Carrier state of enterotoxigenic Escherichia coli virulence markers in pigs: effects on gut microbiota modulation and immune markers transcription.

Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in pigs at early age, leading to high mortality rates and significant economic losses in the swine industry. ETEC effect on gut microbiota and immune system is mostly studied in diarrheic model under controlled laboratory conditions, however its impact on asymptomatic carriers remains unknown. Thus, we investigated whether ETEC can modulate gut microbiota or regulate the transcription of immune markers in asymptomatic pigs in farm environment. Stool samples from newborn piglets, nursery and growing pigs, and sows were screened for ETEC markers, then submitted to 16S-rDNA sequencing to explore gut microbiota composition in carriers (ETEC+) and non-carriers (ETEC-) animals. We observed a reduced α-diversity in ETEC+ animals (p<0.05), while bacterial compositions were mostly driven by ageing (p>0.05). Prevotella marked ETEC-carrier group, while Rikenellaceae RC9 gut group was a marker for a healthy gut microbiota, suggesting being biomarker candidates for surveillance and supplementation purposes. Furthermore, we observed transcription regulation of il6 and tff2 genes in ETEC+ in newborn and nursery stages, respectively. Our findings indicate that ETEC presence modulate gut microbiota and the immune response in asymptomatic pigs; nevertheless, further studies using a probabilistic design must be performed to assess the effect of ETEC presence on gut imbalance in pigs despite the age bias.

Influence of Zika virus on the cytotoxicity, cell adhesion, apoptosis and inflammatory markers of glioblastoma cells.

Glioblastoma (GBM) is one of the most common types of brain tumor in adults. Despite the availability of treatments for this disease, GBM remains one of the most lethal and difficult types of tumors to treat, and thus, a majority of patients die within 2 years of diagnosis. Infection with Zika virus (ZIKV) inhibits cell proliferation and induces apoptosis, particularly in developing neuronal cells, and thus could potentially be considered an alternative for GBM treatment. In the present study, two GBM cell lines (U-138 and U-251) were infected with ZIKV at different multiplicities of infection (0.1, 0.01 and 0.001), and cell viability, migration, adhesion, induction of apoptosis, interleukin levels and CD14/CD73 cell surface marker expression were analyzed. The present study demonstrated that ZIKV infection promoted loss of cell viability and increased apoptosis in U-138 cells, as measured by MTT and triplex assay, respectively. Changes in cell migration, as determined by wound healing assay, were not observed; however, the GBM cell lines exhibited an increase in cell adhesion when compared with non-tumoral cells (Vero). The Luminex immunoassay showed a significant increase in the expression levels of IL-4 specifically in U-251 cells (MOI 0.001) following exposure to ZIKV. There was no significant change in the expression levels of IFN-γ upon ZIKV infection in the cell lines tested. Furthermore, a marked increase in the percentage of cells expressing the CD14 surface marker was observed in both GBM cell lines compared with in Vero cells; and significantly increased CD73 expression was observed particularly in U-251 cells, when compared with uninfected cells. These findings indicate that ZIKV infection could lead to reduced cell viability, elevated CD73 expression, improved cellular adherence, and higher rates of apoptosis in glioblastoma cells. Further studies are required to explore the potential use of ZIKV in the treatment of GBM.

Alternative amplicon-PCR protocol for maximizing bacterial and fungal sequencing in low-biomass samples.

Determining bacterial and fungal communities from low-biomass samples remains a challenge for high-throughput sequencing. Due to the low microbial load and host contamination, some sites, including the female upper reproductive tract and the lower respiratory tract, were even considered sterile until recent years. Despite efforts to improve sampling and DNA isolation protocols, some samples provide insufficient microbial DNA input for library preparation and sequencing. Herein, we propose an alternative amplicon-PCR protocol to be used in bacterial and fungal sequencing in low-biomass samples, targeting 16S-rDNA and the internal transcribed spacer region (ITS), respectively. Similar to a nested-PCR, we performed two sequential PCR reactions to maximise the target amplicon. We compared metagenomic results from the original Illumina protocol (Protocol 1 - P1) and the alternative one (Protocol 2 - P2), using a mock community and clinical samples with different microbial loads. Our findings showed no significant differences in data generated by P1 and P2, indicating that the second amplification round does not bias the microbiota diversity rates. Thus, the alternative protocol can be applied for low-biomass samples when the original protocol results in spurious output, preventing library preparation and sequencing.

Incipient Parallel Evolution of SARS-CoV-2 Deltacron Variant in South Brazil.

With the coexistence of multiple lineages and increased international travel, recombination and gene flow are likely to become increasingly important in the adaptive evolution of SARS-CoV-2. These processes could result in genetic introgression and the incipient parallel evolution of multiple recombinant lineages. However, identifying recombinant lineages is challenging, and the true extent of recombinant evolution in SARS-CoV-2 may be underestimated. This study describes the first SARS-CoV-2 Deltacron recombinant case identified in Brazil. We demonstrate that the recombination breakpoint is at the beginning of the Spike gene. The 5' genome portion (circa 22 kb) resembles the AY.101 (Delta), and the 3' genome portion (circa 8 kb nucleotides) is most similar to the BA.1.1 (Omicron). Furthermore, evolutionary genomic analyses indicate that the new strain emerged after a single recombination event between lineages of diverse geographical locations in December 2021 in South Brazil. This Deltacron, AYBA-RS, is one of the dozens of recombinants described in 2022. The submission of only four sequences in the GISAID database suggests that this lineage had a minor epidemiological impact. However, the recent emergence of this and other Deltacron recombinant lineages (XD, XF, and XS) suggests that gene flow and recombination may play an increasingly important role in the COVID-19 pandemic. We explain the evolutionary and population genetic theory that supports this assertion, concluding that this stresses the need for continued genomic surveillance. This monitoring is vital for countries where multiple variants are present, as well as for countries that receive significant inbound international travel.

Comparative Analysis of the Upper Respiratory Bacterial Communities of Pigs with or without Respiratory Clinical Signs: From Weaning to Finishing Phase.

A prospective study was conducted to identify bacterial communities in the nasal and laryngeal cavities of pigs with or without clinical signs of respiratory disease in a longitudinal fashion, from weaning to the finishing phase. Nasal and laryngeal swabs were collected from asymptomatic pigs (n = 30), as well as from pigs with clinical signs of respiratory disease (n = 30) at the end of the weaning (T1-33 days) phase, end of the nursery phase (T2-71 days), and finishing (T3-173 days). Total DNA was extracted from each sample, and the V4 hypervariable region of the 16S rRNA gene was amplified and sequenced with the Illumina MiSeq platform. Principal coordinates analysis indicated no significant differences between the nasal and laryngeal bacterial communities. Nevertheless, the microbiota composition in the upper respiratory tract (URT) was clearly distinct between animals, with or without signs of respiratory disease, particularly at post-weaning and the end of nursery. In pigs with clinical signs of respiratory disease, Actinobacillus, Streptococcus Porphyromonas, Veillonella, and an unclassified genus of Pasteurellaceae were more abundant than in pigs with no signs. Metabolic prediction identified 28 differentially abundant pathways, mainly related to carbohydrate, energy, amino acid, anaerobic, and nucleotide metabolism in symptomatic pigs (especially in T2). These findings provide evidence that the composition of the URT bacterial microbiota differs significantly when comparing pigs with or without respiratory clinical signs after weaning, and this difference is maintained in the nursery phase; such differences, however, were not evident at the finishing phase.

Cows' reproductive performances and parity order influences the cervicovaginal fungal community.

Fungi have already been described as etiological agents of reproductive diseases such as endometritis and infertility in cows. However, few studies have been developed to elucidate the entire cervicovaginal fungal communities in cows. Therefore, our study aimed to characterize the fungal community present in the cervix of cows with different reproductive performances. Cervicovaginal mucus was collected from 36 Angus breed cows (1.5-12 years old) on a commercial beef cattle ranch. Twenty-one cows had a history of infertility in the year prior to the collection, showing early return to estrus. Ten cows were sampled at 60-70 days postpartum being considered fertile cows. Additionally, five non-sexually active heifers were employed as control group. Ascomycota and Basidiomycota were the predominant fungal phyla in the analyzed animals. Diversity metrics of the cervicovaginal fungal community revealed statistical differences in the composition of the fungal community among infertile cows, fertile cows and non-sexually active heifers. In addition, the cervicovaginal fungal microbiota had significative increased richness and evenness in nulliparous cows and non-sexually active heifers, while in multiparous cows a decreased richness and evenness of the fungal microbiota were identified. These results provide an unprecedented understanding of the cervicovaginal fungal structure associated with infertility and parity order.

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.

Emergence of the novel SARS-CoV-2 lineage VUI-NP13L and massive spread of P.2 in South Brazil.

In this study, we analyzed 340 whole genomes of SARS-CoV-2, which were sampled between April and November 2020 in 33 cities of Rio Grande do Sul, South Brazil. We demonstrated the circulation of two novel emergent lineages, VUI-NP13L and VUI-NP13L-like, and five major lineages that had already been assigned (B.1.1.33, B.1.1.28, P.2, B.1.91, B.1.195). P.2 and VUI-NP13L demonstrated a massive spread in October 2020. Constant and consistent genomic surveillance is crucial to identify newly emerging SARS-CoV-2 lineages in Brazil and to guide decision making in the Brazilian Public Healthcare System.

Viral DNA genomes in sera of farrowing sows with or without stillbirths.

A study was conducted to investigate the serum virome of sows with and without stillbirths after farrowing. Sera from sows with at least one stillbirth or with normal litters were collected immediately after farrowing. Viral DNA was extracted from serum pools and submitted to high throughput sequencing. No differences in the proportion of virus-related reads were found in both groups (p > 0.05). A variety of viral DNA genomes were identified, mostly representative of three viral families: Anelloviridae, Circoviridae and Smacoviridae. Besides, a number of novel unclassified circular Rep-encoding single stranded DNA (CRESS DNA) viruses were also identified. These findings suggest that the presence of such viral genomes in sows' sera bears no correlation with stillbirths' occurrence; it seems likely that these constitute part of the normal serum microbiome of sows at farrowing.

Full-Genome Sequence of a Reassortant H1N2 Influenza A Virus Isolated from Pigs in Brazil.

In this study, the full-genome sequence of a reassortant H1N2 swine influenza virus is reported. The isolate has the hemagglutinin (HA) and neuraminidase (NA) genes from human lineage (H1-δ cluster and N2), and the internal genes (polymerase basic 1 [PB1], polymerase basic 2 [PB2], polymerase acidic [PA], nucleoprotein [NP], matrix [M], and nonstructural [NS]) are derived from human 2009 pandemic H1N1 (H1N1pdm09) virus.