118 research outputs found

    Changes on the viral capsid surface during the evolution of porcine circovirus type 2 (PCV2) from 2009 till 2018 may lead to a better receptor binding

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    Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD). Three major PCV2 genotypes (PCV2a, PCV2b, and PCV2d) have been identified globally. Despite their worldwide distribution, the prevalence and genetic evolution of PCV2 in Belgium has not previously been determined. In this study, 319 samples from animals suffering from diseases likely to be associated with PCV2 were collected from 2009 to 2018 and analysed by virus titration. The overall prevalence of PCV2 in PCVAD-suspected cases was 15.7 per cent (50/319). The phylogenetic analysis demonstrated that at least three genotypes (PCV2a, PCV2b, and PCV2d) circulated in Belgium from 2009 till 2018, and that PCV2 evolved from PCV2a to PCV2b and from PCV2d-1 to PCV2d-2. Sequence comparison among the forty-three PCV2 isolates showed that they had 89.7-100 per cent nucleotide-sequence and 88.5-100 per cent amino-acid-sequence identities. Three amino acid sites were under positive selection. Three-dimensional analysis of genotype-specific amino acids revealed that most of the mutations were on the outside of the cap protein with a few conserved mutations present on the inner side. Mutations toward more basic amino acids were found on the upper and tail parts of two connecting capsid proteins which form one big contact region, most probably involved in receptor binding. The lower part was relatively conserved. This polarity change together with the formation of an extruding part drive the virus to a more efficient GAG receptor binding. Taken together, these results showed a genotype shift from PCV2a to PCV2b and later on from PCV2d-1 to PCV2d-2, and a PCV2 evolution toward a better receptor binding capacity

    Porcine rotavirus mainly infects primary porcine enterocytes at the basolateral surface

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    Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular difusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifcally infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) signifcantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our fndings demonstrate that integrity of the intestinal epithelium is crucial in the host’s innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface

    High quality MinION and Flongle long-read nanopore genome assemblies of Mycoplasma bovis using taxon-specific training of the Bonito basecaller

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    Mycoplasma bovis is a major and primary bovine pathogen causing respiratory and reproductive disorders, mastitis and arthritis. Due to its persistent nature it is difficult to combat infections on farms. No effective vaccine is able to prevent M. bovis infection, leaving antimicrobials as the only first-line treatment. Nevertheless, therapy with antimicrobials is rarely efficient since increased resistance to many commercially available antimicrobials has been reported. An accurate diagnosis including antimicrobial resistance profiling is required to combat infections with working antibiotics, but this cannot be achieved fast enough with classical diagnostics. Here we developed a nanopore-based workflow allowing M. bovis species typing and Antimicrobial Resistance (AMR) Profiling applicable in point-of-care settings in control of M. bovis infections. Our new diagnostic tool was verified with 100 field strains of M. bovis for which whole genome sequencing and MIC testing was performed for practice-relevant antibiotics. Besides whole genome species typing, Single Nucleotide Polymorphism (SNP) analysis was performed to associate strain-specific genetic markers with its phenotypic AMR antibiogram. Raw fast5 outputs ranged from 5.4 Gb up to 17.2 Gb, with an average N50 of 5.5 ± 1.3 Kb per run with 11 M. bovis strains. Furthermore, including the M. bovis PG45 type strain within every run as internal control, inter-run accuracy reached up to 99.95% sequence identity. Since computation time presents a new bottleneck in this new workflow, we exploited a GPU-based bioinformatics pipeline speeding up full bioinformatics analysis to be completed within 10 hours for 11 M. bovis genomes. This new M. bovis diagnostics pipeline delivers a high accurate species identification along with an accurate genotypic antibiogram. This will be accelerated even further to facilitate proper antimicrobial therapy selection for the rapid control of bovine mycoplasmosis

    Role of porcine amino peptidase N and sialic acids in porcine coronavirus infections in primary porcine enterocytes

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    Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been reported to use aminopeptidase N (APN) as a cellular receptor. Recently, the role of APN as a receptor for PEDV has been questioned. In our study, the role of APN in PEDV and TGEV infections was studied in primary porcine enterocytes. After seven days of cultivation, 89% of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to PEDV and TGEV. A significant increase of PEDV and TGEV infection was correlated with a higher expression of APN, which was indicative that APN plays an important role in porcine coronavirus infections. However, PEDV and TGEV infected both APN positive and negative enterocytes. PEDV and TGEV Miller showed a higher infectivity in APN positive cells than in APN negative cells. In contrast, TGEV Purdue replicated better in APN negative cells. These results show that an additional receptor exists, different from APN for porcine coronaviruses. Subsequently, treatment of enterocytes with neuraminidase (NA) had no effect on infection efficiency of TGEV, implying that terminal cellular sialic acids (SAs) are no receptor determinants for TGEV. Treatment of TGEV with NA significantly enhanced the infection which shows that TGEV is masked by SAs

    Role of Porcine Aminopeptidase N and Sialic Acids in Porcine Coronavirus Infections in Primary Porcine Enterocytes.

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    Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been reported to use aminopeptidase N (APN) as a cellular receptor. Recently, the role of APN as a receptor for PEDV has been questioned. In our study, the role of APN in PEDV and TGEV infections was studied in primary porcine enterocytes. After seven days of cultivation, 89% of enterocytes presented microvilli and showed a two- to five-fold higher susceptibility to PEDV and TGEV. A significant increase of PEDV and TGEV infection was correlated with a higher expression of APN, which was indicative that APN plays an important role in porcine coronavirus infections. However, PEDV and TGEV infected both APN positive and negative enterocytes. PEDV and TGEV Miller showed a higher infectivity in APN positive cells than in APN negative cells. In contrast, TGEV Purdue replicated better in APN negative cells. These results show that an additional receptor exists, different from APN for porcine coronaviruses. Subsequently, treatment of enterocytes with neuraminidase (NA) had no effect on infection efficiency of TGEV, implying that terminal cellular sialic acids (SAs) are no receptor determinants for TGEV. Treatment of TGEV with NA significantly enhanced the infection which shows that TGEV is masked by SAs

    TRIPLE DIVIDENDS OF WATER CONSUMPTION CHARGES IN SOUTH AFRICA

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    The South African government is exploring ways to address water scarcity problems by introducing a water resource management charge on the quantity of water used in sectors such as irrigated agriculture, mining and forestry. It is expected that a more efficient water allocation, lower use and a positive impact on poverty can be achieved. This paper reports on the validity of these claims by applying a computable general equilibrium model to analyse the triple dividend of water consumption charges in South Africa: reduced water use, more rapid economic growth, and a more equal income distribution. It is shown that the appropriate, budget-neutral combination of water charges, particularly on irrigated agriculture and coal mining, and reduced indirect taxes, particularly on food, would yield triple dividends.water scarcity, water charges, triple dividend, poverty alleviation, computable general equilibrium model

    Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in Belgium, January 2015

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    Porcine epidemic diarrhea virus (PEDV) is a member of the family Coronaviridae and can cause severe outbreaks of diarrhea in piglets from different age groups. Here, we report the complete genome sequence (28,028 nt) of a PEDV strain isolated during a novel outbreak in Belgium

    Genome sequences of equine herpesvirus 1 strains from a European outbreak of neurological disorders linked to a horse gathering in Valencia, Spain, in 2021

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    Five equine herpesvirus 1 (EHV-1) genome sequences with links to an EHV-1 outbreak with neurological disorders after a horse gathering in Valencia, Spain, in February 2021, were determined. All strains showed the closest relationships to strains from Belgium and the United Kingdom, indicating a common source of infection

    Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses

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    Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen-and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79-1683 and WSU 79-1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79-1683 still replicated significantly more efficient compared to FCoV WSU 79-1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats
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