Differential proteomics of Zika virus (ZIKV) infection reveals molecular changes potentially involved in immune system evasion by a Brazilian strain of ZIKV.

Zika virus (ZIKV) is an arbovirus that was responsible for multiple outbreaks from 2007 to 2015. It has been linked to cases of microcephaly in Brazil in 2015, among other neurological disorders. Differences among strains might be the reason for different clinical outcomes of infection. To evaluate this hypothesis, we performed a comparative proteomic analysis of Vero cells infected with the African strain MR766 (ZIKVAFR) and the Brazilian strain 17 SM (ZIKVBR). A total of 550 proteins were identified as differentially expressed in ZIKVAFR- or ZIKVBR-infected cells compared to the control. The main findings included upregulation of immune system pathways (neutrophil degranulation and adaptive/innate immune system) and potential activation of immune-system-related pathways by ZIKVAFR (mTOR, JAK-STAT, NF-κB, and others) compared with the ZIKVBR/control. In addition, phagocytosis by macrophages and engulfment of leukocytes were activated in ZIKVAFR infection. An in vivo analysis using an immunocompetent C57BL/6N mouse model identified interstitial pneumonia with neutrophil infiltration in the lungs only in mice infected with ZIKVBR at 48 hours postinfection, with a significant amount of virus detected. Likewise, only animals infected with ZIKVBR had viral material in the cytoplasm of lung macrophages. These results suggest that activation of the immune system by ZIKVAFR infection may lead to faster viral clearance by immune cells.

Evolution of the spontaneous sourdoughs microbiota prepared with organic or conventional whole wheat flours from South Brazil.

The purpose of this study was to compare the composition and stability of bacteria and fungi communities during the propagation of sourdoughs prepared with organic or conventional whole wheat (Triticum aestivum) flours from South Brazil. Sourdoughs were prepared and samples were collected during different fermentation times (0 to 216 h). Total DNA of sourdough samples were extracted and the 16S rRNA gene and Internal Transcribed Spacer region were sequenced by MiSeq-Illumina. A total of 43 and 56 OTUs were identified and defined as core taxa in the bacterial and fungal communities, respectively. The analysis revealed increases in the relative abundances of the lactic acid (Pediococcus pentosaceus, Weissella hellenica and Limosilactobacillus pontis) and acetic acid bacteria (Gluconobacter frateurii and Acetobacter tropicalis) during the sourdough propagation. The filaments fungi, Alternaria tenuissima, Fusarium culmorum, Fusarium petersiae and Microdochium seminicola remained more stable in organic than conventional during propagation cycles. After 216 h of fermentation, both sourdoughs were dominated by acid- and salt-tolerant yeast Issatchenkia orientalis (syn Pichia kudriavzevii, and Candida glycerinogenes). In conclusion, there were no significant differences in microbial communities among the sourdough samples. This study revealed that both flours contain autochthonous LAB, AAB, and yeasts with biotechnological applications in sourdough bread-making.

Short-chain fatty acid acetate triggers antiviral response mediated by RIG-I in cells from infants with respiratory syncytial virus bronchiolitis.

BACKGROUND: Gut microbiota-derived short-chain fatty-acid (SFCA) acetate protects mice against RSV A2 strain infection by increasing interferon-ß production and expression of interferon-stimulated genes (ISGs). However, the role of SFCA in RSV infection using strains isolated from patients is unknown. METHODS: We first used RSV clinical strains isolated from infants hospitalized with RSV bronchiolitis to investigate the effects of in vitro SCFA-acetate treatment of human pulmonary epithelial cells. We next examined whether SCFA-acetate treatment is beneficial in a mouse model of RSV infection using clinical isolates. We sought to investigate the relationship of gut microbiota and fecal acetate with disease severity among infants hospitalized with RSV bronchiolitis, and whether treating their respiratory epithelial cells with SCFA-acetate ex-vivo impacts viral load and ISG expression. We further treated epithelial cells from SARS-CoV-2 infected patients with SCFA-acetate. FINDINGS: In vitro pre-treatment of A549 cells with SCFA-acetate reduced RSV infection with clinical isolates and increased the expression of RIG-I and ISG15. Animals treated with SCFA-acetate intranasally recovered significantly faster, with reduction in the RSV clinical isolates viral load, and increased lung expression of IFNB1 and the RIG-I. Experiments in RIG-I knockout A549 cells demonstrated that the protection relies on RIG-I presence. Gut microbial profile was associated with bronchiolitis severity and with acetate in stool. Increased SCFA-acetate levels were associated with increasing oxygen saturation at admission, and shorter duration of fever. Ex-vivo treatment of patients' respiratory cells with SCFA-acetate reduced RSV load and increased expression of ISGs OAS1 and ISG15, and virus recognition receptors MAVS and RIG-I, but not IFNB1. These SCFA-acetate effects were not found on cells from SARS-CoV-2 infected patients. INTERPRETATION: SCFA-acetate reduces the severity of RSV infection and RSV viral load through modulation of RIG-I expression. FUNDING: FAPERGS (FAPERGS/MS/CNPq/SESRS no. 03/2017 - PPSUS 17/2551-0001380-8 and COVID-19 20/2551-0000258-6); CNPq 312504/2017-9; CAPES) - Finance Code 001.

Zika virus exposure affects neuron-glia communication in the hippocampal slices of adult rats.

Zika virus (ZIKV) infection during pregnancy was associated with microcephaly in neonates, but clinical and experimental evidence indicate that ZIKV also causes neurological complications in adults. However, the changes in neuron-glial communication, which is essential for brain homeostasis, are still unknown. Here, we report that hippocampal slices from adult rats exposed acutely to ZIKV showed significant cellular alterations regarding to redox homeostasis, inflammatory process, neurotrophic functions and molecular signalling pathways associated with neurons and glial cells. Our findings support the hypothesis that ZIKV is highly neurotropic and its infection readily induces an inflammatory response, characterized by an increased expression and/or release of pro-inflammatory cytokines. We also observed changes in neural parameters, such as adenosine receptor A2a expression, as well as in the release of brain-derived neurotrophic factor and neuron-specific enolase, indicating plasticity synaptic impairment/neuronal damage. In addition, ZIKV induced a glial commitment, with alterations in specific and functional parameters such as aquaporin 4 expression, S100B secretion and glutathione synthesis. ZIKV also induced p21 senescence-associated gene expression, indicating that ZIKV may induce early senescence. Taken together, our results indicate that ZIKV-induced neuroinflammation, involving nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor κB (NFκB) pathways, affects important aspects of neuron-glia communication. Therefore, although ZIKV infection is transient, long-term consequences might be associated with neurological and/or neurodegenerative diseases.

Clinicopathological characteristics and papillomavirus types in cutaneous warts in bovine.

Thirty-one bovine cutaneous warts were submitted to macroscopic and histological analyses and to molecular analyses to partial amplification and sequencing of the L1 gene of bovine papillomavirus (BPV). Viral types detected were BPV1 (52%), BPV2 (29%), BPV6 (16%) and BPV10 (3%). BPV2 had lower frequency in papilloma in comparison to that in fibropapilloma (p = 0.002).

Zika Virus Infection of Human Mesenchymal Stem Cells Promotes Differential Expression of Proteins Linked to Several Neurological Diseases.

The recent microcephaly outbreak in Brazil has been associated with Zika virus (ZIKV) infection. The current understanding of damage caused by ZIKV infection is still unclear, since it has been implicated in other neurodegenerative and developmental complications. Here, the differential proteome analysis of human mesenchymal stem cells (hMSC) infected with a Brazilian strain of ZIKV was identified by shotgun proteomics (MudPIT). Our results indicate that ZIKV induces a potential reprogramming of the metabolic machinery in nucleotide metabolism, changes in the energy production via glycolysis and other metabolic pathways, and potentially inhibits autophagy, neurogenesis, and immune response by downregulation of signaling pathways. In addition, proteins previously described in several brain pathologies, such as Alzheimer's disease, autism spectrum disorder, amyotrophic lateral sclerosis, and Parkinson's disease, were found with altered expression due to ZIKV infection in hMSC. This potential link between ZIKV and several neuropathologies beyond microcephaly is being described here for the first time and can be used to guide specific follow-up studies concerning these specific diseases and ZIKV infection.

The intestinal virome of malabsorption syndrome-affected and unaffected broilers through shotgun metagenomics.

Malabsorption syndrome (MAS) is an economically important disease of young, commercially reared broilers, characterized by growth retardation, defective feather development and diarrheic faeces. Several viruses have been tentatively associated to such syndrome. Here, in order to examine potential associations between enteric viruses and MAS, the faecal viromes of 70 stool samples collected from diseased (n = 35) and healthy (n = 35) chickens from seven flocks were characterized and compared. Following high-throughput sequencing, a total of 8,347,319 paired end reads, with an average of 231 nt, were generated. Through analysis of de novo assembled contigs, 144 contigs > 1000 nt were identified with hits to eukaryotic viral sequences, as determined by GenBank database. A number of known and unknown representatives of Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, as well as novel uncharacterized CRESS-DNA viruses, were identified. However, the distribution of sequence reads of viral genomes identified in diseased or healthy birds revealed no statistically significant differences. These findings indicate no association between the occurrence of MAS and enteric viruses. The viral genomes reported in the present study, including a variety of novel viruses, seem part of the normal intestinal microbiota of chickens.

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.

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.

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.

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.

Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5 / Soroneutralização com diferentes tipos e subtipos de herpesvírus bovinos 1 e 5

The serum neutralization (SN) test is the gold standard method to measure neutralizing antibodies to bovine herpesviruses. However, in view of the further subdivisions of bovine herpesviruses in types/subtypes, defining which virus to use at challenge in SN tests may be difficult. In view of that, this study was carried out to re-evaluate (SN) sensitivity with different types/subtypes of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) as challenge viruses. Bovine sera (n=810) were collected from two distinct geographic regions and tested by SN with three type 1 viruses (BoHV-1.1 strains "Los Angeles" and "EVI123/98"; BoHV-1.2a strain "SV265/96") and three type 5 viruses (BoHV-5a strain "EVI88/95"; BoHV-5b strain "A663" and BoHV-5c "ISO97/95"). SN tests were performed with a 1 hour incubation of the serum-virus mixtures at 37ºC against 100 TCID50 of each of the viruses. SN sensitivity varied greatly depending on the challenge virus used in the test. The highest sensitivity (327 positive/810 total sera tested; 40.37 percent) was attained when the positive results to the six viruses were added together. No association could be found between any particular type or subtype of virus and the sensitivity of the test. When positive results to each single strain were considered, SN sensitivity varied from 41.7 percent to 81.7 percent, depending on the virus and the geographic region of origin of the sera. Variation was detected even when challenge viruses belonged to the same subtype, where disagreement between positive results reached 41 percent...

O teste de soroneutralização (SN) é o método padrão para a mensuração de anticorpos neutralizantes para herpesvírus bovinos. Entretanto, com as subdivisões propostas destes agentes em tipos e subtipos, a definição de qual amostra utilizar como virus de desafio à SN pode ser difícil. Em vista disso, este estudo foi realizado para re-avaliar a sensibilidade de testes de SN utilizando diferentes tipos e subtipos de herpesvírus bovinos tipos 1 (BoHV-1) e 5 (BoHV-5) como amostras de desafio. Soros bovinos (n=810) foram coletados de duas regiões geográficas distintas e testados frente a amostras do tipo 1 (BoHV-1.1: amostras "Los Angeles" e "EVI123/98", BoHV-1.2a: amostra "SV265/96") e três amostras do tipo 5 (BoHV-5a: "EVI88/95"; BoHV-5b: "A663" e BoHV-5c "ISO97/95"). Os testes de SN foram realizados com incubação de 1 hora a 37ºC da mistura soro-vírus, frente a 100 doses infectantes para 50 por cento dos cultivos celulares (DICC50) de cada um dos vírus. A sensibilidade da SN variou grandemente em função do vírus utilizado no teste. A maior sensibilidade (327 soros positivos/810 soros testados; 40.37 por cento) foi alcançada quando os resultados positivos frente aos seis diferentes vírus foram somados. Nenhuma associação foi detectada entre determinado tipo/subtipo de vírus e a sensibilidade do teste. Quando resultados positivos frente a cada vírus foram considerados isoladamente, a sensibilidade da SN variou entre 41,7 por cento a 81,7 por cento, dependendo do vírus de desafio e da região geográfica de origem das amostras de soro. Variação foi detectada mesmo quando as amostras de desafio pertenciam a um mesmo subtipo; a discrepância entre os resultados positivos atingiu até 41 por cento. Estes resultados indicam que testes de SN contra amostras isoladas de vírus podem apresentar uma sensibilidade notadamente baixa; o emprego de diferentes amostras de vírus de desafio pode aumentar consideravelmente a sensibilidade da prova...

Soroprevalência de herpesvírus bovinos tipos 1 e/ou 5 no Estado do Rio Grande do Sul / Seroprevalence of bovine herpesvirus types 1 and/or 5 in the state of Rio Grande do Sul

Este estudo objetivou estimar a prevalência de anticorpos contra os herpesvírus bovinos tipos 1 e 5 (BoHV-1 e BoHV-5) no Estado do Rio Grande do Sul (RS), Brasil, frente a diferentes cepas de BoHV-1 e BoHV-5. As amostras de soro utilizadas foram extraídas de uma amostragem mais ampla, desenhada para estimar a prevalência de brucelose bovina no Estado. Todos os soros foram coletados de vacas com idade igual ou superior a 24 meses de idade, não vacinadas contra herpesvírus bovinos, de rebanhos de corte e leite. O cálculo amostral foi baseado em uma expectativa de prevalência média de infecção de 33 por cento, considerando-se um erro padrão não superior a 1 por cento e um intervalo de confiança de 95 por cento. Com base nesse cálculo foram examinados 2.200 soros, provenientes de 390 propriedades e 158 municípios. Os soros foram analisados na busca de anticorpos contra BoHV-1 e BoHV-5 pela técnica de soroneutralização (SN), executada frente a quatro cepas de vírus distintas: EVI123/98 e Los Angeles (BoHV-1.1); EVI88/95 (BoHV-5a) e A663 (BoHV-5b). A prevalência média de anticorpos contra o BoHV-1 e BoHV-5 nos animais amostrados foi de 29,2 por cento (642/2200); animais soropositivos foram identificados em 57,7 por cento (225/390) dos rebanhos. As estimativas de prevalência variaram de acordo com a cepa e/ou vírus utilizado para o desafio nos testes de SN. A prevalência e a sensibilidade mais altas foram obtidas quando os resultados positivos à SN frente aos quatro vírus distintos foram somados. O uso de somente um vírus de desafio na SN levaria a redução de sensibilidade de 20,4 por cento a 34,6 por cento quando comparada com os resultados positivos combinados. Estes achados evidenciam que anticorpos contra BoHV-1 e BoHV-5 estão amplamente difundidos nos rebanhos do RS, embora a prevalência em distintas regiões geográficas seja bastante variada. Os resultados obtidos nas estimativas de prevalência foram fortemente afetados pelas diferentes ...

This study was carried out to estimate the prevalence of antibodies to bovine herpesviruses types 1(BoHV-1) and 5 (BoHV-5) in the state of Rio Grande do Sul (RS), Brazil, by testing serum samples against different BoHV-1 and BoHV-5 strains. The sera examined were obtained from a larger sample designed to estimate the prevalence of bovine brucellosis within the state. All sera were collected from cows 24 months or older, not vaccinated to bovine herpesviruses, from both dairy and beef herds. The number of samples to be tested was calculated based on an estimated prevalence of infection of 33 percent, with an average standard deviation of £1 percent and a 95 percent limit of agreement. Sera from 2.200 cattle from 390 farms distributed in 158 counties were tested by serum neutralization (SN) tests in search for antibodies to the following strains: BoHV-1.1 (strains EVI123/98 and Los Angeles), BoHV-5a (strain EVI88/95) and BoHV-5b (strain A663). The overall seroprevalence to BoHV-1 and BoHV-5 in the sampled herds was 29.2 percent (642/2.200); seropositive animals were detected in 225 (57.7 percent) of the sampled farms. Prevalence estimates varied according to the virus used for challenge in SN tests. The highest prevalence and sensitivity were attained when positive SN results against the four different strains were added together. The use of only one virus for challenge in SN tests would lead to a loss in sensitivity from 20.4 percent to 34.6 percent when compared to the combined SN-positive results. These findings provide evidence that antibodies to BoHV-1 and BoHV-5 are largely spread in dairy and beef herds in RS, although prevalence in distinct geographic regions is quite variable. The results were strongly affected by the virus strains used for challenge in SN testing. This must be taken into account when performing serologic tests to detect BoHV-1 and BoHV-5 antibodies. As SN test is not capable of discriminating between antibody ...