An ELISA to Detect Antibodies to Bovine Alphaherpesviruses 1 and 5 and Bubaline Alphaherpesvirus 1 in Cattle Sera.

Bovine alphaherpesvirus 1 (subtypes 1.1, 1.2a, and 1.2b), type 5 (subtypes 5a, 5b, and 5c), and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity (κ = 0.93; PPV = 95.0%; NPV = 97.3%). The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses.

Complete genome characterization of porcine circovirus 3 recovered from wild boars in Southern Brazil.

In the present study, the complete nucleotide sequence of porcine circovirus 3 (PCV3) recovered from wild boars lymph nodes is described. The full genome was named PCV3-wb/Br/RS and comprises 2,000 nucleotides with two open reading frames (ORFs) with a stem-loop motif in intergenic region. The ORFs are oriented in opposite directions and encode the putative capsid (Cap) and replicase (Rep) proteins. Based on amino acid motif analysis, PCV3-wb/Br/RS as well as most of the sequences from wild boars are classified as PCV3b. Phylogenetic analysis including 97 PCV3 sequences available in databases showed that the PCV3-wb/Br/RS genome is more closely related to genomes recovered in Spain, China, Germany and Denmark. Phylogenetic inferences among PCV3-wb/Br/RS and other circoviruses confirmed that these seem to have a most recent common ancestor with bat-associated circoviruses. In addition, PCV3 infection was investigated by real-time PCR in a cohort of 80 wild boars in Southern Brazil. A total of 29 animals (36.3%) were PCV3-positive leading the conclusion that PCV3 is circulating in the wild boar population in Southern Brazil. The role played by PCV3-like infections in wild boars and the risk these could pose to commercial swine production within that region remains to be further investigated.

Viral metagenomics in Brazilian Pekin ducks identifies two gyrovirus, including a new species, and the potentially pathogenic duck circovirus.

Viral metagenomics coupled to high-throughput sequencing has provided a powerful tool for large-scale detection of known and unknown viruses associated to distinct hosts and environments. Using this approach, known and novel viruses have been characterized from sylvatic and commercial avian hosts, increasing our understanding of the viral diversity in these species. In the present work we applied an exploratory viral metagenomics on organs (spleen, liver and bursa of Fabricious) of Pekin ducks from Southern Brazil. The virome contained sequences related to a known duck pathogen (duck circovirus) and a number of other circular ssDNA viruses. Additionally, we detected avian gyrovirus 9 (to date detected only in human feces) and one new avian gyrovirus species, to which is proposed the name avian gyrovirus 13 (GyV13). This study is expected to contribute to the knowledge of the viral diversity in Pekin ducks.

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.

Biofilm formation by Salmonella Enteritidis and Salmonella Typhimurium isolated from avian sources is partially related with their in vivo pathogenicity.

Salmonella Enteritidis and Salmonella Typhimurium are among the most prevalent serotypes isolated from salmonellosis outbreaks and poultry. Salmonella spp. have the capacity to form biofilms on several surfaces, which can favour survival in hostile environments, such as slaughterhouses. Salmonella strains present differences in pathogenicity. However, there is little information regarding the pathogenicity of S. Enteritidis and S. Typhimurium isolated from avian sources and their relationship to biofilm production. The aim of this study was to use a novel pathogenicity index and a biofilm production assay to evaluate their relationships within these serotypes. In addition, we detected the presence of the spiA and agfA genes in these strains. Biofilm formation was investigated at two temperatures (37 °C and 28 °C) using microtiter plate assay, and the results were compared with the individual pathogenicity index of each strain. PCR was used to detect spiA and agfA, virulence genes associated with biofilm production. S. Enteritidis and S. Typhimurium strains were capable of producing biofilm at 37 °C and 28 °C. Sixty-two percent and 59.5% of S. Enteritidis and 73.8% and 46.2% of S. Typhimurium produced biofilm at 37 °C and 28 °C, respectively. Biofilm production at 37 °C was significantly higher in both serotypes. Only S. Enteritidis was capable of adhering strongly at both temperatures. Biofilm production was related to pathogenicity index only at 28 °C for S. Enteritidis. spiA and agfA were found in almost all strains and were not statistically associated with biofilm production.

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.

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.

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.