Evolutionary Enhancement of Zika Virus Infectivity in Aedes aegypti Mosquitoes

Zika virus (ZIKV) remained obscure until the recent explosive outbreaks in French Polynesia (2013-2014) and South America (2015-2016). Phylogenetic studies have shown that ZIKV has evolved into African and Asian lineages. The Asian lineage of ZIKV was responsible for the recent epidemics in the Americas. However, the underlying mechanisms through which ZIKV rapidly and explosively spread from Asia to the Americas are unclear. Non-structural protein 1 (NS1) facilitates flavivirus acquisition by mosquitoes from an infected mammalian host and subsequently enhances viral prevalence in mosquitoes. Here we show that NS1 antigenaemia determines ZIKV infectivity in its mosquito vector Aedes aegypti, which acquires ZIKV via a blood meal. Clinical isolates from the most recent outbreak in the Americas were much more infectious in mosquitoes than the FSS13025 strain, which was isolated in Cambodia in 2010. Further analyses showed that these epidemic strains have higher NS1 antigenaemia than the FSS13025 strain because of an alanine-to-valine amino acid substitution at residue 188 in NS1. ZIKV infectivity was enhanced by this amino acid substitution in the ZIKV FSS13025 strain in mosquitoes that acquired ZIKV from a viraemic C57BL/6 mouse deficient in type I and II interferon (IFN) receptors (AG6 mouse). Our results reveal that ZIKV evolved to acquire a spontaneous mutation in its NS1 protein, resulting in increased NS1 antigenaemia. Enhancement of NS1 antigenaemia in infected hosts promotes ZIKV infectivity and prevalence in mosquitoes, which could have facilitated transmission during recent ZIKV epidemics

The molecular diversity of transcriptional factor TfoX is a determinant in natural transformation in Glaesserella parasuis

Natural transformation is a mechanism by which a particular bacterial species takes up foreign DNA and integrates it into its genome. The swine pathogen Glaesserella parasuis (G. parasuis) is a naturally transformable bacterium. The regulation of competence, however, is not fully understood. In this study, the natural transformability of 99 strains was investigated. Only 44% of the strains were transformable under laboratory conditions. Through a high-resolution melting curve and phylogenetic analysis, we found that genetic differences in the core regulator of natural transformation, the tfoX gene, leads to two distinct natural transformation phenotypes. In the absence of the tfoX gene, the highly transformable strain SC1401 lost its natural transformability. In addition, when the SC1401 tfoX gene was replaced by the tfoX of SH0165, which has no natural transformability, competence was also lost. These results suggest that TfoX is a core regulator of natural transformation in G. parasuis, and that differences in tfoX can be used as a molecular indicator of natural transformability. Transcriptomic and proteomic analyses of the SC1401 wildtype strain, and a tfoX gene deletion strain showed that differential gene expression and protein synthesis is mainly centered on pathways related to glucose metabolism. The results suggest that tfoX may mediate natural transformation by regulating the metabolism of carbon sources. Our study provides evidence that tfoX plays an important role in the natural transformation of G. parasuis

A Comparative Transcriptomic Analysis Reveals That HSP90AB1 Is Involved in the Immune and Inflammatory Responses to Porcine Deltacoronavirus Infection

PDCoV is an emerging enteropathogenic coronavirus that mainly causes acute diarrhea in piglets, seriously affecting pig breeding industries worldwide. To date, the molecular mechanisms of PDCoV-induced immune and inflammatory responses or host responses in LLC-PK cells in vitro are not well understood. HSP90 plays important roles in various viral infections. In this study, HSP90AB1 knockout cells (HSP90AB1KO) were constructed and a comparative transcriptomic analysis between PDCoV-infected HSP90AB1WT and HSP90AB1KO cells was conducted using RNA sequencing to explore the effect of HSP90AB1 on PDCoV infection. A total of 1295 and 3746 differentially expressed genes (DEGs) were identified in PDCoV-infected HSP90AB1WT and HSP90AB1KO cells, respectively. Moreover, most of the significantly enriched pathways were related to immune and inflammatory response-associated pathways upon PDCoV infection. The DEGs enriched in NF-κB pathways were specifically detected in HSP90AB1WT cells, and NF-κB inhibitors JSH-23, SC75741 and QNZ treatment reduced PDCoV infection. Further research revealed most cytokines associated with immune and inflammatory responses were upregulated during PDCoV infection. Knockout of HSP90AB1 altered the upregulated levels of some cytokines. Taken together, our findings provide new insights into the host response to PDCoV infection from the transcriptome perspective, which will contribute to illustrating the molecular basis of the interaction between PDCoV and HSP90AB1

BAK-Mediated Pyroptosis Promotes Japanese Encephalitis Virus Proliferation in Porcine Kidney 15 Cells

As a zoonotic virus, Japanese Encephalitis virus (JEV) poses a serious threat to human health and the breeding industry. Regarding the mechanism and complications of tissue inflammation caused by JEV, such as encephalitis and orchitis, there is no effective drug treatment currently, and the mechanism of occurrence has not been thoroughly studied. Therefore, it is necessary to study the mechanism of the inflammatory pathway caused by JEV. As one of the key proteins regulating cell death, BCL2 antagonist/killer (BAK) is also a necessary prerequisite for the release of cellular inflammatory factors. We found that after JEV infection, BAK-knockdown cells died less than normal cells, and the transcription levels of inflammatory factors such as TNF, IFNα, and IL-1β and their corresponding regulatory genes were also significantly reduced. By further verifying protein expression on the cell death pathway, it was found that pyroptotic activation and virus titer were also significantly reduced in BAK.KD cells, suggesting that JEV proliferation might be related to BAK-induced cell death. From our data, we could conclude that JEV utilized the BAK-promoted pyroptotic pathway to release more virions after the final Gasdermin D-N (GSDMD-N) protein pore formation for the purpose of JEV proliferation. Therefore, the study of the endogenous cell death activator protein BAK and the final release pathway of JEV, is expected to provide some new theoretical basis for future research on the screening of targeted drugs for the treatment of inflammatory diseases caused by JEV

CD38 Enhances TLR9 Expression and Activates NLRP3 Inflammasome after Porcine Parvovirus Infection

(1) Background: Porcine Parvovirus (PPV) is a single-stranded DNA virus without envelope which causes great harm in relation to porcine reproductive disorders in clinic. Cluster of Differentiation 38 (CD38) is a transmembrane protein widely existing in mammals. Its various functions make it a very popular research object, including in the viral infection field. (2) Methods: Western blotting and an EdU Cell Proliferation Kit were used to evaluate the effect of CD38-deficient cells. Relative quantitative real-time RT-PCR was used to detect the transcription levels of cytokines after PPV infection. The renilla luciferase reporter gene assay was used to verify the activation function of CD38 on downstream factors. The fluorescence probe method was used to detect the level of intracellular reactive oxygen species (ROS). (3) Results: This study found that the loss of CD38 function inhibited the up-regulated state of Toll-like Receptor 9 (TLR9), Interferon-α (IFN-α), and Myxovirus Resistance 1 (Mx1) after PPV infection. The luminescence of the group transfected with both CD38 expression plasmid and TLR9 promoter renilla luciferase reporter plasmid was significantly up-regulated compared with the control, suggesting that CD38 may activate the promoter of TLR9. In addition, CD38 deficiency not only activated the transcription of Sirtuin-1 (SIRT1), but also inhibited ROS level and the transcription of NLR Family Pyrin Domain Containing 3 (NLRP3). (4) Conclusion: (i) CD38 may participate in the TLR9/IFN-α/Mx1 pathway by activating the expression of TLR9 after PPV infected PK-15 cells; (ii) CD38 may activate the NLRP3/CASP1 pathway by increasing ROS level; (iii) CD38 deficiency activates the expression of SIRT1 and can prevent the normal proliferation of PPV

Metal Ion Periplasmic-Binding Protein YfeA of <i>Glaesserella parasuis</i> Induces the Secretion of Pro-Inflammatory Cytokines of Macrophages via MAPK and NF-κB Signaling through TLR2 and TLR4

The YfeA gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system Yfe, encodes the substrate-binding subunit of the iron, zinc, and manganese transport system in bacteria. As a potential vaccine candidate in Glaesserella parasuis, the functional mechanisms of YfeA in the infection process remain obscure. In this study, vaccination with YfeA effectively protected the C56BL6 mouse against the G. parasuis SC1401 challenge. Bioinformatics analysis suggests that YfeA is highly conserved in G. parasuis, and its metal-binding sites have been strictly conserved throughout evolution. Stimulation of RAW 264.7 macrophages with YfeA verified that toll-like receptors (TLR) 2 and 4 participated in the positive transcription and expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The activation of TLR2 and TLR4 utilized the MyD88/MAL and TRIF/TRAM pairs to initiate TLRs signaling. Furthermore, YfeA was shown to stimulate nuclear translocation of NF-κB and activated diverse mitogen-activated protein (MAP) kinase signaling cascades, which are specific to the secretion of particular cytokine(s) in murine macrophages. Separate blocking TLR2, TLR4, MAPK, and RelA (p65) pathways significantly decreased YfeA-induced pro-inflammatory cytokine production. In addition, YfeA-stimulated RAW 264.7 produces the pro-inflammatory hallmark, reactive oxygen species (ROS). In conclusion, our findings indicate that YfeA is a novel pro-inflammatory mediator in G. parasuis and induces TLR2 and TLR4-dependent pro-inflammatory activity in RAW 264.7 macrophages through P38, JNK-MAPK, and NF-κB signaling pathways

Characterization, phylogenetic analysis, and pathogenicity of a novel genotype 2 porcine Enterovirus G

Enterovirus G belongs to the family Picornaviridae and are associated with a variety of animal diseases. We isolated and characterized a novel EV-G2 strain, CHN-SCMY2021, the first genotype 2 strain isolated in China. CHN-SCMY2021 is about 25 nm diameter with morphology typical of picornaviruses and its genome is 7341 nucleotides. Sequence alignment and phylogenetic analysis based on VP1 indicated that this isolate is a genotype 2 strain. The whole genome similarity between CHN-SCMY2021 and other EV-G genotype 2 strains is 78.3–86.4%, the greatest similarity is to EVG/Porcine/JPN/Iba26–506/2014/G2 (LC316792.1). Recombination analysis indicated that CHN-SCMY2021 resulted from recombination between 714,171/CaoLanh_VN (KT265894.2) and LP 54 (AF363455.1). Except for ST cells, CHN-SCMY2021 has a broad spectrum of cellular adaptations, which are susceptible to BHK-21, PK-15, IPEC-J2, LLC-PK and Vero cells. In piglets, CHN-SCMY2021 causes mild diarrhea and thinning of the intestinal wall. The virus was mainly distributed to intestinal tissue but was also found in heart, liver, spleen, lung, kidney, brain, and spinal cord. CHN-SCMY2021 is the first systematically characterized EV-G genotype 2 strain from China, our results enrich the information on the epidemiology, molecular evolution and pathogenicity associated with EV-G

Dynamic tubulation of mitochondria drives mitochondrial network formation

Mitochondria form networks. Formation of mitochondrial networks is important for maintaining mitochondrial DNA integrity and interchanging mitochondrial material, whereas disruption of the mitochondrial network affects mitochondrial functions. According to the current view, mitochondrial networks are formed by fusion of individual mitochondria. Here, we report a new mechanism for formation of mitochondrial networks through KIF5B-mediated dynamic tubulation of mitochondria. We found that KIF5B pulls thin, highly dynamic tubules out of mitochondria. Fusion of these dynamic tubules, which is mediated by mitofusins, gives rise to the mitochondrial network. We further demonstrated that dynamic tubulation and fusion is sufficient for mitochondrial network formation, by reconstituting mitochondrial networks in vitro using purified fusion-competent mitochondria, recombinant KIF5B, and polymerized microtubules. Interestingly, KIF5B only controls network formation in the peripheral zone of the cell, indicating that the mitochondrial network is divided into subzones, which may be constructed by different mechanisms. Our data not only uncover an essential mechanism for mitochondrial network formation, but also reveal that different parts of the mitochondrial network are formed by different mechanisms.National Natural Science Foundation of China [31123004, 31030043, 30971484, 31321003]; National Basic Research Program of China (973 Program) [2011CB910100]SCI(E)PubMed中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)[email protected]; [email protected]

Porcine Deltacoronavirus (PDCoV) Entry into PK-15 Cells by Caveolae-Mediated Endocytosis

(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-&beta;-cyclodextrin (M&beta;CD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets