TK/gI/gE/11K Genes Is Not Involved in the Virulence Enhancement of Highly Virulent Pseudorabies Virus

The large-scale outbreaks of Pseudorabies in china since 2011 in vaccine-immunized pig farms were caused by the highly virulent Pseudorabies virus (PRV). To investigate the factors involved in the virulence enhancement of the variant PRV, four recombinant viruses with the virulence gene’s replacement, gI/gE/11K and TK/gI/gE/11K, between the variant strain HN1201 and the classical strain Fa were constructed based on bacterial artificial chromosome infectious clones. Compared with their parental strain, the viral titers of the recombinant PRV strains are not strongly influenced by the replacement of TK/gI/gE/11K, while as previously reported, the strain HN1201 and its derived viruses showed the higher mortality, the severer clinical symptoms and tissues damage than that of strain Fa and its derived strains. In summary, the TK/gI/gE/11K genes of variant strain HN1201 showed no contribute to its virulence enhancement compares with the classical strain.

Natural Transformation in a Classical-Biotype Vibrio cholerae Strain

ABSTRACT Vibrio cholerae causes the gastrointestinal illness cholera, which spreads throughout the globe in large pandemics. The current pandemic is caused by O1 El Tor biotype strains, whereas previous pandemics were caused by O1 classical biotype strains. El Tor V. cholerae is noted for its ability to acquire exogenous DNA through chitin-induced natural transformation, which has been exploited for genetic manipulation of El Tor strains in the laboratory. In contrast, the prototypical classical strain O395 lacks this ability, which was suspected to be due to a mutation in the regulatory gene hapR. HapR and the regulator TfoX control expression of a third competence regulator, QstR. We found that artificial induction of both TfoX and QstR in the presence of HapR in O395 was required for efficient DNA uptake. However, natural transformation in the classical strain is still orders of magnitude below that of an El Tor strain. O395 expressing HapR could also undergo natural transformation after growth on chitin, which could be increased by artificial induction of TfoX and/or QstR. A plasmid that expresses both TfoX and QstR was created that allowed for consistent DNA uptake in O395 carrying a hapR plasmid. This technique was also used to facilitate cotransformation into O395 of unmarked DNA (ΔlacZ, ΔflaA, ΔflgG) for multiplex genome editing by natural transformation (MuGENT). These results demonstrate that the classical biotype O395 strain is functionally capable of DNA uptake, which allows for the rapid genetic manipulation of its genome. IMPORTANCE Natural transformation (uptake of exogenous DNA) in Vibrio cholerae has contributed to the evolution of these human pathogens. Classical biotype V. cholerae strains were responsible for the first six cholera pandemics but were replaced by El Tor biotype V. cholerae in the current pandemic. This study demonstrates that classical V. cholerae is functionally capable of natural transformation, but inactivation of the transformation regulator HapR and inherent levels of transformation that are lower than those of El Tor V. cholerae suggest that the classical biotype may be less able to utilize natural transformation for horizontal gene transfer.

P782 Septal flash is a prevalent and early dyssynchrony marker in transcatheter aortic valve replacement-induced left bundle branch block

BACKGROUND New-onset left bundle branch block (LBBB) is a frequent complication after transcutaneous aortic valve replacement (TAVR). LBBB is associated with echocardiographic dyssynchrony in heart failure patients, but this has not been thoroughly investigated in acute LBBB following TAVR. PURPOSE This study aims to assess the timing and incidence of echocardiographic dyssynchrony in acute TAVR-induced LBBB patients. METHODS The study enrolled all TAVR-induced LBBB patients at our Hospital between January 2013 and May 2019. Presence of LBBB was scored within 72 hours following TAVR. Dyssynchrony was assessed by: 1/ presence of septal flash (SF), 2/ interventricular mechanical delay (IVMD, the difference between left and right ventricular pre-ejection interval using pulsed wave Doppler; cut-off ≥40 ms) and 3/ presence of ‘classical dyssynchronous strain pattern’ assessed with speckle tracking (Figure 1). As a control, these three LBBB-related dyssynchrony markers were assessed and compared to LBBB patients without TAVR (non-TAVR patients) in normal ànd reduced EF, but all having SF. RESULTS Out of 134 consecutive TAVR procedures, 30 (22%) were complicated by acute LBBB. SF was present in 72% of TAVR-induced LBBB patients, with a median time from TAVR to SF diagnosis of 24 hours. However, only 1 (5%) of these TAVR patients exhibited a classical dyssynchronous contraction strain pattern (Figure 1), despite presence of SF. Finally, the IVMD values in these TAVR-LBBB patients did not meet the ‘dyssynchrony’ cut-off. As a control, we compared these dyssynchrony parameters in patients with non-TAVR related LBBB with normal and reduced EF, all exhibiting SF. A classical strain pattern was present in 33% of non-TAVR LBBB patients with preserved left ventricular ejection fraction (LV EF) (p = 0.17), and in 80% of non-TAVR LBBB patients with reduced LV EF ≤30% (p < 0.001). The IVMD in non-TAVR LBBB patients with SF and preserved LV EF was 52 ms (p = 0.002) and 57 ms in non-TAVR LBBB patients with SF and LV EF ≤30% (p = 0.009). CONCLUSION SF as dyssynchrony marker develops early after TAVR-induced LBBB and is much more prevalent than the classical strain-based dyssynchrony pattern and IVMD. Our findings from the control population suggest that progressive LBBB-induced LV remodeling (septal thinning and relative lateral thickening) may be required for a ‘classical dyssynchrony strain pattern’ or significant IVMD to occur in TAVR-LBBB patients, but longitudinal follow-up studies in TAVR-LBBB patients are required as proof-of-concept. Abstract P782 Figure 1

The stronger downregulation of in vitro and in vivo innate antiviral responses by a very virulent strain of infectious bursal disease virus (IBDV), compared to a classical strain, is mediated, in part, by the VP4 protein

IBDV is economically important to the poultry industry. Very virulent (vv) strains cause higher mortality rates than other strains for reasons that remain poorly understood. In order to provide more information on IBDV disease outcome, groups of chickens (n=18) were inoculated with the vv strain, UK661, or the classical strain, F52/70. Birds infected with UK661 had a lower survival rate (50%) compared to F52/70 (80%). There was no difference in peak viral replication in the bursa of Fabricius (BF), but the expression of chicken IFNβ, MX1 and IL-8 was significantly lower in the BF of birds infected with UK661 compared to F52/70 (p<0.05) as quantified by RTqPCR, and this trend was also observed in DT40 cells infected with UK661 or F52/70 (p<0.05). The induction of expression of type I IFN in DF-1 cells stimulated with polyI:C (measured by an IFN-β luciferase reporter assay) was significantly reduced in cells expressing ectopic VP4 from UK661 (p<0.05), but was higher in cells expressing ectopic VP4 from F52/70. Cells infected with a chimeric recombinant IBDV carrying the UK661-VP4 gene in the background of PBG98, an attenuated vaccine strain that induces high levels of innate responses (PBG98-VP4UK661) also showed a reduced level of IFNα and IL-8 compared to cells infected with a chimeric virus carrying the F52/70-VP4 gene (PBG98-VP4F52/70), and birds infected with PBG98-VP4UK661 also had a reduced expression of IFNα in the BF compared to birds infected with PBG98-VP4F52/70. Taken together, these data demonstrate that UK661 induced the expression of lower levels of anti-viral type I IFN and proinflammatory genes than the classical strain in vitro and in vivo and this was, in part, due to strain-dependent differences in the VP4 protein.

Impact of Classical Strain Improvement of Penicillium rubens on Amino Acid Metabolism during β-Lactam Production

ABSTRACT To produce high levels of β-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for β-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production. IMPORTANCE Penicillium rubens is an important industrial producer of β-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.

Homogenization of frame lattices leading to second gradient models coupling classical strain and strain-gradient terms

We determine in the framework of static linear elasticity the homogenized behavior of three-dimensional periodic structures made of welded elastic bars. It has been shown that such structures can be modeled as discrete systems of nodes linked by extensional, flexural/torsional interactions corresponding to frame lattices and that the corresponding homogenized models can be strain-gradient models, i.e., models whose effective elastic energy involves components of the first and the second gradients of the displacement field. However, in the existing models, there is no coupling between the classical strain and the strain-gradient terms in the expression of the effective energy. In the present article, under some assumptions on the positions of the nodes of the unit cell, we show that classical strain and strain-gradient strain terms can be coupled. In order to illustrate this coupling we compute the homogenized energy of a particular structure that we call asymmetrical pantographic structure.