Transcriptome analysis of Kluyveromyces marxianus under succinic acid stress and development of robust strains.

Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: • Global gene transcription of K. marxianus is changed by succinic acid (SA) • Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA • Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.

Phylogenetic analysis of porcine circovirus 3 circulating in Canadian pigs.

INTRODUCTION: Porcine circovirus 3 (PCV3) has been detected in pigs worldwide and associated with several clinical signs. METHODS: To investigate the genetic diversity of PCV3 strains circulating in Canada, 44 PCV3 positive samples from Saskatchewan (2/44), Manitoba (2/44), Quebec (4/44), Alberta (11/44) and Ontario (25/44) submitted to diagnostic laboratories in Canada between 2019 and 2021 were sequenced and analyzed. RESULTS: Phylogenetic analysis of capsid genes showed that all of the 44 Canadian strains classified into PCV3a and segregated into seven lineages with common amino acid changes observed at A24V, R27K, N56D, T77S, Q98R, L150I (F) and R168K positions. CONCLUSION: Future studies are required to determine whether the polymorphisms in capsid proteins, as revealed in this study, could be associated with differences in the pathogenicity or antigenicity of PCV3 strains. This is the first phylogenetic analysis of PCV3 strains among different provinces in Canada.

The nasal viromes of cattle on arrival at western Canadian feedlots and their relationship to development of bovine respiratory disease.

Bovine respiratory disease (BRD) has a complex pathogenesis and aetiology, being the costliest disease affecting the cattle industry in North America. In this study, we applied Nanopore-based viral metagenomic sequencing to explore the nasal virome of cattle upon arrival at feedlot and related the findings to the development of BRD. Deep nasal swabs (DNS) from 310 cattle for which BRD outcomes were known (155 cattle developed BRD within 40 days and 155 remained healthy) were included. The most prevalent virus in on-arrival samples was bovine coronavirus (BCV) (45.2%, 140/310), followed by bovine rhinitis virus B (BRBV) (21.9%, 68/310), enterovirus E (EVE) (19.6%, 60/310), bovine parainfluenza virus 3 (BPIV3) (10.3%, 32/310), ungulate tetraparvovirus 1 (UTPV1) (9.7%, 30/310) and influenza D virus (7.1%, 22/310). No relationship was found between BRD development and the number of viruses detected, the presence of any specific individual virus or combination of viruses. Bovine kobuvirus (BKV) was detected in 2.6% of animals (8/310), being the first report of this virus in Canada. Results of this study demonstrate the diversity of viruses in bovine DNS collected upon arrival at feedlot and highlights the need for further research into prediction of BRD development in the context of mixed infections.

Equine small intestinal angiomatosis.

Multiple red, raised nodules multifocally distributed along the serosal surface of the normal and the nonviable jejunum were identified in a 24-year-old neutered male horse undergoing surgery for removal of the strangulating lipoma around the jejunum. Histologically, these nodules consisted of many significantly and variably dilated, blood-filled vascular channels lined by a single layer of flattened, well-differentiated endothelial cells with occasional thrombi within a mildly thickened fibrous stroma. A diagnosis of intestinal angiomatosis was proposed. To the best of the authors' knowledge, this is the second report of small intestinal angiomatosis in a horse.

Angiomatose du petit intestin équin. De multiples nodules rouges surélevés distribués de manière multifocale le long de la surface séreuse du jéjunum viable et non-viable furent identifiés chez un cheval mâle castré âgé de 24 ans soumis à une chirurgie pour le retrait d'un lipome étranglant autour du jéjunum. Histologiquement, ces nodules consistaient en de nombreux canaux vasculaires remplis de sang dilatés de manière significative et variable, et tapissés par une couche unique de cellules endothéliales aplaties et bien différenciées avec à l'occasion des thrombi à l'intérieur d'un stroma fibreux légèrement épaissi. Un diagnostic d'angiomatose intestinale fut proposé. Au meilleur de la connaissance des auteurs, ceci constitue le deuxième rapport d'angiomatose du petit intestin chez un cheval.(Traduit par Dr Serge Messier).

Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples.

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What's In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

The pulmonary virome, bacteriological and histopathological findings in bovine respiratory disease from western Canada.

The aetiology and pathogenesis of bovine respiratory disease (BRD) are complex and involve the interplay of infectious agents, management and environmental factors. Previous studies of BRD focused on ante-mortem samples from the upper respiratory tract and identified several unconventional viruses. The lung, however, is the primary location where significant BRD lesions are usually found and is a common post-mortem diagnostic specimen. In this study, results of high-throughput virome sequencing, bacterial culture, targeted real-time PCR and histological examination of 130 bovine pneumonic lungs from western Canadian cattle were combined to explore associations of microorganisms with different types of pneumonia. Fibrinous bronchopneumonia (FBP) was the predominant type of pneumonia (46.2%, 60/130) and was associated with the detection of Mannheimia haemolytica. Detection of Histophilus somni and Pasteurella multocida was associated with suppurative bronchopneumonia (SBP) and concurrent bronchopneumonia and bronchointerstitial pneumonia (BP&BIP), respectively. Sixteen viruses were identified, of which bovine parvovirus 2 (BPV2) was the most prevalent (11.5%, 15/130) followed by ungulate tetraparvovirus 1 (UTPV1, 8.5%, 11/130) and bovine respiratory syncytial virus (BRSV, 8.5%, 11/130). None of these viruses, however, were significantly associated with a particular type of pneumonia. Unconventional viruses such as influenza D virus (IDV) and bovine rhinitis B virus (BRBV) were detected, although sparsely, consistent with our previous findings in upper respiratory tract samples. Taken together, our results show that while virus detection in post-mortem lung samples is of relatively little diagnostic value, the strong associations of H. somni and M. haemolytica with SBP and FBP, respectively, indicate that histopathology can be useful in differentiating bacterial aetiologies.

Respiratory viruses identified in western Canadian beef cattle by metagenomic sequencing and their association with bovine respiratory disease.

Bovine respiratory disease (BRD) causes considerable economic losses in North America. The pathogenesis involves interactions between bacteria, viruses, environment and management factors. Primary viral infection can increase the risk of secondary fatal bacterial infection. The objective of this study was to use metagenomic sequencing to characterize the respiratory viromes of paired nasal swabs and tracheal washes from western Canadian feedlot cattle, with or without BRD. A total of 116 cattle (116 nasal swabs and 116 tracheal washes) were analysed. The presence of influenza D virus (IDV), bovine rhinitis A virus (BRAV), bovine rhinitis B virus (BRBV), bovine coronavirus (BCV) and bovine respiratory syncytial virus (BRSV) was associated with BRD. Agreement between identification of viruses in nasal swabs and tracheal washes was generally weak, indicating that sampling location may affect detection of infection. This study reported several viruses for the first time in Canada and provides a basis for further studies investigating candidate viruses important to the prevention of BRD.

Characterization of the interactome of the porcine reproductive and respiratory syndrome virus glycoprotein-5.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry, causing reproductive failure in sows and respiratory disorders in piglets. Glycosylated protein 5 (GP5) is a major envelope protein of the virus. It is essential for virus particle assembly and involved in viral pathogenesis. In the present study, we identified the host cellular proteins that interact with GP5 by performing immunoprecipitation in MARC-145 cells infected by a recombinant PRRSV containing a FLAG-tag insertion in GP5. In total, 122 cellular proteins were identified by LC-MS/MS. Gene Ontology and KEGG databases were used to map these proteins to different cellular processes, locations and functions. Interestingly, 10.24% of identified cellular proteins were involved in the process of translation. Follow up experiments demonstrated that expression of GP5 in transfected cells led to inhibition of translation of reporter genes. Interaction between GP5 and ATP synthase subunit alpha (ATP5A) was further confirmed by co-immunoprecipitation suggesting a possible role of GP5 in regulation of ATP production in cells. These data contribute to a better understanding of GP5's role in viral pathogenesis and virus-host interactions.

Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with tubulin.

Porcine reproductive and respiratory syndrome virus (PRRSV) encodes the small envelope (E) protein which is a minor structural component of the virion that is important for virus infectivity. To better understand the biological functions of the E protein, we studied interactions between E and PRRSV cellular proteins. Using immunoprecipitation-coupled mass spectrometry approach, we previously identified tubulin-α as an interacting partner of E. In this study, we confirmed this interaction using co-immunoprecipitation and co-localization assays. In addition, we demonstrated that the 25-residue C-terminal endodomain of E was essential for its interaction with tubulin-α. Over-expression of the E protein in cultured cells led to microtubule depolymerisation. Similarly, we observed that microtubule depolymerisation occurs in MARC-145 cells at the late stage of PRRSV replication. Also, depolymerisation of microtubules by colcemid significantly inhibited PRRSV replication in MARC-145 cells at early time points but the effect was not as dramatic at the late stage of infection. These data suggest that PRRSV infection of MARC-145 cells requires the microtubules network to facilitate early phase of infection whereas microtubules depolymerisation occurs at the late stage of PRRSV replication. Interaction between E and tubulin-α may contribute to microtubules depolymerisation.

GP5 expression in Marc-145 cells inhibits porcine reproductive and respiratory syndrome virus infection by inducing beta interferon activity.

The major neutralizing epitope of porcine reproductive and respiratory syndrome virus (PRRSV) is mainly located on virus glycoprotein 5 (GP5). Immunization with exogenous GP5 or exposure to native GP5 by means of DNA immunization can provide some degree of immune protection to PRRSV infection in pigs. However, during PRRSV infection in pigs, the production of neutralization antibodies induced by GP5 is delayed or suppressed. This suggests that the synthesis of GP5 is late than some PRRSV proteins or other PRRSV proteins interfering with the function of GP5 in inducing host responses during virus infection. Here, to exclude the impacts of the other PRRSV proteins and determine the role of GP5 in the replication of PRRSV in vitro, a Marc-145 cell line stably expressing GP5 (Marc-145-GP5(Flag)) was constructed. Cell proliferation and cell apoptosis measurements indicated that the expression of GP5 in Marc-145 cells did not disturb the cells' viability. Following infection with different PRRSV strains PRRSV replication in Marc-145-GP5(Flag) cells was inhibited significantly. Type I interferon assay results showed that beta interferon (IFN-ß) in the Marc-145-GP5(Flag) cells were increased at mRNA and protein levels. When siRNA was introduced into the cells to knock down IFN-ß mRNA, PRRSV infectivity of these cells was recovered. These data suggest that early GP5 expression is not favorable for further infection by PRRSV, because it not only stimulates production of neutralization antibodies in pigs, but also induces IFN-ß production in host cells. Therefore, GP5 is an important protein in the induction of self-protection responses from the host.