Flaviviruses are known to cause a variety of diseases in humans in different parts of the world. There are very limited numbers of antivirals to combat flavivirus infection, and therefore new drug targets must be explored. The flavivirus NS2B-NS3 proteases are responsible for the cleavage of the flavivirus polyprotein, which is necessary for productive viral infection and for causing clinical infections; therefore, they are a promising drug target for devising novel drugs against different flaviviruses. This review highlights the structural details of the NS2B-NS3 proteases of different flaviviruses, and also describes potential antiviral drugs that can interfere with the viral protease activity, as determined by various studies. Moreover, optimized in vitro reaction conditions for studying the NS2B-NS3 proteases of different flaviviruses may vary and have been incorporated in this review. The increasing availability of the in silico and crystallographic/structural details of flavivirus NS2B-NS3 proteases in free and drug-bound states can pave the path for the development of promising antiflavivirus drugs to be used in clinics. However, there is a paucity of information available on using animal cells and models for studying flavivirus NS2B-NS3 proteases, as well as on the testing of the antiviral drug efficacy against NS2B-NS3 proteases. Therefore, on the basis of recent studies, an effort has also been made to propose potential cellular and animal models for the study of flavivirus NS2B-NS3 proteases for the purposes of exploring flavivirus pathogenesis and for testing the efficacy of possible drugs targets, in vitro and in vivo.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the β2-microglobulin (β2M) gene ( B2M ) and reduced cellular levels of β2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of β2M expression. Exogenous expression of Nsp4 downregulated β2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M . These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4.

The mucosal immune system of the respiratory tract possesses an effective “defense barrier” against the invading pathogenic microorganisms; therefore, the lungs of healthy organisms are considered to be sterile for a long time according to the strong pathogens-eliminating ability. The emergence of next-generation sequencing technology has accelerated the studies about the microbial communities and immune regulating functions of lung microbiota during the past two decades. The acquisition and maturation of respiratory microbiota during childhood are mainly determined by the birth mode, diet structure, environmental exposure and antibiotic usage. However, the formation and development of lung microbiota in early life might affect the occurrence of respiratory diseases throughout the whole life cycle. The interplay and crosstalk between the gut and lung can be realized by the direct exchange of microbial species through the lymph circulation, moreover, the bioactive metabolites produced by the gut microbiota and lung microbiota can be changed via blood circulation. Complicated interactions among the lung microbiota, the respiratory viruses, and the host immune system can regulate the immune homeostasis and affect the inflammatory response in the lung. Probiotics, prebiotics, functional foods and fecal microbiota transplantation can all be used to maintain the microbial homeostasis of intestinal microbiota and lung microbiota. Therefore, various kinds of interventions on manipulating the symbiotic microbiota might be explored as novel effective strategies to prevent and control respiratory diseases.

Mosquitoes harbour a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns, and significant economic losses across the globe. Viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms in the vicinity of Shanghai, China, to explore the viral community carried by mosquitoes. The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms, which comprised of more than 48 viral taxonomic families, specific to vertebrates, invertebrates, plants, fungi, bacteria and protozoa. In addition, a considerable number of viral reads were related to viruses that are not classified by host. The read sequences related to animal viruses included parvoviruses, anelloviruses, circoviruses, flavivirus, rhabdovirus and seadornaviruses, which might be taken up by mosquitoes from viremic animal hosts during blood feeding. Notably, sample G1 contained the most abundant sequence related to Banna virus, which is of public health interest because it causes encephalitis in humans. Furthermore, non-classified viruses also shared considerable virus sequences in all the samples, presumably belonging to unexplored virus category. Overall, the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms, which is a potential source of diseases for mammals including humans and animals. These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.

Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen but the cell signaling pathways that drive T cell responses regulating antifungal immunity are incompletely understood. Notch is a key signaling pathway regulating T cell development, and differentiation and functional responses of mature T cells in the periphery. The targeting of Notch signaling within T cells has been proposed as a potential treatment for alloimmune and autoimmune disorders, but it is unknown whether disturbances to T cell immunity may render these patients vulnerable to fungal infections. To elucidate the role of Notch signaling during fungal infections, we infected mice expressing the pan-Notch inhibitor dominant negative mastermind-like within mature T cells with C. neoformans. Inhibition of T cell–restricted Notch signaling increased fungal burdens in the lungs and CNS, diminished pulmonary leukocyte recruitment, and simultaneously impaired Th1 and Th2 responses. Pulmonary leukocyte cultures from T cell Notch-deprived mice produced less IFN-γ, IL-5, and IL-13 than wild-type cells. This correlated with lower frequencies of IFN-γ–, IL-5–, and IL-13–producing CD4⁺ T cells, reduced expression of Th1 and Th2 associated transcription factors, Tbet and GATA3, and reduced production of IFN-γ by CD8⁺ T cells. In contrast, Th17 responses were largely unaffected by Notch signaling. The changes in T cell responses corresponded with impaired macrophage activation and reduced leukocyte accumulation, leading to diminished fungal control. These results identify Notch signaling as a previously unappreciated regulator of Th1 and Th2 immunity and an important element of antifungal defenses against cryptococcal infection and CNS dissemination.

Getah virus (GETV), in the genus Alphavirus and the family Togaviridae, has been detected throughout the world. GETV causes high morbidity and mortality in newborn piglets, entailing serious economic losses. Therefore, the experimental work on GETV detection is necessary. However, due to the influence of a variety of unavoidable factors, the ELISA test for the primary screening of animal diseases has low accuracy in detection results. Therefore, we optimized a recombinant E2 (rE2) protein-based enzyme-linked immunosorbent assay (ELISA) for the detection of GETV antibodies in pig serum. The E2 protein was successfully expressed and purified with SDS-PAGE. A Western blotting analysis of sera from infected pigs showed strong reaction with a viral antigen of ~46 KDa corresponding to the E2 glycoproteins. By using chessboard titration and comparing the P/N values, we found that the optimal concentration of coated antigen was found to be 24.5 μg/mL, and the optimal dilution of serum specimens was 1:100. The best working dilution of the horseradish peroxidase (HRP)-conjugated goat anti-pig immunoglobulin (IgG) was 1:5000. The optimal coating conditions were 12 h at 4 °C. The optimal incubation conditions for serum specimens, blocking, and reaction with the secondary antibody were all 1 h at 37 °C. We also investigated the seroprevalence of GETV in 133 serum specimens collected in Eastern China, and 37.59% of the samples tested positive for anti-GETV IgG antibodies, indicating that the seroprevalence of GETV is high in pig populations in China. The seroprevalence was significantly lower in spring (April; 24.24%, 16/66) than in autumn (October; 50.75%, 34/67), which suggested that the presence of anti-GETV antibodies in pigs was seasonal. In conclusion, we improved an rE2 ELISA that detected pig antibodies against GETV after experimental and natural infections. This should be useful in the diagnosis and surveillance of GETV infections.