Repurposing of approved antiviral drugs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a promising strategy to treat Coronavirus disease 2019 (COVID-19) patients. Previously we reported our hypothesis that the antiviral drugs with high lung distributions might benefit COVID-19 patients by reducing viral loads. So far, chloroquine, lopinavir, hydroxychloroquine, azithromycin, favipiravir, ribavirin, darunavir, remdesivir, and umifenovir have been tested in COVID-19 clinical trials. Here we validated our hypothesis by comparing the pharmacokinetics profiles of these drugs and their capabilities of reducing viral load in clinical trials. According to bulk RNA and single cell RNA sequencing analysis, we found that high expression of both angiotensin converting enzyme 2 (ACE2) and transmembrane Serine Protease 2 (TMPRSS2) makes the lung and intestine vulnerable to SARS-CoV-2. Hydroxychloroquine, chloroquine, and favipiravir, which were highly distributed to the lung, were reported to reduce viral loads in respiratory tract of COVID-19 patients. Conversely, drugs with poor lung distributions, including lopinavir/ritonavir, umifenovir and remdesivir, were insufficient to inhibit viral replication. Lopinavir/ritonavir might inhibit SARS-CoV-2 in the GI tract according to their distribution profiles. We concluded here that the antiviral drugs should be distributed straight to the lung tissue for reducing viral loads in respiratory tract of COVID-19 patients. Additionally, to better evaluate antiviral effects of drugs that target the intestine, the stool samples should also be collected for viral RNA test in the future.

Avian leukosis virus (ALV) is detrimental to poultry health and causes substantial economic losses from mortality and decreased performance. Because tumorigenesis is a complex mechanism, the regulatory architecture of the immune system is likely to include the added dimensions of modulation by miRNAs and long-noncoding RNA (lncRNA). To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and the associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were infected with ALV-J or maintained as non-injected controls. Spleen samples were collected at 40 days post injection (dpi), and sequenced. There were 864 genes, 7 miRNAs and 17 lncRNAs differentially expressed between infected and non-infected birds. The combined analysis of the 4 RNA expression datasets revealed that ALV infection is detected by pattern-recognition receptors (TLR9 and TLR3) leading to a type-I IFN mediated innate immune response that is modulated by IRF7 and IRF1. Co-expression network analysis of mRNA with miRNA, lncRNA and virus genes identified key elements within the complex networks utilized during ALV response. The integration of information from the host transcriptomic, epigenetic and virus response also has the potential to provide deeper insights into other host-pathogen interactions.

Avian leukosis virus (ALV) is oncogenic retrovirus that not only causes immunosuppression but also enhances the host's susceptibility to secondary infection. Exosomes play vital role in the signal transduction cascades that occur in response to viral infection. We want to explore the function of exosomes in the spread of ALV and the body's subsequent immunological response. RNA-sequencing and the isobaric tags for relative and absolute quantitation (iTRAQ) method were used to detect differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in exosomes secreted by macrophage cells in response to injection with ALV subgroup J (ALV-J). RNA-sequencing identified 513 DEGs in infected cells, with specific differential regulation in mRNA involved in tight junction signaling, TNF signaling, salmonella infection response, and immune response, among other important cellular processes. Differential regulation was observed in 843 lncRNAs, with particular enrichment in those lncRNA targets involved in Rap1 signaling, HTLV-I infection, tight junction signaling, and other signaling pathways. A total of 50 DEPs were identified in the infected cells by iTRAQ. The proteins enriched are involved in immune response, antigen processing, the formation of both MHC protein and myosin complexes, and transport. Combined analysis of the transcriptome and proteome revealed that there were 337 correlations between RNA and protein enrichment, five of which were significant. Pathways that were enriched on both the RNA and protein levels were involved in pathways in cancer, PI3K-Akt signaling pathway, Endocytosis, Epstein-Barr virus infection. These data show that exosomes are transmitters of intercellular signaling in response to viral infection. Exosomes can carry both viral nucleic acids and proteins, making it possible for exosomes to be involved in the viral infection of other cells and the transmission of immune signals between cells. Our sequencing results confirme previous studies on exosomes and further find exosomes may cause immunosuppression and immune tolerance.

The H5 highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, we constructed a replication-competent recombinant influenza A virus of clade 2.3.4.4 Н5N1 expressing the clade 2.3.2.1 H5 HA1 protein from a tricistronic NS segment. We used a truncated NS1 protein of 73 amino acids combined with a heterologous dimerization domain to increase protein stability. H5 HA1 and nuclear export information were fused in frame with a truncated NS1 open reading frame, separated by 2A self-processing sites. The resulting PR8-H5-NS1(73)H5 stably expressed clade 2.3.4.4 H5 HA and clade 2.3.2.1 H5 HA1 proteins and exhibited similar in vitro growth kinetics as the parental PR8-2344H5 virus. PR8-H5-NS1(73)H5 induced specific hemagglutination-inhibition (HI) antibody against clade 2.3.4.4 H5 that was comparable to that of the combination vaccine of PR8-2344H5 and PR8-2321H5. HI antibody titers were significantly lower against clade 2.3.2.1 H5 virus than with the combination vaccine. PR8-H5-NS1(73)H5 completely protected chickens from both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs challenge. Our results suggested that PR8-H5-NS1(73)H5 was highly immunogenic and efficacious against both clade 2.3.4.4 and clade 2.3.2.1 H5 HPAIVs in chickens.

The H5 and H7N9 subtypes of highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, a replication competent recombinant influenza A virus of the Í5N1 subtype expressing the H7 HA1 protein from a tri-cistronic NS segment was constructed. A heterologous dimerization domain was used to combine with the truncated NS1 protein of 73 amino acids to increase protein stability. H7 HA1, nuclear export protein coding region, and the truncated NS1 were fused in-frame into a single open reading frame via 2A self-cleaving peptides. The resulting PR8-H5-NS1(73)H7 stably expressed the H5 HA and H7 HA1 proteins, and exhibited similar growth kinetics as the parental PR8-H5 virus in vitro. PR8-H5-NS1(73)H7 induced specific hemagglutination inhibition (HI) antibody against H5, which was comparable to that of the combination vaccine of PR8-H5 and PR8-H7. The HI antibody titers against H7 virus were significantly lower than that by the combination vaccine. PR8-H5-NS1(73)H7 completely protected chickens from challenge with both H5 and H7 HPAIVs. These results suggest that PR8-H5-NS1(73)H7 is highly immunogenic and efficacious against both H5 and H7N9 HPAIVs in chickens. Highlights: - PR8-H5-NS1(73)H7 simultaneously expressed two HA proteins of different avian influenza virus subtypes. - PR8-H5-NS1(73)H7 was highly immunogenic in chickens. - PR8-H5-NS1(73)H7 provided complete protection against challenge with both H5 and H7N9 HPAIVs.

China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies.

HIV-1 is dependent upon cellular co-factors to mediate its replication cycle in CD4(+) T cells and macrophages, the two major cell types infected by the virus in vivo. One critical co-factor is Cyclin T1, a subunit of a general RNA polymerase II elongation factor known as P-TEFb. Cyclin T1 is targeted directly by the viral Tat protein to activate proviral transcription. Cyclin T1 is up-regulated when resting CD4(+) T cells are activated and during macrophage differentiation or activation, conditions that are also necessary for high levels of HIV-1 replication. Because Cyclin T1 is a subunit of a transcription factor, the up-regulation of Cyclin T1 in these cells results in the induction of cellular genes, some of which might be HIV-1 co-factors. Using shRNA depletions of Cyclin T1 and transcriptional profiling, we identified 54 cellular mRNAs that appear to be Cyclin T1-dependent for their induction in activated CD4(+) T Jurkat T cells and during differentiation and activation of MM6 cells, a human monocytic cell line. The promoters for these Cyclin T1-dependent genes (CTDGs) are over-represented in two transcription factor binding sites, SREBP1 and ARP1. Notably, 10 of these CTDGs have been reported to be involved in HIV-1 replication, a significant over-representation of such genes when compared to randomly generated lists of 54 genes (p value<0.00021). The results of siRNA depletion and dominant-negative protein experiments with two CTDGs identified here, CDK11 and Casein kinase 1 gamma 1, suggest that these genes are involved either directly or indirectly in HIV-1 replication. It is likely that the 54 CTDGs identified here include novel HIV-1 co-factors. The presence of CTDGs in the protein space that was available for HIV-1 to sample during its evolution and acquisition of Tat function may provide an explanation for why CTDGs are enriched in viral co-factors.

Genomic imprinting is an epigenetic regulation that leads to gene expression in a parent-of-origin specific manner. AFF3, the central component of the Super Elongation Complex-like 3 (SEC-L3), is enriched at both the intergenic-differentially methylated region (IG-DMR) and the Meg3 enhancer within the imprinted Dlk1-Dio3 locus to regulate the allele-specific gene expression in this locus. The localization of AFF3 to IG-DMR requires ZFP57. However, how AFF3 functions at the Meg3 enhancer in maintaining allele-specific gene expression remains unclear. Here, we demonstrate that AFF3 is associated with the Krüppel-like zinc finger protein ZFP281 in mouse embryonic stem (ES) cells. ZFP281 recruits AFF3 to the Meg3 enhancer within the imprinted Dlk1-Dio3 locus, thus regulating the allele-specific expression of the Meg3 polycistron. Our genome-wide analyses further identify ZFP281 as a critical factor generally associating with AFF3 at enhancers and functioning together with AFF3 in regulating the expression of a subset of genes. Our study suggests that different zinc finger proteins can recruit AFF3 to different regulatory elements and differentially regulate the function of AFF3 in a context-dependent manner.

Mumps incidence in mainland China remains at a high level. Genotype F has been the predominant genotype of mumps virus (MuV) in the last 20 years in mainland China. To better understand the genetic characteristics of MuV in China, the sequences of the Small Hydrophobic (SH), Hemagglutinin-Neuraminidase (HN) and Fusion (F) genes of MuVs of genotype F collected during 2001-2015 were determined. The evolutionary rates of the HN and F genes were similar (0.5 × 10-3 substitutions/site/year) whereas the SH gene evolutionary rate was three times faster. The most recent common ancestor of genotype F was traced back to 1980. Four lineages were identified within HN and F MuV sequences. A phylogeographic analysis indicated that the genotype F viruses originally spread from the Liaoning and Shandong provinces followed by a spread to the South and East of China. This study provides important genetic baseline data for the development of prevention and control measures of mumps.

Glioma is the most common type of malignant tumor in the central nervous system with an unfavorable prognosis and limited treatment. In this study, we are devoted to addressing the prognostic value of DNA damage repair-related genes in low-grade glioma (LGG). We plotted the landscape of DNA damage repair (DDR)-related genes and identified SMC4 as an independent prognostic marker with integrated bioinformatics analysis, which is overexpressed in different histologic subtypes of glioma. We observed that SMC4 expression is elevated in recurrent LGG patients or those with advanced histologic staging. SMC4 depletion inhibits proliferation and induces increased replication damage in LGG cells. Lastly, we predicted and validated the transcription modulation of SMC4 by a transcription factor, MYB, at the -976bp~ -837bp of the SMC4 promoter region in LGG cells. Together, our study identified SMC4 as a potential prognostic biomarker for LGG patients, which functions to promote cell proliferation by repairing replication damage and the expression of SMC4 could be transcriptionally regulated by MYB.

The activation of innate antiviral immunity is a promising approach for combatting viral infections. In this study, we screened Chinese herbs that activated human immunity and identified coptisine as a potent inhibitor of the influenza virus with an EC50 of 10.7 μM in MDCK cells. The time of an addition assay revealed that pre-treatment with coptisine was more effective at reducing viral replication than co-treatment or post-treatment. Our bulk RNA-sequencing data showed that coptisine upregulated the p21 signaling pathway in MDCK cells, which was responsible for its antiviral effects. Specifically, coptisine increased the expression of p21 and FOXO1 in a dose-dependent manner while leaving the MELK expression unchanged. Docking analysis revealed that coptisine likely inhibited MELK activity directly by forming hydrogen bonds with ASP-150 and GLU-87 in the catalytic pocket. These findings suggest that coptisine may be a promising antiviral agent that regulates the p21 signaling pathway to inhibit viral replication.

Advanced lung cancers carrying Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation remain a group that lacks effective treatments. Receptor activator of nuclear factorκB ligand (RANKL) has been demonstrated to drive malignant phenotypes in lung cancer; however, its role in KRAS-mutant (mt) lung adenocarcinoma (LUAD) is not yet fully elucidated.

To better understand the interaction between SARS-CoV-2 and human host and find potential ways to block the pandemic, one of the unresolved questions is that how the virus economically utilizes the resources of the hosts. Particularly, the tRNA pool has been adapted to the host genes. If the virus intends to translate its own RNA, then it has to compete with the abundant host mRNAs for the tRNA molecules. Translation initiation is the rate-limiting step during protein synthesis. The tRNAs carrying the initiation Methionine (iMet) recognize the start codon termed initiation ATG (iATG). Other normal Met-carrying tRNAs recognize the internal ATGs. The tAI of virus genes is significantly lower than the tAI of human genes. This disadvantage in translation elongation of viral RNAs must be compensated by more efficient initiation rates. In the human genome, the abundance of iMet-tRNAs to Met-tRNAs is five times higher than the iATG to ATG ratio. However, when SARS-CoV-2 infects human cells, the iMet has an 8.5-time enrichment to iATG. We collected 58 virus species and found that the enrichment of iMet is higher in all viruses compared to human. Our study indicates that the genome sequences of viruses like SARS-CoV-2 have the advantage of competing for the iMet-tRNAs with host mRNAs. The capture of iMet-tRNAs allows the fast translation initiation and the reproduction of virus itself, which compensates the lower tAI of viral genes. This might explain why the virus could rapidly translate its own RNA and reproduce itself from the sea of host mRNAs. Meanwhile, our study reminds the researchers not to ignore the mutations related to ATGs.

Angiogenesis constitutes a major mechanism responsible for exercise-induced beneficial effects. Our previous study identified a cluster of differentially expressed extracellular vesicle microRNAs (miRNAs) after exercise and found that some of them act as exerkines. However, whether these extracellular vesicle miRNAs mediate the exercise-induced angiogenesis remains unknown.

Infectious bronchitis virus (IBV), an ongoing emergence enveloped virus with a single-stranded positive-sense RNA genome, belongs to the Gammacoronavirus genus in the Coronaviridae family. IBV-associated tracheitis, nephritis, salpingitis, proventriculitis and egg drop have caused devastating economic losses to poultry industry worldwide. Since the end of 2018, a remarkably increasing number of commercial broilers and layers, vaccinated or not, were infected with IBV in China. Here, we described two IB outbreaks with severe respiratory system or kidney injury in IBV-vaccinated commercial poultry farms in central China. Other possible causative viral pathogens, including avian influenza virus (AIV), Newcastle disease virus (NDV) and Kedah fatal kidney syndrome virus (KFKSV), were excluded by reverse transcription-polymerase chain reaction (RT-PCR), and three virulent IBV strains, HeN-1/China/2019, HeN-2/China/2019 and HeN-101/China/2019, were identified. Although the gross pathologic appearance of these two IB outbreaks was different, the newly identified IBV strains were all closely related to the ck/China/I0529/17 strain and grouped into GI-19 genotype clade based on the sequencing and phylogenetic analysis of the complete S1 genes. Moreover, there are still some evolutionary distance between the newly identified IBV strains, HeN-101/China/2019 in particular, and other GI-19 strains, suggesting that Chinese IBV strains constantly emerge and evolve towards different directions. In conclusion, this study provided an insight of the recently emerging IBV outbreaks in IBV-vaccinated commercial poultry farms and identified the genetic characteristics of three virulent GI-19 IBV strains, which shows the need to carry out proper preventive measures and control strategies.

Hepatitis B virus (HBV) infection causes acute hepatitis B (AHB), chronic hepatitis B (CHB), liver cirrhosis (LC), and eventually hepatocellular carcinoma (HCC). The presence of hepatitis B e antigen (HBeAg) in the serum generally indicates ongoing viral replication and disease progression. However, the mechanism by which HBeAg regulates HBV infection remains unclear. Interferons (IFNs) are pleiotropic cytokines that participate in host innate immunity. After binding to receptors, IFNs activate the JAK/STAT pathway to stimulate expression of IFN-stimulated genes (ISGs), leading to induction of antiviral responses. Here, we revealed that HBeAg represses IFN/JAK/STAT signaling to facilitate HBV replication. Initially, HBeAg stimulates the expression of suppressor of cytokine signaling 2 (SOCS2). Subsequently, SOCS2 impairs IFN/JAK/STAT signaling through reducing the stability of tyrosine kinase 2 (TYK2), downregulating the expression of type I and III IFN receptors, attenuating the phosphorylation and nucleus translocation of STAT1. Finally, SOCS2 inhibits the expression of ISGs, which leads to the repression of IFN action and facilitation of viral replication. These results demonstrate an important role of HBeAg in the regulation of IFN action, and provide a possible molecular mechanism by which HBV resists the IFN therapy and maintains persistent infection.

Although immune checkpoint inhibitors (ICIs) against programmed cell death protein 1 (PD-1) and its ligand PD-L1 have demonstrated potency towards treating patients with non-small cell lung carcinoma (NSCLC), the potential association between Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene substitutions and the efficacy of ICIs remains unclear. In this study, we aimed to find point mutations in the KRAS gene resistant to ICIs and elucidate resistance mechanism.

Increasing evidence shows the African lineage Zika virus (ZIKV) displays a more severe neurovirulence compared to the Asian ZIKV. However, viral determinants and the underlying mechanisms of enhanced virulence phenotype remain largely unknown. Herein, we identify a panel of amino acid substitutions that are unique to the African lineage of ZIKVs compared to the Asian lineage by phylogenetic analysis and sequence alignment. We then utilize reverse genetic technology to generate recombinant ZIKVs incorporating these lineage-specific substitutions based on an infectious cDNA clone of Asian ZIKV. Through in vitro characterization, we discover a mutant virus with a lysine to arginine substitution at position 101 of capsid (C) protein (termed K101R) displays a larger plaque phenotype, and replicates more efficiently in various cell lines. Moreover, K101R replicates more efficiently in mouse brains and induces stronger inflammatory responses than the wild type (WT) virus in neonatal mice. Finally, a combined analysis reveals the K101R substitution promotes the production of mature C protein without affecting its binding to viral RNA. Our study identifies the role of K101R substitution in the C protein in contributing to the enhanced virulent phenotype of the African lineage ZIKV, which expands our understanding of the complexity of ZIKV proteins.

Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/β-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the β-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.