Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice.

Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup's stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.IMPORTANCETo date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.

Echovirus induces autophagy to promote viral replication via regulating mTOR/ULK1 signaling pathway.

Among enteroviruses, echovirus can cause severe illnesses in neonates or infants, with high morbidity and mortality. Autophagy, a central component of host defense mechanisms, can function against diverse infections. In the present study, we investigated the interplay between echovirus and autophagy. We demonstrated that echovirus infection increases LC3-II expression dose-dependently, accompanied by an increased intracellular LC3 puncta level. In addition, echovirus infection induces the formation of autophagosome. These results suggest that echovirus infection induces autophagy machinery. Furthermore, phosphorylated mTOR and ULK1 were both decreased upon echovirus infection. In contrast, both levels of the vacuolar protein sorting 34 (VPS34) and Beclin-1, the downstream molecules which play essential roles in promoting the formation of autophagic vesicles, increased upon virus infection. These results imply that the signaling pathways involved in autophagosome formation were activated by echovirus infection. Moreover, induction of autophagy promotes echovirus replication and viral protein VP1 expression, while inhibition of autophagy impairs VP1 expression. Our findings suggest that autophagy can be induced by echovirus infection via regulating mTOR/ULK1 signaling pathway and exhibits a proviral function, revealing the potential role of autophagy in echovirus infection.

Identification of novel anti-ZIKV drugs from viral-infection temporal gene expression profiles.

Zika virus (ZIKV) infections are typically asymptomatic but cause severe neurological complications (e.g. Guillain-Barré syndrome in adults, and microcephaly in newborns). There are currently no specific therapy or vaccine options available to prevent ZIKV infections. Temporal gene expression profiles of ZIKV-infected human brain microvascular endothelial cells (HBMECs) were used in this study to identify genes essential for viral replication. These genes were then used to identify novel anti-ZIKV agents and validated in publicly available data and functional wet-lab experiments. Here, we found that ZIKV effectively evaded activation of immune response-related genes and completely reprogrammed cellular transcriptional architectures. Knockdown of genes, which gradually upregulated during viral infection but showed distinct expression patterns between ZIKV- and mock infection, discovered novel proviral and antiviral factors. One-third of the 74 drugs found through signature-based drug repositioning and cross-reference with the Drug Gene Interaction Database (DGIdb) were known anti-ZIKV agents. In cellular assays, two promising antiviral candidates (Luminespib/NVP-AUY922, L-161982) were found to reduce viral replication without causing cell toxicity. Overall, our time-series transcriptome-based methods offer a novel and feasible strategy for antiviral drug discovery. Our strategies, which combine conventional and data-driven analysis, can be extended for other pathogens causing pandemics in the future.

CRISPR/Cas13a Trans-Cleavage-Triggered Catalytic Hairpin Assembly Assay for Specific and Ultrasensitive SARS-CoV-2 RNA Detection.

New coronavirus (SARS-CoV-2), which has caused the coronavirus disease 2019 (COVID-19) pandemic, has brought about a huge burden on global healthcare systems. Rapid and early detection is important to prevent the spread of the pandemic. Here, an assay based on CRISPR/Cas13a and catalytic hairpin assembly (CHA), termed as Cas-CHA, was developed for ultrasensitive and specific detection of SARS-CoV-2 RNA. Upon specific recognition of the target, the CRISPR/Cas13a collaterally cleaved a well-designed hairpin reporter and triggered the CHA reaction. Under optimized conditions, the assay detected the SARS-CoV-2 RNA with a wide range of 100 aM to 100 nM and realized a low detection limit of 84 aM. At the same time, the whole detecting process could be completed within 35 min. More importantly, the assay was able to distinguish SARS-CoV-2 RNA from common human coronaviruses and analyze in saliva samples. By the flexible design of crRNA, the assay was expanded to detect other viruses. The clinical sample analysis verified that the proposed assay held a great potential for practical testing.

Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone.

Tick-borne encephalitis virus (TBEV) is a pathogenic virus known to cause central nervous system (CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious cDNA clone of TBEV that was isolated in China (the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1 (NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect (CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.

Crystal structure of a tick-borne flavivirus RNA-dependent RNA polymerase suggests a host adaptation hotspot in RNA viruses.

The RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of nucleic acid polymerases. RdRPs are essential in virus life cycle due to their central role in viral genome replication/transcription processes. However, their contribution in host adaption has not been well documented. By solving the RdRP crystal structure of the tick-borne encephalitis virus (TBEV), a tick-borne flavivirus, and comparing the structural and sequence features with mosquito-borne flavivirus RdRPs, we found that a region between RdRP catalytic motifs B and C, namely region B-C, clearly bears host-related diversity. Inter-virus substitutions of region B-C sequence were designed in both TBEV and mosquito-borne Japanese encephalitis virus backbones. While region B-C substitutions only had little or moderate effect on RdRP catalytic activities, virus proliferation was not supported by these substitutions in both virus systems. Importantly, a TBEV replicon-derived viral RNA replication was significantly reduced but not abolished by the substitution, suggesting the involvement of region B-C in viral and/or host processes beyond RdRP catalysis. A systematic structural analysis of region B-C in viral RdRPs further emphasizes its high level of structure and length diversity, providing a basis to further refine its relevance in RNA virus-host interactions in a general context.

Sugar Protectants Improve the Thermotolerance and Biocontrol Efficacy of the Biocontrol Yeast, Candida oleophila.

A variety of sugar compounds have been used as additives to protect various biocontrol yeasts from adverse environmental stresses. However, studies on maltose and lactose as sugar protectants are limited, and their protective effect is not clear. In the present study, exposure of the biocontrol yeast Candida oleophila cells to 45°C for 10 min, while immersed in either 5 or 10% (w/v) maltose or lactose, provided a significant protective effect. The addition of maltose and lactose significantly enhanced enzyme activity and gene expression of catalase, thioredoxin reductase, and glutathione reductase, relative to cells that have been immersed in sterile distilled water (controls) exposed to 45°C. In addition, C. oleophila cells suspended in maltose and lactose solutions also exhibited higher viability and ATP levels, relative to control cells. Notably, the biocontrol efficacy of C. oleophila against postharvest diseases of apple fruit was maintained after the yeast was exposed to the high temperature treatment while immersed in maltose and lactose solutions. These results demonstrate the potential of maltose and lactose as sugar protectants for biocontrol agent against heat stress.