Virus discovery in cultured portunid crabs: Genomic, phylogenetic, histopathological and microscopic characterization of a reovirus and a new bunyavirus.

Portunid crabs are distributed worldwide and highly valued in aquaculture. Viral infections are the main limiting factor for the survival of these animals and, consequently, for the success of commercial-scale cultivation. However, there is still a lack of knowledge about the viruses that infect cultured portunid crabs worldwide. Herein, the genome sequence and phylogeny of Callinectes sapidus reovirus 2 (CsRV2) are described, and the discovery of a new bunyavirus in Callinectes danae cultured in southern Brazil is reported. The CsRV2 genome sequence consists of 12 dsRNA segments (20,909 nt) encode 13 proteins. The predicted RNA-dependent RNA polymerase (RdRp) shows a high level of similarity with that of Eriocheir sinensis reovirus 905, suggesting that CsRV2 belongs to the genus Cardoreovirus. The CsRV2 particles are icosahedral, measuring approximately 65 nm in diameter, and exhibit typical non-turreted reovirus morphology. High throughput sequencing data revealed the presence of an additional putative virus genome similar to bunyavirus, called Callinectes danae Portunibunyavirus 1 (CdPBV1). The CdPBV1 genome is tripartite, consisting of 6,654 nt, 3,120 nt and 1,656 nt single-stranded RNA segments that each encode a single protein. Each segment has a high identity with European shore crab virus 1, suggesting that CdPBV1 is a new representative of the family Cruliviridae. The putative spherical particles of CdPBV1 measure ∼120 nm in diameter and present a typical bunyavirus morphology. The results of the histopathological analysis suggest that these new viruses can affect the health and, consequently, the survival of C. danae in captivity. Therefore, the findings reported here should be used to improve prophylactic and pathogen control practices and contribute to the development and optimization of the production of soft-shell crabs on a commercial scale in Brazil.

Characterization of novel of temperate phages of Staphylococcus aureus isolated from bovine milk.

Bovine mastitis is an important disease in dairy cows, and Staphylococcus aureus is the most prevalent microorganism. Bacteriophages are considered an alternative to treat bacterial infections due to antimicrobial resistance crisis. In this study, we isolated and characterized novel S. aureus temperate phages, namely B_UFSM4 and B_UFSM5, from bovine milk. The complete genomes of B_UFSM4 and B_UFSM5 have 41.396 bp and 41.829 bp, respectively. The viruses have double-stranded DNA and linear architecture. Phylogenic similarity was observed by proteome with Staphylococcus phage phiPV83, CN125 and JS01. Therefore, the phages were classified into the family Siphoviridae, genus Biseptimavirus and order Caudovirales. In the host range, the B_UFSM4 and B_UFSM5 had lytic activity of 45.8% and 54.16%, respectively, inclusive on isolates from Staphylococcus sciuri and Rothia terrae. Thus, in this study, species novel of S. aureus temperate phages was isolated and characterized, these phages reveal similarities to each other; however, they are distinct from other species of S. aureus phages of the family Siphoviridae.

The virome of the white-winged vampire bat Diaemus youngi is rich in circular DNA viruses.

In the Neotropical region, the white-winged vampire bat (Diaemus youngi) is the rarest of the three species of vampire bats. This bat species feeds preferentially on bird blood, and there is limited information on the viruses infecting D. youngi. Hence, this study aimed to expand the knowledge about the viral diversity associated with D. youngi by sampling and pooling the lungs, liver, kidneys, heart, and intestines of all animals using high-throughput sequencing (HTS) on the Illumina MiSeq platform. A total of three complete and 10 nearly complete circular virus genomes were closely related to gemykrogvirus (Genomoviridae family), smacovirus (Smacoviridae family), and torque teno viruses (TTVs) (Anelloviridae family). In addition, three sequences of bat paramyxovirus were detected and found to be closely related to viruses reported in Pomona roundleaf bats and rodents. The present study provides a snapshot of the viral diversity associated with white-winged vampire bats and provides a baseline for comparison to viruses detected in future outbreaks.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs.

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named 'Suismacovirus', comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses' genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

Detection of multiple viruses in oropharyngeal samples from Brazilian free-tailed bats (Tadarida brasiliensis) using viral metagenomics.

In this study, we analyzed the viral population in oropharyngeal samples from T. brasiliensis using a viral metagenomic approach. Genomes corresponding to members of the families Circoviridae, Genomoviridae, Herpesviridae, Paramyxoviridae, Coronaviridae, and Astroviridae were detected. This study provides the first preliminary understanding of the oropharyngeal virome of T. brasiliensis, which may guide the discovery and isolation of novel viruses in the future and highlights the need for continuing investigations in this regard.

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.

Investigation on porcine circovirus type 3 in serum of farrowing sows with stillbirths.

Since its first identification in 2016, porcine circovirus 3 (PCV3) has been detected in healthy and/or diseased swine in many countries worldwide. In a previous study by our group, PCV3 was detected in sera of sows which had at least one stillborn piglet in the last parturition. As such, it became important to investigate if the presence of PCV3 in sows' sera could be associated to the occurrence of stillbirths. With that aim, the frequency of PCV3 infections and viral DNA loads in sows' sera was investigated through a real-time quantitative PCR in 89 serum samples of just farrowed sows with or without stillbirths. PCV3 genomes were identified in most samples, with genome loads ranging between less than 10 to 200,000 copies per mL of serum. No significant differences were observed either in the frequency of infection or PCV3 viral loads in sows with or without stillbirths. Thus, no association could be established between PCV3 infection of sows at farrowing and stillbirths' occurrence.

The virome of an endangered stingless bee suffering from annual mortality in southern Brazil.

Meliponiculture - the management of stingless bee colonies - is an expanding activity in Brazil with economic, social and environmental potential. However, unlike in apiculture, the pathogens that impact on meliponiculture remain largely unknown. In southern Brazil, every year at the end of the summer, managed colonies of the stingless bee Melipona quadrifasciata manifest a syndrome that eventually leads to collapse. Here we characterize the M. quadrifasciata virome using high-throughput sequencing, with the aim of identifying potentially pathogenic viruses, and test whether they are related to the syndrome outbreaks. Two paired viromes are explored, one from healthy bees and another from unhealthy ones. Each virome is built from metagenomes assembled from sequencing reads derived either from RNA or DNA. A total of 40 621 reads map to viral contigs of the unhealthy bees' metagenomes, whereas only 11 reads map to contigs identified as viruses of healthy bees. The viruses showing the largest copy numbers in the virome of unhealthy bees belong to the family Dicistroviridae - common pathogenic honeybee viruses - as well as Parvoviridae and Circoviridae, which have never been reported as being pathogenic in insects. Our analyses indicate that they represent seven novel viruses associated with stingless bees. PCR-based detection of these viruses in individual bees (healthy or unhealthy) from three different localities revealed a statistically significant association between viral infection and symptom manifestation in one meliponary. We conclude that although viral infections may contribute to colony collapses in the annual syndrome in some meliponaries, viruses spread opportunistically during the outbreak, perhaps due to colony weakness.

Bovine papillomavirus 24: a novel member of the genus Xipapillomavirus detected in the Amazon region.

Bovine papillomaviruses (BPVs) have been described as etiologic agents of cutaneous and mucosal papillomas in cattle. In the present study, we describe a new BPV that was detected in a cutaneous papilloma from a cow. Phylogenetic analysis suggests that this virus belong to the genus Xipapillomavirus, and we refer to it here as BPV type 24 (BPV24). Coinfection with members of the genera Epsilonpapillomavirus and Deltapapillomavirus in a cutaneous papilloma from a different animal was also detected, and the full genomes of these viruses were sequenced. Both papillomas were from cattle within Acre State in the Amazon region of Brazil. The data presented here demonstrate the utility of using high-throughput methods to indentify coinfections and allow the characterization of new genomes.

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.

Faecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular ssDNA viruses.

This study is focused on the identification of the faecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with blastx revealed that 279 contigs (4 %) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes. In addition, a range of circular replication-associated protein encoding DNA viruses were also identified. The characterization of the faecal virome of healthy chickens described here not only provides a description of the viruses encountered in such niche but should also represent a baseline for future studies comparing viral populations in healthy and diseased chicken flocks. Moreover, it may also be relevant for human health, since chickens represent a significant proportion of the animal protein consumed worldwide.

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 sequence of bubaline alphaherpesvirus 1 (BuHV1) isolated in Australia in 1972.

Bubaline alphaherpesvirus 1 (BuHV1) is a member of the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. To date, no full genome sequence of BuHV has been published. Here, we report the complete genome sequence of bubaline alphaherpesvirus 1 (BuHV1) strain b6 (BuHV1-b6), isolated from a water buffalo (Bubalus bubalis) in 1972 in Australia. The virus was multiplied in MDBK cells, and the DNA was extracted and subjected to high-throughput sequencing. The reads were aligned and combined into a single genome sequence, with bovine alphaherpesvirus 5 (BoHV5) strain SV507/99 (accession number NC005261) as a reference. The BuHV1-b6 genome is a linear double-stranded DNA molecule, 137,452 bp long, with a GC content of 76.8%. The genome consists of two unique sequences: a long, or UL, sequence (103,818 bp) and a short, or US, sequence (9,586 bp), with the latter being flanked by inverted IR and TR elements of 12,024 bp each. The arrangement is typical of herpesvirus genomes of the D-type. The overall sequence has a 92.2% similarity at the nucleotide level to the reference BoHV5 strain. Our report provides a significant landmark in the history of herpesviruses, represented by the genome sequence of this 44-year-old virus isolate.

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.

Ungulate copiparvovirus 2 in healthy and postweaning multisystemic wasting syndrome-affected pigs.

A SYBR Green-based real-time polymerase chain reaction (qPCR) was designed to detect Ungulate copiparvovirus 2, also known as porcine parvovirus 4 (PPV4). The test was applied to search for PPV4 DNAemia in sera from 1- to 4-month-old pigs displaying signs of postweaning multisystemic wasting syndrome (PMWS), as well as in sera from healthy swine at equivalent age and in sera from older healthy animals (>6 months old). High levels of PPV4 DNA were detected in PMWS-affected pigs. The mean viral DNA load in PMWS-affected pigs was 5.2 × 107 copies/mL, whereas in young healthy pigs it was 1.4 × 105 copies/mL (P ≤ 0.001). Although the copy numbers were lower in younger PMWS-affected individuals, this result sheds some light on the possible association between PPV4 viral load detection in this group and the immune impairment caused by PMWS.

Ungulate copiparvovirus 1 (bovine parvovirus 2): characterization of a new genotype and associated viremia in different bovine age groups.

A novel bovine parvovirus 2 (BPV2) genotype comprising 5394 nt was identified by next generation sequencing from sera of healthy cattle at different age groups farmed in the state of Rio Grande do Sul, Brazil. The genome organization of new BPV2 genotype retains the two ORFs typical of members of the Parvovirinae with 86.4 % of overall nucleotide sequence identities in comparison to other members of the subfamily. Phylogenetic analysis revealed similar clustering with two previously described bovine BPV2 within the genus Copiparvovirus. No significant differences (P ≥ 0.05) were detected in the distribution of BPV2 infection in cattle at different age groups. This is the third complete or near complete genome sequence of BPV2 reported to date and may contribute to a better understanding of the biology of copiparvoviruses and its interactions with the host.

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.

Chicken parvovirus viral loads in cloacal swabs from malabsorption syndrome-affected and healthy broilers.

Chicken parvovirus (ChPV) has been associated with malabsorption syndrome (MAS) in broilers. However, the participation of this virus in such syndrome is unclear, since it may be detected in diseased and healthy chickens. In the course of these studies, it was argued whether ChPV genome loads might be correlated to the occurrence of MAS. To check such a hypothesis, a SYBR green-based quantitative polymerase chain reaction was developed to detect and quantify ChPV genomes. Cloacal swabs from 68 broilers with MAS and 59 from healthy animals were collected from different poultry farms. Genomes of ChPV were detected in all samples, regardless of their health status. However, viral genome loads in MAS-affected broilers were significantly higher (1 × 105 genome copies per 100 ng DNA) than in healthy animals (1.3 × 103 GC/100 ng DNA). These findings indicate that there is an association between high ChPV genome loads and the occurrence of MAS in broilers.

Molecular detection and characterization of BK and JC polyomaviruses in urine samples of renal transplant patients in Southern Brazil.

The human polyomaviruses JC (JCPyV) and BK (BKPyV) are widespread in the human population. Following the primary infection, virus reactivation may lead to nephropathy and graft rejection in renal transplant patients. This study was carried out to access the presence of BKPyV and JCPyV DNA in urine samples collected from renal transplant patients (n = 92) and healthy individuals (n = 88) in Porto Alegre, Rio Grande do Sul. The samples were submitted to a nested PCR. A significantly higher frequency (P < 0.001) of BKPyV was found in renal transplant patients (65.2%) in comparison to the control group (32.9%). JCPyV was detected equally in both groups. Phylogenetic analysis of both BKPyV and JCPyV amplicons demonstrates the presence of the BKPyV subtypes I and II, whereas for JCPyV, four different groups are found (1, 2, 3, and 4).