencing of the seven PCR products revealed homology with EcPV-2. This study confirms the occurrence of EcPV-2 infection in Brazilian horses. Moreover, the results presented here provide useful information concerning the phylogeny from the viruses detected in our samples. We hope to encourage further studies on this novel agent, contributing to its characterization, and, possibly, to the eventual development of preventive measurements, including a possible vaccine.

Seroprevalence of bovine vaccinia in cows and its correlation with the productive profile of affected farms in Distrito Federal, Brazil.

Bovine vaccinia (BV) is an infectious disease caused by Vaccinia virus (VACV) characterized by vesicular and exanthematic lesions, mainly in cattle. Although BV has been described in some Brazilian regions in the last decades, official information regarding the current prevalence in bovine herds of Midwestern Brazil is lacking. Thus, the current study aimed to estimate the seroprevalence and risk factors associated with BV in cattle in the Distrito Federal (DF), Brazil. Sera of 312 cows of 64 herds were tested by virus-neutralizing test for VACV antibodies. Herd and animal seroprevalence were estimated to be 33.3% (CI 95%: 18.2-48.3%) and 10.6% (CI 95%: 1.0-20.2%), respectively. Seropositive cows were detected in dairy, beef, and mixed-purpose farms. The results of an epidemiological questionnaire showed that no risk factor analyzed was positively associated with seropositivity to VACV. There was no significant association between type of milking (manual/mechanic) and seropositivity to VACV; however, most seropositive cows were present in farms with high daily milk production and high number of lactating and adult cows. Our results indicate that VACV circulates in many regions of DF with considerable prevalence in dairy cows. Control measures to restrict VACV circulation and consequences of the infection may be advisable.