In vitro inhibition of feline coronavirus replication by small interfering RNAs

Infection with virulent biotypes of feline coronavirus (FCoV) can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. In this study we demonstrate the ability of small interfering RNA (siRNA) mediated RNA interference (RNAi) to inhibit the replication of virulent FCoV strain FIPV WSU 79-1146 in an immortalised feline cell line. A panel of eight synthetic siRNAs targeting four different regions of the FCoV genome were tested for antiviral effects. Efficacy was determined by qRT-PCR of intracellular viral genomic and messenger RNA, TCID50 infectivity assay of extracellular virus, and direct IFA for viral protein expression. All siRNAs demonstrated an inhibitory effect on viral replication in vitro. The two most effective siRNAs, targeting the untranslated 5' leader sequence (L2) and the nucleocapsid gene (N1), resulted in a >95% reduction in extracellular viral titre. Further characterisation of these two siRNAs demonstrated their efficacy when used at low concentrations and in cells challenged with high viral loads. Taken together these findings provide important information for the potential therapeutic application of RNAi in treating FIP.     

Possible methionine biosynthesis inhibition by pyrimidinamine fungicides

The broad-spectrum fungicide CGA 219417 inhibits mycelial growth of Botrytis cinerea Pers ex Fr. Pseudocercosporella herpotrichoides (Fron.) Deighton and Helminthosporium oryzae B. de Haan on defined media lacking amino acids. The growth inhibition of B. cinerea is reversed by the addition of a mixture of 19 amino acids at a concentration of 100 μM each or by the addition of methionine or homocysteine in concentrations of 100 μM or 1 mM, respectively. In the case of B. cinerea, the reversal of growth inhibition by methionine could also be shown for pyrimethanil and mepanipyrim. These findings suggest that the pyrimidinamine fungicides inhibit the biosynthesis of methionine in phytopathogenic fungi.     

Glyoxalase 1 expression is downregulated in preimplantation blastocysts of diabetic rabbits

In a diabetic pregnancy, an altered maternal metabolism led to increased formation of reactive α‐dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) in the reproductive organs and embryos. The enzyme glyoxalase (GLO) 1 detoxifies reactive α‐dicarbonyls thus protecting cells against malfunction or modifications of proteins by advanced glycated end products (AGEs). The aim of this study was to analyse the influence of a maternal insulin‐dependent diabetes mellitus (IDD) on GLO1 expression and activity in preimplantation embryos in vivo and human trophoblast cells (Ac‐1M88) in vitro. Maternal diabetes was induced in female rabbits by alloxan before conception and maintained during the preimplantation period. GLO1 expression and activity were investigated in 6‐day‐old blastocysts from healthy and diabetic rabbits. Furthermore, blastocysts and human trophoblast cells were exposed in vitro to hyperglycaemia, GO and MGO and analysed for GLO1 expression and activity. During gastrulation, GLO1 was expressed in all compartments of the rabbit blastocyst. Maternal diabetes decreased embryonic GLO1 protein amount by approx. 30 per cent whereas the enzymatic activity remained unchanged, indicating that the specific GLO1 activity increases along with metabolic changes. In in vitro cultured embryos, neither hyperglycaemia nor MGO and GO had an effect on GLO1 protein amount. In human trophoblast cells, a stimulating effect on the GLO1 expression was shown in the highest GO concentration, only. Our data show that maternal diabetes mellitus affects the specific activity of GLO1, indicating that GLO1 was post‐translationally modified due to changes in metabolic processes in the preimplantation embryos.     

Perceptions and attitudes of swine exhibitors towards recommendations for reducing zoonotic transmission of influenza A viruses

Since 2011, there have been 468 cases of variant influenza A virus (IAV) reported in the United States, many of which were associated with youth swine exhibition. In an effort to mitigate risk associated with exposure to IAV in swine, the “Measures to Minimize Influenza Transmission at Swine Exhibitions” (MtM) was developed for show organizers, volunteers and exhibitors. These recommendations are updated annually; however, it is not clear if youth swine exhibitors are aware of the recommendations; support the recommendations; and would be willing to practise recommended behaviours. Therefore, a cross‐sectional survey method was used to assess swine exhibitor perceptions and their adoption of swine production practices aimed at reducing the transmission of IAV at the human–animal interface. In addition, the survey asked participants their state of residence and the number of shows they would attend in 2017. In all, 155 participants who showed swine on a regular basis (x? = 11 shows per year), from at least 18 states within the US, completed the survey. At least, 67% of participants believed each statement was a good recommendation, with 6 of 11 recommendations being supported by >90% of participants. When asked if recommendations could be implemented, 65%–94% of respondents agreed, and 21%–89% of participants had already implemented each recommendation, respectively. Although significant efforts have been made to increase signage at swine exhibitions (warning of risks associated with eating/drinking in animal areas), a majority of respondents report eating/drinking in the barn and are unwilling to change their behaviours. This study provides evidence that developing and disseminating static recommendations to reduce zoonotic disease transmission is not enough to change human behaviour to prevent future variant IAV infections associated with swine exhibitions.     

Detection of Toxoplasma gondii in Crassostrea spp. oysters cultured in an estuarine region in eastern Amazon

The aim of the present study was to detect DNA of Toxoplasma gondii in Crassostrea spp. oysters cultured in the state of Pará, Brazil. A total of 400 oysters were directly collected from a fixed rack system. Gills, gastrointestinal tract (GIT) and intervalvular liquid were separated and grouped into pool samples of 10 animals, resulting in 40 samples each of gills, GIT and intervalvular liquid. DNA extraction was performed using a commercial kit, and T. gondii DNA was detected by nested PCR using the primers Toxo3 and Toxo4, which produced an amplification product of 155 bp of the T. gondii gene B1. Nucleotide sequencing was performed for positive samples, and the obtained sequences were identified by comparison with sequences in GenBank. The DNA of T. gondii was detected in 5.8% (7/120) of the pool samples, of which 7.5% (3/40) was in the GIT, 5% (2/40) in the gills, and 5% (2/40) was in the intervalvular liquid. The obtained sequences presented 100% identity and overlap with T. gondii DNA sequences. This is the report of detection of T. gondii DNA in oysters from genus Crassostrea spp. originating from the state of Pará, eastern Amazon.