Cattle influenza D virus in Brazil is divergent from established lineages.

Influenza D virus (IDV) is endemic in cattle on several continents and can also infect a wide range of hosts. IDV was first detected in a bovine respiratory disease outbreak associated with bovine alphaherpesvirus 1 in Brazil. Sequence analysis of partial segments showed that the virus is phylogenetically divergent from previously described IDVs from other continents. As the first molecular description of IDV in South America, this can be a first step toward investigating IDV infections in cattle in Brazil and surrounding countries in which the beef industry is economically important.

Synthesizing the connections between environmental disturbances and zoonotic spillover.

Zoonotic spillover is a phenomenon characterized by the transfer of pathogens between different animal species. Most human emerging infectious diseases originate from non-human animals, and human-related environmental disturbances are the driving forces of the emergence of new human pathogens. Synthesizing the sequence of basic events involved in the emergence of new human pathogens is important for guiding the understanding, identification, and description of key aspects of human activities that can be changed to prevent new outbreaks, epidemics, and pandemics. This review synthesizes the connections between environmental disturbances and increased risk of spillover events based on the One Health perspective. Anthropogenic disturbances in the environment (e.g., deforestation, habitat fragmentation, biodiversity loss, wildlife exploitation) lead to changes in ecological niches, reduction of the dilution effect, increased contact between humans and other animals, changes in the incidence and load of pathogens in animal populations, and alterations in the abiotic factors of landscapes. These phenomena can increase the risk of spillover events and, potentially, facilitate new infectious disease outbreaks. Using Brazil as a study model, this review brings a discussion concerning anthropogenic activities in the Amazon region and their potential impacts on spillover risk and spread of emerging diseases in this region.

Insights into the origin and diversification of bovine viral diarrhea virus 1 subtypes.

In this study, we performed phylogenetic and evolutionary analysis on bovine viral diarrhea virus 1 (BVDV-1) sequences to investigate the origin and temporal diversification of different BVDV-1 subtypes. Dated phylogenies using the complete polyprotein sequence were reconstructed, and the time of the most recent common ancestor (tMRCA) was estimated. The results demonstrated that BVDV-1 subtypes clustered into two phylogenetic clades, where the predominant subtypes worldwide grouped together. In the temporal analysis, the tMRCA of BVDV-1 was 1336, and the diversification into different subtypes appears to have occurred around 363 years ago. The present results help to elucidate the origins of BVDV-1 subtypes and the dynamics of ruminant pestiviruses.

A new highly divergent copiparvovirus in sheep.

The subfamily Parvovirinae within the family Parvoviridae consists of viruses that can infect a wide range of vertebrate hosts and cause effects ranging from severe disease to asymptomatic infection. In the present study, high-throughput sequencing (HTS) was utilized to analyze samples obtained from an abortion outbreak in a sheep flock to identify a putative viral etiology. A highly divergent nearly complete parvovirid genome sequence, approximately 4.9 kb in length, was determined. The nonstructural protein (NS1) amino acid (aa) sequence of this virus shared less than 30% identity with those of other copiparvoviruses and less than 22% identity with those of members of other genera in the subfamily Parvovirinae. Phylogenetically, this virus, which we have provisionally named "sheep copiparvovirus 1", formed a cluster with copiparvovirus sequences and should be classified as a member of a new species in the genus Copiparvovirus.

Evaluation of the serum virome in calves persistently infected with Pestivirus A, presenting or not presenting mucosal disease.

Bovine viral diarrhea virus 1, reclassified as Pestivirus A, causes an economically important cattle disease that is distributed worldwide. Pestivirus A may cause persistent infection in that calves excrete the virus throughout their lives, spreading the infection in the herd. Many persistently infected (PI) calves die in the first 2 years of life from mucosal disease (MD) or secondary infections, probably as a consequence of virus-induced immune depression. Here, high-throughput sequencing (HTS) was applied for evaluation of the total virome in sera of (i) PI calves displaying clinically apparent MD (n = 8); (ii) PI calves with no signs of MD (n = 8); and (iii) control, Pestivirus A-free calves (n = 8). All the groups were collected at the same time and from the same herd. Serum samples from calves in each of the groups were pooled, submitted to viral RNA/DNA enrichment, and sequenced by HTS. Viral genomes of Pestivirus A, Ungulate erythroparvovirus 1, bosavirus (BosV), and hypothetical circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses were identified. Specific real-time PCR assays were developed to determine the frequency of occurrence of such viruses in each of the groups. The absolute number of distinct viral genomes detected in both PI calf groups was higher than in the control group, as revealed by higher number of reads, contigs, and genomes, representing a wider range of taxons. Genomes representing members of the family Parvoviridae, such as U. erythroparvovirus 1 and BosV, were most frequently detected in all the three groups of calves. Only in MD-affected PI calves, we found two previously unreported Hypothetical single-stranded DNA genomes clustered along with CRESS-DNA viruses. These findings reveal that parvoviruses were the most frequently detected viral genomes in cattle serum; its frequency of detection bears no statistical correlation with the status of calves in relation to Pestivirus A infection, since clinically normal or MD-affected/non-affected PI calves were infected with similar U. erythroparvovirus 1 genome loads. Moreover, MD-affected PI calves were shown to support viremia of CRESS-DNA viral genomes; however, the meaning of such correlation remains to be established.

Genomic and antigenic relationships between two 'HoBi'-like strains and other members of the Pestivirus genus.

'HoBi'-like viruses comprise a putative new species within the genus Pestivirus of the family Flaviviridae. 'HoBi'-like viruses have been detected worldwide in batches of fetal calf serum, in surveillance programs for bovine pestiviruses and from animals presenting clinical signs resembling bovine viral diarrhea virus (BVDV)-associated diseases. To date, few complete genome sequences of 'HoBi'-like viruses are available in public databases. Moreover, detailed analyses of such genomes are still scarce. In an attempt to expand data on the genetic diversity and biology of pestiviruses, two genomes of 'HoBi'-like viruses recovered from Brazilian cattle were described and characterized in this study. Analysis of the whole genome and antigenic properties of these two new 'HoBi'-like isolates suggest that these viruses are genetically close to recognized pestiviruses. The present data provide evidence that 'HoBi'-like viruses are members of the genus Pestivirus and should be formally recognized as a novel species.

Genomic characterization of a bovine viral diarrhea virus subtype 1i in Brazil.

Bovine viral diarrhea virus 1 (BVDV-1) belongs to the genus Pestivirus within the family Flaviviridae. Based on the 5' untranslated region (UTR) sequence, BVDV-1 can be divided into at least 17 subtypes (1a though 1q). BVDV-1i is an uncommon subtype that has been reported in the United Kingdom and Uruguay. Here, we report the complete genome sequence of the first subtype 1i BVDV-1 (strain ACM/BR/2016) isolated from cattle in southern Brazil. The genome is 12,231 nt in length and contains a single ORF that encodes a polyprotein of 3,896 amino acids, flanked by 5' and 3'UTRs of 325 and 220 nt, respectively. Phylogenetic inferences based on the whole genome, the 5'UTR, and the Npro region showed that strain ACM/BR/2016 is closely related to previously characterized BVDV-1i members. Its 5'UTR shares the highest nucleotide identity (90.5%) with BVDV-1i strains from United Kingdom, and its Npro is most closely related to that of a Uruguayan strain (90.6%). To the best of our knowledge, this is the first BVDV-1i strain from which the whole genome has been completely sequenced and characterized. The complete genome of a BVDV-1i will help future studies on pestivirus evolution and heterogeneity.

Genome characterization of a bovine papillomavirus type 5 from cattle in the Amazon region, Brazil.

Papillomaviruses are small and complex viruses with circular DNA genome that belongs to the Papillomavirus family, which comprises at least 39 genera. The bovine papillomavirus (BPV) causes an infectious disease that is characterized by chronic and proliferative benign tumors that affect cattle worldwide. In the present work, the full genome sequence of BPV type 5, an Epsilonpapillomavirus, is reported. The genome was recovered from papillomatous lesions excised from cattle raised in the Amazon region, Northern Brazil. The genome comprises 7836 base pairs and exhibits the archetypal organization of the Papillomaviridae. This is of significance for the study of BPV biology, since currently available full BPV genome sequences are scarce. The availability of genomic information of BPVs can provide better understanding of the differences in genetics and biology of papillomaviruses.

Complete genome sequence of Deltapapillomavirus 4 (bovine papillomavirus 2) from a bovine papillomavirus lesion in Amazon Region, Brazil.

The complete genome sequence of bovine papillomavirus 2 (BPV2) from Brazilian Amazon Region was determined using multiple-primed rolling circle amplification followed by Illumina sequencing. The genome is 7,947 bp long, with 45.9% GC content. It encodes seven early (E1, E2,E4, E5, E6,E7, and E8) and two late (L1 and L2) genes. The complete genome of a BPV2 can help in future studies since this BPV type is highly reported worldwide although the lack of complete genome sequences available.

Genetic characterization of Amazonian bovine papillomavirus reveals the existence of four new putative types.

Papillomaviruses are small and complex viruses that belong to the Papillomaviridae family, which comprises 39 genera. The bovine papillomavirus (BPV) causes an infectious disease that is characterized by chronic and proliferative benign tumors that affect cattle worldwide. Different genotypes of BPVs can cause distinct skin and mucosal lesions and the immunity they raise has low cross-protection. This report aimed to genotype BPVs in cattle from Northern Brazil based on nucleotide partial sequences of the L1 ORF. Skin wart samples from 39 bovines clinically and histopathologically diagnosed as cutaneous papillomatosis from Acre and Rondônia States were analyzed. The results revealed four already reported BPV types (BPVs 1, 2, 11, and 13), nine putative new BPV subtypes and four putative new BPV types as well as two putative new BPV types that were already reported. To our knowledge, this is the first record of BPVs from the Brazilian Amazon region that identified new possible BPV types and subtypes circulating in this population. These findings point to the great genetic diversity of BPVs that are present in this region and highlight the importance of this knowledge before further studies about vaccination are attempted.

HoBi-like Pestivirus Is Highly Prevalent in Cattle Herds in the Amazon Region (Northern Brazil).

Pestiviruses are globally distributed and cause substantial economic losses to the cattle industry. In Brazil, the country with the world's largest cattle population, pestivirus infections are well described in some regions, such as in the south, where a high frequency of BVDV-2 is described and contrasts with the high prevalence of HoBi-like pestivirus (HoBiPeV) in the northeast. However, there is a lack of information about pestiviruses in the Amazon Region, in northern Brazil, with a cattle population estimated at 55.7 million head, which has a significant impact on the international livestock market. Therefore, this study investigated the seroprevalence and genetic variability of ruminant pestiviruses in 944 bovine serum samples from four states in northern Brazil: Pará (PA), Amapá (AP), Roraima (RR), and Amazonas (AM). Our results showed that 45.4% of the samples were seropositive (19.8% for BVDV-1, 14.1% for BVDV-2, and 20.9% for HoBiPeV). All samples were tested by RT-qPCR, and three were positive and classified as HoBiPeV in a phylogenetic analysis. These serological and molecular results contrast with those from other regions of the world, suggesting that the northern Brazilian states have a high prevalence of all bovine pestiviruses including HoBiPeV.

An outbreak of visna-maedi in a flock of sheep in Southern Brazil.

Visna-maedi is a multisystemic and progressive inflammatory disease caused by a non-oncogenic retrovirus (Visna-maedi virus, VMV). An outbreak of visna-maedi occurred in Southern Brazil in sheep with clinical signs of blindness and stumbling gait. At post-mortem examination, all animals had similar lesions, including heavy non-collapsed lungs and multifocal yellow areas in the cerebral white matter, affecting mainly the periventricular region. These lesions corresponded histologically to lymphocytic interstitial pneumonia and histiocytic periventricular encephalitis surrounding areas of necrosis, in addition to significant demyelination in the brain. Serology was performed in all the sheep from the flock and 14% were seropositive for VMV. The presence of VMV was confirmed through PCR and partial sequencing of the 5'LTR. Sequencing demonstrated that the virus had 89.7 to 90.0% of nucleotide identity with VMV strains reported in the USA. This is the first description of clinical disease related to VMV in Brazil leading to economic losses. This study calls for the need to implement control measures to prevent the spread of small ruminant lentiviruses in Brazil.

Detection of human Mastadenovirus C in wild guinea pigs (Cavia aperea aperea) feces.

The Adenoviridae family is composed by a high diversity of viruses that are extremely resistant in environment and are frequently excreted in animal reservoir feces for long periods. The knowledge of adenovirus (AdV) diversity among wild species may be important for the understanding of the epidemiology of putative emerging diseases. Cavia aperea aperea, commonly known as wild guinea pigs, wild cavies, or preas, are small herbivorous rodents widely distributed throughout South America and classified in Caviidae family, as well as domestic guinea pigs and capybaras. In order to investigate their potential role as reservoir of zoonotic agents, the present study aimed to verify the presence of AdV in fecal samples of 14 preas from Northeast Brazil. When submitted to nested PCR, two out of 14 samples (14.28%) were positive for AdV and classified as human Mastadenovirus C (HAdV-C) using DNA sequencing and phylogenetic analysis. Wild guinea pigs are synanthropic rodents that live in close contact with humans. The investigation of viral agents in rodents is important due to their potential role as reservoirs of human and animal pathogens. Moreover, the present work presents the first known evidence of HAdV in wild guinea pig stool samples, which may represent both the impact of anthropogenic pollution to wild animals and an important knowledge in terms of human health.

Virome characterization in serum of healthy show pigs raised in Oklahoma demonstrated great diversity of ssDNA viruses.

In the United States, show pigs are raised to compete in agricultural events. These animals are usually raised in small herds with extensive human, domestic, and wild animal contact. Therefore, pathogen monitoring in this animal category is critical for improved disease surveillance and preparedness. This study characterized the virome of healthy show pigs using high-throughput sequencing using pooled serum samples from 2018 or 2019 (200 samples each pool). Results demonstrated the presence of DNA viral families (Parvoviridae, Circoviridae, and Herpesviridae) and RNA families (Arteriviridae, Flaviviridae, and Retroviridae). Twenty-three viral species were identified, including the first detection of porcine bufavirus in the US. Moreover, important swine pathogens identified included porcine reproductive and respiratory syndrome virus, atypical porcine pestivirus, and porcine circovirus (PCV). Additionally, complete coding genomes of 17 viruses from the Parvoviridae, Anelloviridae, and Circoviridae families were retrieved and included the first near full-length genomes of US Ungulate bocaparvovirus 3 species.

Virome Characterization in Commercial Bovine Serum Batches-A Potentially Needed Testing Strategy for Biological Products.

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies' established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols.

Bovine leukemia viral DNA found on human breast tissue is genetically related to the cattle virus.

Bovine leukemia virus (BLV) infection is widespread in cattle and associated with B cell lymphoma. In a previous study we demonstrated that bovine leukemia viral DNA was detected in human breast tissues and significantly associated with breast cancer. Our current study aimed to determine whether BLV DNA found in humans and cattle at the same geographical region were genetically related. DNA was extracted from the breast tissue of healthy (n = 32) or cancerous women patients (n = 27) and from the blood (n = 30) of cattle naturally infected with BLV, followed by PCR-amplification and partial nucleotide sequencing of the BLV env gene. We found that the nucleotide sequence identity between BLV env gene fragments obtained from human breast tissue and cattle blood ranged from 97.8 to 99.7% and grouped into genotype 1. Thus, our results further support the hypothesis that this virus might cause a zoonotic infection.

Evaluation of cardioprotective effects of carvedilol in dogs receiving doxorubicin chemotherapy: A prospective, randomized, double-blind, placebo controlled pilot study.

The aim of this pilot study was to evaluate the cardioprotective effects of carvedilol in dogs receiving doxorubicin chemotherapy and provide suggestions to future studies based on results and limitations of our study. Thirteen dogs were randomized into two experimental groups: 6 dogs in carvedilol group and 7 dogs in placebo group. In carvedilol group, 0.39 mg/kg ± 0.04 twice-daily oral carvedilol was started on the day of the first doxorubicin treatment and continued throughout the chemotherapy protocol until the final cardiological evaluation. Cardiological evaluations were performed before the first doxorubicin administration and then 10 to 15 days after each subsequent dose. Troponin I and oxidative stress tests were performed with serum collected from dogs at the initial and final cardiological evaluation. Carvedilol produced some echocardiographic and electrocardiographic changes (reduced E velocity and E/IVRT ratio, as well reduced heart rate and increased PR and QT interval) due to its beta-block effect. In placebo group Doppler study showed a significant increase in mitral flow deceleration time (EDT), as well increased amplitude of the S wave in the right, and R wave in the left, precordial chest leads. There were significant difference in the EDT, E/IVRT and A' velocity, as well heart rate, PR interval and R wave in V4/CV6LU precordial chest lead between groups. In conclusion, some indexes of diastolic function and in precordial chest leads were less affected by doxorubicin in carvedilol than in control group. This suggests that carvedilol may have a beneficial effect in canine cancer patients receiving doxorubicin.

Serologic evidence of West Nile virus and Saint Louis encephalitis virus in horses from Southern Brazil.

Flaviviruses as West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Ilhéus virus (ILHV), and Rocio virus (ROCV) are previously reported in different Brazilian regions, but studies in Southern Brazil are still scarce. To improve the information regarding flaviviruses in Southern Brazil, horse serum samples were analyzed using RT-qPCR and a commercial ELISA-Ab against WNV followed by PRNT75. All 1000 samples analyzed by real-time RT-PCR resulted negative. The 465 subsampled samples were analyzed by a commercial ELISA-Ab against WNV, and the 18.5% (86/465) positive samples were further analyzed by PRNT75. In the PRNT75, 13/86 and 2/86 horses were positive for SLEV and WNV, respectively. It was observed that 5.8% (13/226) of the farms presented at least one positive animal for SLEV in PRNT75, whereas 0.9% (2/226) for WNV. Apart from the lower seroprevalences identified when compared to data previously reported in other Brazilian regions, our results suggest that public health professionals must be aware of the presence of these potential zoonotic pathogens.

Survey for pestiviruses in backyard pigs in southern Brazil.

The Pestivirus genus comprises species that affect animal health and productivity worldwide. Members of the Suidae family are hosts for classical swine fever virus (CSFV), an important pathogen tracked by the World Organization for Animal Health (OIE). However, swine are also susceptible to other pestivirus species that can result in disease or compromise CSFV detection. We searched for pestivirus infection in swine sera collected from 320 backyard pig herds in southern Brazil. We used reverse-transcription PCR primers for Bungowannah virus; atypical porcine pestivirus (APPV); and a panpestivirus pair that detects bovine viral diarrhea virus (BVDV)-1, -2, and HoBi-like pestivirus (HoBiPeV), border disease virus (BDV), and CSFV. Two samples were positive using the panpestivirus primer pair and were classified as BVDV-1d and -2a, respectively. Serum samples were tested for virus neutralization against BVDV-1a, -1b, and -2 strains, resulting in 28 (4.4%) positive samples. Of those, 16 samples had the highest titers against BVDV-1a (2), BVDV-1b (5), and BVDV-2 (9). Our results indicate that Bungowannah virus, APPV, CSFV, BDV, and HoBiPeV have not been circulating in these specific backyard swine populations. However, ruminant pestiviruses were detected and must be considered in future pestivirus control programs conducted in Brazil.

Canine papillomavirus type 16 associated to squamous cell carcinoma in a dog: virological and pathological findings.

Papillomaviruses (PVs) are circular double-stranded DNA virus belonging to Papillomaviridae family. During the infection cycle, PVs translate proteins that can influence cell growth and differentiation, leading to epidermal hyperplasia and papillomas (warts) or malignant neoplasms. Canis familiaris papillomaviruses (CPVs) have been associated with different lesions, such as oral and cutaneous papillomatosis, pigmented plaques, and squamous cell carcinomas (SCCs). Here, we report a clinical case of a mixed bred female dog with pigmented plaques induced by CPV16 (Chipapillomavirus 2) that progressed to in situ and invasive SCCs. Gross and histological findings were characterized, and the lesions were mainly observed in ventral abdominal region and medial face of the limbs. In situ hybridization (ISH) revealed strong nuclear hybridization signals in the neoplastic epithelial cells, as well as in the keratinocytes and koilocytes of the pigmented viral plaques. The full genome of the CPV16 recovered directly from the lesions was characterized, and the phylogenetic relationships were determined. The identification of oncoprotein genes (E5, E6, and E7) by high throughput sequencing (HTS) and their expected domains are suggestive of the malignant transformation by CPV16.