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.

Antigenic relationships between Caprine alphaherpesvirus 1 (CpHV-1) and Bovine alphaherpesvirus 1 (BoHV-1) and experimental CpHV-1 infection of kids and calves.

Caprine alphaherpesvirus 1 (CpHV-1) is a worldwide pathogen of goats and is closely related to Bovine alphaherpevirus 1 (BoHV-1). We herein studied the antigenic relationships of CpHV-1 with BoHV-1 and investigated the pathogenesis of CpHV-1 in kids and calves. Monoclonal antibody reactivity revealed that CpHV-1 and BoHV-1 share immunogenic epitopes in the major envelope glycoproteins gB, gC and gD. The antigenic relationship was further demonstrated by virus-neutralizing assays, in which CpHV-1 and BoHV-1 antisera presented varied degrees of cross-neutralization against the respective heterologous viruses. Although cross-neutralization was observed between both viruses and the heterologous antisera, BoHV-1 antisera neutralized CpHV-1 with higher efficiency than CpHV-1 antisera neutralized BoHV-1. Hence, the antigenic cross-reactivity between CpHV-1 and BoHV-1 should be considered upon serologic testing of goats and cattle in regions where the two viruses co-circulate. Intranasal (IN) inoculation of CpHV-1 (WI13-46 isolate) in seven seronegative kids resulted in efficient viral replication in the respiratory tract. Additionally, mild to moderate systemic and respiratory signs were observed, including apathy, hyperthermia, nasal discharge and respiratory distress. Dexamethasone administration to the inoculated kids between days 36 and 40 pi did not result in virus shedding in nasal secretions. However, latent infection had been established, as evidenced by the detection of CpHV-1 DNA in trigeminal ganglia and olfactory bulbs of kids euthanized at day 67 pi. Contrasting with the outcome of infection in kids, IN inoculation of CpHV-1 in calves did not result in productive infection as no virus replication or shedding were detected, and the animals did not develop clinical signs nor seroconverted. The animal experiments demonstrated that CpHV-1 was able to produce respiratory disease in kids, but did not replicate to detectable levels in calves.

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.

Glycoprotein-G-gene-based molecular and phylogenetic analysis of rabies viruses associated with a large outbreak of bovine rabies in southern Brazil.

A large outbreak of hematophagous-bat-associated bovine rabies has been occurring in Rio Grande do Sul (RS), the southernmost Brazilian state, since 2011, with official estimates exceeding 50,000 cattle deaths. The present article describes a genetic characterization of rabies virus (RABV) recovered from 59 affected cattle and two sheep, from 56 herds in 16 municipalities (2012-2016). Molecular analysis was performed using the nucleotide (nt) and predicted amino acid (aa) sequences of RABV glycoprotein G (G). A high level of nt and aa sequence identity was observed among the examined G sequences, ranging from 98.4 to 100%, and from 97.3 to 100%, respectively. Likewise, high levels of nt and aa sequence identity were observed with bovine (nt, 99.8%; aa, 99.8%) and hematophagous bat (nt, 99.5%; aa, 99.4%) RABV sequences from GenBank, and lower levels were observed with carnivore RABV sequences (nt, 92.8%; aa, 88.1%). Some random mutations were observed in the analyzed sequences, and a few consistent mutations were observed in some sequences belonging to cluster 2, subcluster 2b. The clustering of the sequences was observed in a phylogenetic tree, where two distinct clusters were evident. Cluster 1 comprised RABV sequences covering the entire study period (2012 to 2016), but subclusters corresponding to different years could be identified, indicating virus evolution and/or introduction of new viruses into the population. In some cases, viruses from the same location obtained within a short period grouped into different subclusters, suggesting co-circulation of viruses of different origins. Subcluster segregation was also observed in sequences obtained in the same region during different periods, indicating the involvement of different viruses in the cases at different times. In summary, our results indicate that the outbreaks occurring in RS (2012 to 2016) probably involved RABV of different origins, in addition to a possible evolution of RABV isolates within this period.

Respiratory and neurological disease in rabbits experimentally infected with equid herpesvirus 1.

Equid herpesvirus type 1 (EHV-1) is an important pathogen of horses worldwide, associated with respiratory, reproductive and/or neurological disease. A mouse model for EHV-1 infection has been established but fails to reproduce some important aspects of the viral pathogenesis. Then, we investigated the susceptibility of rabbits to EHV-1 aiming at proposing this species as an alternative model for EHV-1 infection. Weanling rabbits inoculated intranasal with EHV-1 Kentucky D (10(7) TCID50/animal) shed virus in nasal secretions up to day 8-10 post-inoculation (pi), presented viremia up to day 14 pi and seroconverted to EHV-1 (virus neutralizing titers 4 to 64). Most rabbits (75%) developed respiratory disease, characterized by serous to hemorrhagic nasal discharge and mild to severe dyspnea. Some animals (20%) presented neurological signs as circling, bruxism and opisthotonus. Six animals died during acute disease (days 3-6); infectious virus and/or viral DNA were detected in the lungs, trigeminal ganglia (TG), olfactory bulbs (OBs) and cerebral cortex/brain (CC). Histological examination showed necrohemorrhagic, multifocal to coalescent bronchointerstitial pneumonia and diffuse alveolar edema. In two rabbits euthanized at day 50 pi, latent EHV-1 DNA was detected in the OBs. Dexamethasone administration at day 50 pi resulted in virus reactivation, demonstrated by virus shedding, viremia, clinical signs, and increase in VN titers and/or by detection of virus DNA in lungs, OBs, TGs and/or CC. These results demonstrate that rabbits are susceptible to EHV-1 infection and develop respiratory and neurological signs upon experimental inoculation. Thus, rabbits may be used to study selected aspects of EHV-1 biology and pathogenesis, extending and complementing the mouse model.

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.

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.

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.

Bovine herpesvirus-5 infection in a rabbit experimental model: immunohistochemical study of the cellular response in the CNS.

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 µl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 10(7.5) TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways.

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.

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.

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.

Vaccinia viruses isolated from cutaneous disease in horses are highly virulent for rabbits.

Two genotypically distinct Vaccinia viruses (VACV), named P1V and P2V, were isolated from an outbreak of cutaneous disease in horses in Southern Brazil. We herein investigated the susceptibility of rabbits, a proposed animal model, to P1V and P2V infection. Groups of weanling rabbits were inoculated intranasally (IN) with P1V or P2V at low (10(2.5) TCID50), medium (10(4.5)TCID50), or high titer (10(6.5)TCID50). Rabbits inoculated with medium and high titers shed virus in nasal secretions and developed serous to hemorrhagic nasal discharge and severe respiratory distress, followed by progressive apathy and high lethality. Clinical signs appeared around days 3-6 post-inoculation (pi) and lasted up to the day of death or euthanasia (around days 5-10). Virus shedding and clinical signs were less frequent in rabbits inoculated with low virus titers. Viremia was detected in all groups, with different frequencies. Viral DNA was detected in the feces of a few animals inoculated with P1V and P2V, low titer, and with P2V at high titer. Gross necropsy findings and histological examination showed diffuse interstitial fibrousing pneumonia with necrosuppurative bronchopneumonia and intestinal liquid content. Neutralizing antibodies were detected in all inoculated animals surviving beyond day 9 pi. These results show that rabbits are highly susceptible to VACV isolated from horses, and develop severe respiratory and systemic disease upon IN inoculation. Thus, rabbits may be used to study selected aspects of VACV infection and disease.

Isolation and identification of feline calicivirus and feline herpesvirus in Southern Brazil.

Feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1) are the two primary causes of upper respiratory tract disease in cats. The aim of this study was to demonstrate the distribution of FCV and FHV-1 among the feline population of several counties in Rio Grande do Sul State, Brazil. To this end, conjunctival and nasal swabs were collected from 302 cats from different locations, including households, breeding catteries, veterinary clinics, animal hospitals and experimental research facilities. The samples were collected between July 2006 to June 2009. The virus isolation was performed in CRFK cells and, subsequently, the identification was confirmed by PCR. FCV, FHV-1, or both were isolated from 55 cats from 28 different locations. FCV alone was isolated from 52.7% (29/55) of the animals that tested positively, FHV-1 alone was isolated from 38.2% (21/55) of the animals that tested positively, and co-infection were detected in 9.1% (5/55) of the animals that tested positively. Virus detection was more prevalent in cats that were less than 1 year old, among animals that shared a living space with other cats, and females. FCV and FHV-1 were isolated from vaccinated cats. In addition, both viruses were isolated from cats that showed no signs of disease. The results suggest that a carrier state is common for both viruses in the evaluated population. A search for other causes of respiratory disease in that population is necessary; and further studies relating to the molecular characterization of viruses and vaccine efficacy are also necessary.

Genome Sequence of a Brazilian Bovine Enterovirus.

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.