Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library.

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

Mutational antigenic landscape of prevailing H9N2 influenza virus hemagglutinin spectrum.

H9N2 influenza viruses are globally endemic in birds, and a sharp increase in human infections with H9N2 occurred during 2021 to 2022. In this study, we assess the antigenic and pathogenic impact of 23 hemagglutinin (HA) amino acid mutations. Our study reveals that three specific mutations, labeled R164Q, N166D, and I220T, are responsible for the binding of antibodies with escape mutations. Variants containing R164Q and I220T mutations increase viral replication in avian and mammalian cells. Furthermore, T150A and I220T mutations are found to enhance viral replication in mice, indicating that these mutations may have the potential to adapt mammals. Structure analysis reveals that residues 164 and 220 bearing R164Q and I220T mutations increase interactions with the surrounding residues. Our findings enrich current knowledge about the risk assessment regarding which predominant HA immune-escape mutations of H9N2 viruses may pose the greatest threat to the emergence of pandemics in birds and humans.

HVT-vectored H7 vaccine protects chickens from lethal infection with the highly pathogenic H7N9 Avian influenza virus.

Since mid-2016, the highly pathogenic H7N9 subtype avian influenza virus (AIV) has threatened both public health and the poultry industry. Although a vaccination strategy has been deemed imperative to manage the virus, the most commonly used inactivated vaccines today are susceptible to interference from maternal antibodies and associated with an over-reliance on humoral immunity. In response, we developed a recombination vaccine with the herpesvirus of turkeys (HVT) as the vector to squeeze HPAI H7N9 and assessed its protective efficiency in immunized chickens. By inserting an enhanced green fluorescent protein (EGFP) expression cassette (i.e., MCMV+EGFP+SV40 polyA) into the HVT065 and HVT066 positions, we obtained the recombinant HVT expressing EGFP (i.e., rHVT-EGFP). Electroporation and EGFP tags improved the efficiency of transfection compared with transfection using expression plasmids without any fluorescent labeling and traditional liposomes. Using limiting dilution analysis and ultrasonic cell disruption techniques, we screened and purified a cell-bound herpes virus based on rHVT-EGFP and consequently constructed a recombinant HVT expressing the hemagglutinin (HA) of H7N9 (i.e., rHVT-H7HA), which was able to proliferate similarly to the parental strain, stably pass for at least 15 generations in vitro, and replicate stably in multiple organs in vivo. After chickens were immunized with rHVT-H7HA, the average antibody titers reached up to 3 log2 at 35 d post-vaccination and remained stable. Those results suggest that rHVT-H7HA can protect chickens against H7N9 with a dose-independent immune protection rate of 90% and significantly reduce the lung virus titer 4 d post-challenge.

Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I.

Senecavirus A (SVA)-induced porcine idiopathic vesicular disease has caused huge economic losses worldwide. Glucose metabolism in the host cell is essential for SVA proliferation; however, the impact of the virus on glucose metabolism in host cells and the subsequent effects are still unknown. Here, glycolysis induced by SVA is shown to facilitate virus replication by promoting lactate production, which then attenuates the interaction between the mitochondrial antiviral-signaling protein (MAVS) and retinoic acid-inducible gene I (RIG-I). SVA induces glycolysis in PK-15 cells, as indicated by significantly increased expression of hexokinase 2 (HK2), 6-phosphofructokinase (PFKM), pyruvate kinase M (PKM), phosphoglycerate kinase 1 (PGK1), hypoxia-inducible factor-1 alpha (HIF-1α), and superoxide dismutase-2 (SOD2) in a dose-and replication-dependent manner, and enhanced lactate production, while reducing ATP generation. Overexpression of PKM, PGK1, HIF-1α, and PDK3 in PK-15 cells and high glucose concentrations promote SVA replication, while glycolytic inhibitors decrease it. Inhibition of RLR signaling allowed better replication of SVA by promoting lactate production to attenuate the interaction between MAVS and RIG-I, and regulatory effect of glycolysis on replication of SVA was mainly via RIG-I signaling. SVA infection in mice increased expression of PKM and PGK1 in tissues and serum yields of lactate. Mice treated with high glucose and administered sodium lactate showed elevated lactate levels and better SVA replication, as well as suppressed induction of RIG-I, interferon beta (IFNß), IFNα, interferon-stimulated gene 15 (ISG15), and interleukin 6 (IL-6). The inhibitory effect on interferons was lower in mice administered sodium oxamate and low glucose compared to the high glucose, indicating that RLR signaling was inhibited by SVA infection through lactate in vitro and in vivo. These results provide a new perspective on the relationship between metabolism and innate immunity of the host in SVA infection, suggesting that glycolysis or lactate may be new targets against the virus.

That H9N2 avian influenza viruses circulating in different regions gather in the same live-poultry market poses a potential threat to public health.

H9N2 avian influenza viruses are endemic and persistent in China, but those that are prevalent in different provinces are also causes of wide epidemics, related to the spread of wild birds and the cross-regional trade in live poultry. For the past 4 years, beginning in 2018, we have sampled a live-poultry market in Foshan, Guangdong, in this ongoing study. In addition to the prevalence of H9N2 avian influenza viruses in China during this period, we identified isolates from the same market belonging to clade A and clade B, which diverged in 2012-2013, and clade C, which diverged in 2014-2016, respectively. An analysis of population dynamics revealed that, after a critical divergence period from 2014 to 2016, the genetic diversity of H9N2 viruses peaked in 2017. Our spatiotemporal dynamics analysis found that clade A, B, and C, which maintain high rates of evolution, have different prevalence ranges and transmission paths. Clades A and B were mainly prevalent in East China in the early stage, and then spread to Southern China, becoming epidemic with clade C. Strains from different regions converge at the same live-poultry market to communicate, which may be one reasons the H9N2 viruses are difficult to eradicate and increasingly dominant throughout China. Selection pressure and molecular analysis have demonstrated that single amino acid polymorphisms at key receptor binding sites 156, 160, and 190 under positive selection pressure, suggesting that H9N2 viruses are undergoing mutations to adapt to new hosts. Live-poultry markets are important because people who visit them have frequent contact with poultry, H9N2 viruses from different regions converge at these markets and spread through contact between live birds and humans, generating increased risks of human exposure to these viruses and threatening public health safety. Thus, it is important to reducing the cross-regional trade of live poultry and strengthening the monitoring of avian influenza viruses in live-poultry markets to reduce the spread of avian influenza viruses.

USP1-Associated Factor 1 Modulates Japanese Encephalitis Virus Replication by Governing Autophagy and Interferon-Stimulated Genes.

Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus that can cause central nervous system diseases in humans and animals. Host factors attempt to limit virus replication when the viruses invade the host by using various strategies for replication. It is essential to clarify the host factors that affect the life cycle of JEV and explore its underlying mechanism. Here, we found that USP1-associated factor 1 (UAF1; also known as WD repeat-containing protein 48) modulated JEV replication. We found that JEV propagation significantly increased in UAF1-depleted Huh7 cells. Moreover, we found that knockdown of UAF1 activated cell autophagic flux in further functional analysis. Subsequently, we demonstrated that autophagy can be induced by JEV, which promotes viral replication by inhibiting interferon-stimulated gene (ISG) expression in Huh7 cells. The knockdown of UAF1 reduced ISG expression during JEV infection. To explore the possible roles of autophagy in UAF1-mediated inhibition of JEV propagation, we knocked out ATG7 to generate autophagy-deficient cells and found that depletion of UAF1 failed to promote JEV replication in ATG7 knockout cells. Moreover, in ATG7-deficient Huh7 cells, interference with UAF1 expression did not lead to the induction of autophagy. Taken together, these findings indicate that UAF1 is a critical regulator of autophagy and reveal a mechanism by which UAF1 knockdown activates autophagy to promote JEV replication. IMPORTANCE Host factors play an essential role in virus replication and pathogenesis. Although UAF1 is well known to form complexes with ubiquitin-specific proteases, little is known about the function of the UAF1 protein itself. In this study, we confirmed that UAF1 is involved in JEV replication. Notably, we discovered a novel function for UAF1 in regulating autophagy. Furthermore, we demonstrated that UAF1 modulated JEV replication through its autophagy regulation. This study is the first description of the novel function of UAF1 in regulating autophagy, and it clarifies the underlying mechanism of the antiviral effect of UAF1 against JEV. These results provide a new mechanistic insight into the functional annotation of UAF1 and provide a potential target for increasing virus production during vaccine production.

Highly efficient and robust π-FISH rainbow for multiplexed in situ detection of diverse biomolecules.

In the unprecedented single-cell sequencing and spatial multiomics era of biology, fluorescence in situ hybridization (FISH) technologies with higher sensitivity and robustness, especially for detecting short RNAs and other biomolecules, are greatly desired. Here, we develop the robust multiplex π-FISH rainbow method to detect diverse biomolecules (DNA, RNA, proteins, and neurotransmitters) individually or simultaneously with high efficiency. This versatile method is successfully applied to detect gene expression in different species, from microorganisms to plants and animals. Furthermore, we delineate the landscape of diverse neuron subclusters by decoding the spatial distribution of 21 marker genes via only two rounds of hybridization. Significantly, we combine π-FISH rainbow with hybridization chain reaction to develop π-FISH+ technology for short nucleic acid fragments, such as microRNA and prostate cancer anti-androgen therapy-resistant marker ARV7 splicing variant in circulating tumour cells from patients. Our study provides a robust biomolecule in situ detection technology for spatial multiomics investigation and clinical diagnosis.

Key amino acid position 272 in neuraminidase determines the replication and virulence of H5N6 avian influenza virus in mammals.

Avian influenza H5N6 virus not only wreaks economic havoc in the poultry industry but also threatens human health. Strikingly, as of August 2022, 78 human beings were infected with H5N6, and the spike in the number of human infections with H5N6 occurred during 2021. In the life cycle of influenza virus, neuraminidase (NA) has numerous functions, especially viral budding and replication. Here, we found that NA-D272N mutation became predominant in H5N6 viruses since 2015 and significantly increased the viral replication and virulence in mice. D272N mutation in NA protein increased viral release from erythrocytes, thermostability, early transcription, and accumulation of NA protein. Particularly, the dominant 272 residue switch from N to S has occurred in wild bird-origin H5N6 viruses since late 2016 and N272S mutation induced significantly higher levels of inflammatory cytokines in infected human cells. Therefore, comprehensive surveillance of bird populations needs to be enhanced to monitor mammalian adaptive mutations of H5N6 viruses.

N6-methyladenosine modification of viral RNA and its role during the recognition process of RIG-I-like receptors.

N6-methyladenosine (m6A) is the most abundant RNA chemical modification in eukaryotes and is also found in the RNAs of many viruses. In recent years, m6A RNA modification has been reported to have a role not only in the replication of numerous viruses but also in the innate immune escape process. In this review, we describe the viruses that contain m6A in their genomes or messenger RNAs (mRNAs), and summarize the effects of m6A on the replication of different viruses. We also discuss how m6A modification helps viral RNAs escape recognition by exogenous RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), during viral invasion. Overall, the goal of our review is to summarize how m6A regulates viral replication and facilitates innate immune escape. Furthermore, we elaborate on the potential of m6A as a novel antiviral target.

A Characterization and an Evolutionary and a Pathogenicity Analysis of Reassortment H3N2 Avian Influenza Virus in South China in 2019-2020.

Seasonal H3N2 influenza virus has always been a potential threat to public health. The reassortment of the human and avian H3N2 influenza viruses has resulted in major influenza outbreaks, which have seriously damaged human life and health. To assess the possible threat of the H3N2 avian influenza virus to human health, we performed whole-genome sequencing and genetic evolution analyses on 10 H3N2 field strains isolated from different hosts and regions in 2019-2020 and selected representative strains for pathogenicity tests on mice. According to the results, the internal gene cassettes of nine strains had not only undergone reassortment with the H1, H2, H4, H6, and H7 subtypes, which circulate in poultry and mammals, but also with H10N8, which circulates in wild birds in the natural environment. Three reassorted strains were found to be pathogenic to mice, of these one strain harboring MP from H10N8 showed a stronger virulence in mice. This study indicates that reassorted H3N2 AIVs may cross the host barrier to infect mammals and humans, thereby, necessitating persistent surveillance of H3N2 AIVs.

Toosendanin suppresses African swine fever virus replication through upregulating interferon regulatory factor 1 in porcine alveolar macrophage cultures.

African swine fever virus (ASFV) is a highly infectious and lethal swine pathogen that causes severe socio-economic consequences in affected countries. Unfortunately, effective vaccine for combating ASF is unavailable so far, and the prevention and control strategies for ASFV are still very limited. Toosendanin (TSN), a triterpenoid saponin extracted from the medicinal herb Melia toosendan Sieb. Et Zucc, has been demonstrated to possess analgesic, anti-inflammatory, anti-botulism and anti-microbial activities, and was used clinically as an anthelmintic, while the antiviral effect of TSN on ASFV has not been reported. In this study, we revealed that TSN exhibited a potent inhibitory effect on ASFV GZ201801-38 strain in porcine alveolar macrophages (PAMs; EC50 = 0.085 µM, SI = 365) in a dose-dependent manner. TSN showed robust antiviral activity in different doses of ASFV infection and reduced the transcription and translation levels of ASFV p30 protein, viral genomic DNA quantity as well as viral titer at 24 and 48 h post-infection. In addition, TSN did not affect virion attachment and release but intervened in its internalization in PAMs. Further investigations disclosed that TSN played its antiviral role by upregulating the host IFN-stimulated gene (ISG) IRF1 rather than by directly inactivating the virus particles. Overall, our results suggest that TSN is an effective antiviral agent against ASFV replication in vitro and may have the potential for clinical use.

Identification of cytochrome c oxidase subunit 4 isoform 1 as a positive regulator of influenza virus replication.

Human infection with highly pathogenic H5N1 influenza virus causes severe respiratory diseases. Currently, the drugs against H5N1 are limited to virus-targeted inhibitors. However, drug resistance caused by these inhibitors is becoming a serious threat to global public health. An alternative strategy to reduce the resistance risk is to develop antiviral drugs targeting host cell proteins. In this study, we demonstrated that cytochrome c oxidase subunit 4 isoform 1 (COX41) of host cell plays an important role in H5N1 infection. Overexpression of COX41 promoted viral replication, which was inhibited by silencing or knockout the expression of COX41 in the host cell. The ribonucleoproteins (RNPs) of H5N1 were retained in the cell nucleus after knockout cellular COX41. Strikingly, inhibition of cellular COX41 by lycorine, a small-molecule compound isolated from Amaryllidaceae plants, reduced the levels of COX41-induced ROS and phosphorylation of extracellular signal-regulated kinase (ERK) in cells, thus resulting in the blockage of nuclear export of vRNP and inhibition of viral replication. In H5N1-infected mice that were treated with lycorine, we observed a reduction of viral titers and inhibition of pathological changes in the lung and trachea tissues. Importantly, no resistant virus was generated after culturing the virus with the continuous treatment of lycorine. Collectively, these findings suggest that COX41 is a positive regulator of H5N1 replication and might serve as an alternative target for anti-influenza drug development.

Development and Application of a Duplex Droplet Digital Polymerase Chain Reaction Assay for Detection and Differentiation of EP402R-Deleted and Wild-Type African Swine Fever Virus.

African swine fever (ASF) is a highly fatal porcine disease caused by the African swine fever virus (ASFV), and resulting in huge economic losses across the globe. ASF has been raging in China for 3 years, and recently EP402R-deleted ASFV strains emerged, showing sub-acute or chronic symptoms in pigs and providing novel difficulties to monitor and control the disease as EP402R-deleted strains possess no hemadsorption (HAD) ability. In addition, the gene deletion virus with low viral load is prone to results retest or false negative due to the high cycle threshold (Ct) value under the current real-time polymerase chain reaction (PCR) detection method. Thus, a new method is needed to detect and distinguish wild strains and gene-deleted viruses. In this study, a duplex droplet digital polymerase chain reaction (ddPCR) assay based on the ASFV B646L and EP402R genes was established and showed good linearity (R2 > 0.99). The limit of detection for duplex ddPCR was 52 copies per reaction and 8.6 copies per reaction for B646L and EP402R, respectively. No cross-reaction with other porcine viruses [classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine parvovirus (PPV), Japanese encephalitis virus (JEV), and porcine circovirus type 2 (PCV2)] was identified by this assay. In addition, 44 ASFV-suspicious clinical samples as well as EP402R-deleted ASFV were tested in parallel by duplex real-time PCR and ddPCR, indicative of a higher sensitivity which belonged to the duplex ddPCR assay. In summary, this is the first time that duplex ddPCR assay has been successfully developed to provide an efficient method to detect and differentiate ASFV wild-type and gene-deleted strains.

Real-Time Visualization of the Infection and Replication of a Mouse-Lethal Recombinant H9N2 Avian Influenza Virus.

H9N2 avian influenza viruses (AIVs) continuously cross the species barrier to infect mammalians and are repeatedly transmitted to humans, posing a significant threat to public health. Importantly, some H9N2 AIVs were found to cause lethal infection in mice, but little is known about the viral infection dynamics in vivo. To analyze the real-time infection dynamics, we described the generation of a mouse-lethal recombinant H9N2 AIV, an influenza reporter virus (VK627-NanoLuc virus) carrying a NanoLuc gene in the non-structural (NS) segment, which was available for in vivo imaging. Although attenuated for replication in MDCK cells, VK627-NanoLuc virus showed similar pathogenicity and replicative capacity in mice to its parental virus. Bioluminescent imaging of the VK627-NanoLuc virus permitted successive observations of viral infection and replication in infected mice, even following the viral clearance of a sublethal infection. Moreover, VK627-NanoLuc virus was severely restricted by the K627E mutation in PB2, as infected mice showed little weight loss and a low level of bioluminescence. In summary, we have preliminarily established a visualized tool that enables real-time observation of the infection and replication dynamics of H9N2 AIV in mice, which contributes to further understanding the mechanisms underlying the pathogenic enhancement of H9N2 AIV to mice.

Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice.

Since mid-2016, the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic (HP) phenotype in China, raising many concerns about public health and poultry industry. The insertion of a "KRTA" motif at hemagglutinin cleavage site (HACS) occurred in the early stage of HP H7N9 variants. During the co-circulation, the HACS of HP-H7N9 variants were more polymorphic in birds and humans. Although HP-H7N9 variants, unlike the H5 subtype virus, exhibited the insertions of basic and non-basic amino acids, the underlying function of those insertions and substitutions remains unclear. The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China. Then, we generated six H7N9 viruses bearing the PEIPKGR/G, PEVPKGR/G, PEVPKRKRTAR/G, PEVPKGKRTAR/G, PEVPKGKRIAR/G, and PEVPKRKRR/G motifs. Interestingly, after the deletion of threonine and alanine (TA) at HACS, the H7N9 viruses manifested decreased thermostability and virulence in mice, and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence. By contrast, the insertion of non-basic amino acid isoleucine and alanine (IA) decreased the transmissibility in chickens and virulence in mice. Remarkably, the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens, suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice. The ongoing evolution of H7N9 increasingly threatens public health and poultry industry, so, its comprehensive surveillance and prevention of H7N9 viruses should be pursued.

Characterization of four novel H5N6 avian influenza viruses with the internal genes from H5N1 and H9N2 viruses and experimental challenge of chickens vaccinated with current commercially available H5 vaccines.

Since 2014, highly pathogenic avian influenza H5N6 viruses have been responsible for outbreaks in poultry. In this study, four H5N6 virus strains were isolated from faecal samples of sick white ducks and dead chickens in Shandong in 2019. These H5N6 viruses were triple-reassortant viruses that have not been previously characterized. Their HA genes were derived from the H5 viruses and were closely related to the vaccine strain Re-11. Their NA genes all fell into the N6-like lineage and the internal gene were derived from H5N1 and H9N2 viruses. They all showed high pathogenicity in mice and caused lethal infection with high rates of transmission in chickens. Moreover, the SPF chickens inoculated with the currently used H5 (Re-11 and Re-12 strains)/H7 (H7-Re-2 strain) trivalent inactivated vaccines in China were completely protected from these four H5N6 viruses. Our study indicated the necessity of continued surveillance for H5 influenza A viruses and the importance of timely update of vaccine strains in poultry industry.