Harmonic and anharmonic vibrational computations for biomolecular building blocks: Benchmarking DFT and basis sets by theoretical and experimental IR spectrum of glycine conformers.

Advanced vibrational spectroscopic experiments have reached a level of sophistication that can only be matched by numerical simulations in order to provide an unequivocal analysis, a crucial step to understand the structure-function relationship of biomolecules. While density functional theory (DFT) has become the standard method when targeting medium-size or larger systems, the problem of its reliability and accuracy are well-known and have been abundantly documented. To establish a reliable computational protocol, especially when accuracy is critical, a tailored benchmark is usually required. This is generally done over a short list of known candidates, with the basis set often fixed a priori. In this work, we present a systematic study of the performance of DFT-based hybrid and double-hybrid functionals in the prediction of vibrational energies and infrared intensities at the harmonic level and beyond, considering anharmonic effects through vibrational perturbation theory at the second order. The study is performed for the six-lowest energy glycine conformers, utilizing available "state-of-the-art" accurate theoretical and experimental data as reference. Focusing on the most intense fundamental vibrations in the mid-infrared range of glycine conformers, the role of the basis sets is also investigated considering the balance between computational cost and accuracy. Targeting larger systems, a broad range of hybrid schemes with different computational costs is also tested.

Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d.

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity.

Activation of sialoadhesin down-regulates the levels of innate antiviral cytokines in porcine alveolar macrophages in vitro.

Porcine sialoadhesin (pSn) is a crucial porcine reproductive and respiratory syndrome virus (PRRSV) receptor mediating the attachment and internalization of virus into its major target cells, porcine alveolar macrophages (PAMs). However, the role of pSn in innate antiviral immune response has not yet been investigated. In this study, our results showed that PRRSV down-regulated significantly the mRNA levels of IFN-α, IFN-ß, IFN-γ, IFN-λ1, IFN-λ3 and IFN-λ4 and up-regulated significantly the mRNA levels of IL-10 and pSn in infected PAMs in vitro, suggesting that PRRSV infection inhibited the transcription of innate antiviral cytokines in host cells. Our results also showed that selective activation of pSn down-regulated significantly the mRNA levels of IFN-α, IFN-ß, IFN-γ, IFN-λ1, IFN-λ3, IFN-λ4 and TNF-α and up-regulated significantly the mRNA level of IL-10 in PAMs in vitro, suggesting that pSn signaling inhibited the transcription of innate antiviral cytokines. Further results showed that pSn1, pSn2, pSn3, pSn4 and pSn5 domains of pSn were responsible for the inhibition of levels of innate antiviral cytokines. In conclusion, our results suggested that pSn suppressed innate antiviral immune response by down-regulating the levels of innate antiviral cytokines in PAMs. It was possible that PRRSV-pSn interaction may suppress innate antiviral immune response to PRRSV infection by repressing the production of innate antiviral cytokines.