The global succinylation of SARS-CoV-2-infected host cells reveals drug targets.

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.

A Review of the Emerging Poultry Visceral Gout Disease Linked to Avian Astrovirus Infection.

Avian astroviruses, including chicken astrovirus (CAstV), avian nephritisvirus (ANV), and goose astrovirus (GoAstV), are ubiquitous enteric RNA viruses associated with enteric disorders in avian species. Recent research has found that infection of these astroviruses usually cause visceral gout in chicken, duckling and gosling. However, the underlying mechanism remains unknown. In the current article, we review recent discoveries of genetic diversity and variation of these astroviruses, as well as pathogenesis after astrovirus infection. In addition, we discuss the relation between avian astrovirus infection and visceral gout in poultry. Our aim is to review recent discoveries about the prevention and control of the consequential visceral gout diseases in poultry, along with the attempt to reveal the possible producing process of visceral gout diseases in poultry.

Newcastle disease virus RNA-induced IL-1ß expression via the NLRP3/caspase-1 inflammasome.

Newcastle disease virus (NDV) infection causes severe inflammation and is a highly contagious disease in poultry. Virulent NDV strains (GM) induce large quantities of interleukin-1ß (IL-1ß), which is the central mediator of the inflammatory reaction. Excessive expression of IL-1ß exacerbates inflammatory damage. Therefore, exploring the mechanisms underlying NDV-induced IL-1ß expression can aid in further understanding the pathogenesis of Newcastle disease. Here, we showed that anti-IL-1ß neutralizing antibody treatment decreased body temperature and mortality following infection with virulent NDV. We further explored the primary molecules involved in NDV-induced IL-1ß expression from the perspective of both the host and virus. This study showed that overexpression of NLRP3 resulted in increased IL-1ß expression, whereas inhibition of NLRP3 or caspase-1 caused a significant reduction in IL-1ß expression, indicating that the NLRP3/caspase-1 axis is involved in NDV-induced IL-1ß expression. Moreover, ultraviolet-inactivated GM (chicken/Guangdong/GM/2014) NDV failed to induce the expression of IL-1ß. We then collected virus from GM-infected cell culture supernatant using ultracentrifugation, extracted the viral RNA, and stimulated the cells further with GM RNA. The results revealed that RNA alone was capable of inducing IL-1ß expression. Moreover, NLRP3/caspase-1 was involved in GM RNA-induced IL-1ß expression. Thus, our study elucidated the critical role of IL-1ß in the pathogenesis of Newcastle disease while also demonstrating that inhibition of IL-1ß via anti-IL-1ß neutralizing antibodies decreased the damage associated with NDV infection; furthermore, GM RNA induced IL-1ß expression via NLRP3/caspase-1.

Transcriptome Analysis Reveals the Neuro-Immune Interactions in Duck Tembusu Virus-Infected Brain.

The duck Tembusu virus (DTMUV) is a mosquito-borne flavivirus. It causes severe symptoms of egg-drop, as well as neurological symptoms and brain damage in ducks. However, the specific molecular mechanisms of DTMUV-induced neurovirulence and host responses in the brain remain obscure. To better understand the host-pathogen and neuro-immune interactions of DTMUV infection, we conducted high-throughput RNA-sequencing to reveal the transcriptome profiles of DTMUV-infected duck brain. Totals of 117, 212, and 150 differentially expressed genes (DEGs) were identified at 12, 24, and 48 h post infection (hpi). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses uncovered genes and pathways related to the nervous system and immune responses in duck brain. Neuro-related genes, including WNT3A, GATA3, and CHRNA6, were found to be significantly downregulated. RIG-I-like receptors (DHX58, IFIH1) and Toll-like receptors (TLR2 and TLR3) were activated, inducing the expression of 22 interferon stimulated genes (ISGs) and antigen-processing and -presenting genes (TAP1 and TAP2) in the brain. Our research provides comprehensive information for the molecular mechanisms of neuro-immune and host-pathogen interactions of DTMUV.

Host immune responses of pigeons infected with Newcastle disease viruses isolated from pigeons.

Newcastle disease (ND), affecting over 250 bird species, is caused by the Newcastle disease virus (NDV). ND is one of the leading causes of morbidity and mortality in pigeons. Most studies investigating NDV in pigeons have focused on the epidemiology and pathogenicity of the virus. However, the host immune responses in pigeons infected with NDVs remains largely unclear. In this study, we investigated the host immune responses in pigeons infected with two NDV stains, a pigeon paramyxovirus type 1(PPMV-1) strain, GZH14, and a genotype II virus, KP08. Although no mortality was observed upon infection with either virus, obvious neurological effects were observed in the GZH14-infected pigeons but not in the KP08-infected pigeons. Both viruses could replicate in the examined tissues, namely brain, lung, spleen, trachea, kidney, and bursa of Fabricius. The expression level of RIG-I, IL-6, IL-1ß, CCL5, and IL-8 were up-regulated by both viruses in the brain, lung and spleen at 3 and 7 days post-infection. Notably, these proinflammatory cytokines and chemokines showed more intense expression in the brain, when induced by the GZH14 strain than with the KP08 strain. These results indicate that the intense inflammatory responses induced by PPMV-1 in the brain may be a critical determinant of neurological symptoms in pigeons infected with PPMV-1. Our study provides new insight into the pathogenicity of PPMV-1 in pigeons attributable to the host immune responses.

Strength of the repulsive part of the interatomic potential determines fragility in metallic liquids.

The dynamical behaviour of liquids is frequently characterized by the fragility, which can be defined from the temperature dependence of the shear viscosity, η (ref. ). For a strong liquid, the activation energy for η changes little with cooling towards the glass transition temperature, Tg. The change is much greater in fragile liquids, with the activation energy becoming very large near Tg. While fragility is widely recognized as an important concept-believed, for example, to play an important role in glass formation-the microscopic origin of fragility is poorly understood. Here, we present new experimental evidence showing that fragility reflects the strength of the repulsive part of the interatomic potential, which can be determined from the steepness of the pair distribution function near the hard-sphere cutoff. On the basis of an analysis of scattering data from ten different metallic alloy liquids, we show that stronger liquids have steeper repulsive potentials.

Avian Influenza (H7N9) Viruses Co-circulating among Chickens, Southern China.

In April 2017, three avian influenza (H7N9) viruses were isolated from chickens in southern China. Each virus had different insertion points in the cleavage site of the hemagglutinin protein compared to the first identified H7N9 virus. We determined that these viruses were double or triple reassortant viruses.

Generation and evaluation of a genetically attenuated Newcastle disease virus rGM-VIIm as a genotype-matched vaccine.

Despite intensive vaccination campaigns, outbreaks of Newcastle disease (ND) have been frequently reported in China, especially of genotype VII that first emerged in the late 1990s. Given the dire need for vaccines against the circulating genotype VII virus, we developed an alternative method to recover a highly virulent recombinant GM (rGM) virus that involves a T7 system with a hammerhead ribozyme sequence introduced downstream of the T7 promoter. By changing the F0 polybasic cleavage site RRQKR↓F to the monobasic GRQGR↓L, we generated a mutant virus (rGM-VIIm) that was found to be highly attenuated in chickens. The rGM-VIIm virus not only produced fourfold higher hemagglutination assay (HA) titers than the parental virus, but also exhibited genetic stability after 15 continuous passages in specific-pathogen-free (SPF) embryonated eggs. Whether live or inactivated, rGM-VIIm and LaSota vaccines can protect vaccinated birds from GM challenge infection. However, live and inactivated rGM-VIIm vaccines reduced virus shedding of the homologous challenge virus significantly better than the LaSota virus vaccine did. Altogether, our results suggest that rGM-VIIm vaccines could aid in the control of ND in China.

Short communication: antiviral activity of porcine IFN-λ3 against porcine epidemic diarrhea virus in vitro.

A new family of IFNs called type III IFN or IFN-λ has been described, and shown to induce antiviral activity against several viruses in the cell culture. In this study, the molecular cloning, expression, and antiporcine epidemic diarrhea virus (PEDV) activity of porcine IFN-λ3 (poIFN-λ3) were reported. The full-length poIFN-λ3 cDNA sequence encoded 196 amino acids with a 23 amino acid signal peptide. Sequence alignments showed that poIFN-λ3 had an amino acid sequence similarity to Ovis aries (78.1 %), Bos taurus (76.0 %), Tupaia belangeri (71.3 %), Equus caballus (69.9 %), and Homo sapiens (69.9 %). The phylogenetic analysis based on the genomic sequences indicated that poIFN-λ3 is located in the same branch as B. taurus and O. aries IFN-λ3. The poIFN-λ3 without a signal anchor sequence was efficiently expressed in Escherichia coli, and the purified recombinant poIFN-λ3 exhibited significant antiviral effects against PEDV in a dose- and time-dependent manner. This inhibitory effect of poIFN-λ3 on PEDV was observed under three different treatment conditions. The highest inhibition of PEDV was observed in Vero E6 cell cultures pretreated with poIFN-λ3 (prior to PEDV infection). In addition, poIFN-λ3 was able to induce the expression of IFN-stimulated genes, including ISG15, OAS1, and Mx1 in Vero E6 cells. These data demonstrate that poIFN-λ3 has antiviral activity against PEDV and may serve as a useful biotherapeutic candidate to inhibit PEDV or other viruses in swine.

Phylogenetic relationships and pathogenicity variation of two Newcastle disease viruses isolated from domestic ducks in Southern China.

BACKGROUND: Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which is enzootic and causes large economic losses in the poultry sector. Genotype VII and genotype IX NDV viruses were the predominant circulating genotype in China, which may possibly be responsible for disease outbreaks in chicken flocks in recent years. While ducks and geese usually have exhibited inapparent infections. METHODS: In the present study, we investigate the complete genome sequence, the clinicopathological characterization and transmission of two virulent Newcastle disease viruses, SS-10 and NH-10, isolated from domestic ducks in Southern China in 2010. RESULTS: F, and the complete gene sequences based on phylogenetic analysis demonstrated that SS-10 (genotype VII) and NH-10 (genotype IX) belongs to class II. The deduced amino acid sequence was (112)R-R-Q-K/R-R-F(117) at the fusion protein cleavage site. Animal experiment results showed that the SS-10 virus isolated from ducks was highly pathogenic for chickens and geese, but low pathogenic for ducks. It could be detected from spleen, lung, kidney, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. Moreover, it could transmit to chickens, ducks and geese by naive contact. However, the NH-10 virus isolated from ducks could infect some chickens, ducks and geese, but only caused chickens to die. Additionally, it could transmit to the naive contact chickens, ducks, and geese. CONCLUSION: The two NDV isolates exhibited different biological properties with respect to pathogenicity and transmission in chickens, ducks and geese. Therefore, no species-preference exists for chicken, duck or goose viruses and more attention should be paid to the trans-species transmission of VII NDVs between ducks, geese and chickens for the control and eradication of ND.

Interferon regulatory factor 7- (IRF7-) mediated immune response affects Newcastle disease virus replication in chicken embryo fibroblasts.

Interferon regulatory factor 7 (IRF7) is essential for the induction of an antiviral response. Previous studies have shown that virus replication causes the activation or expression of Type I interferon (IFN) in cells, which further activates IFN-stimulated genes (ISGs) to retard virus growth. In this study, after infection of chicken embryo fibroblasts (CEFs) with the lentogenic Newcastle disease virus (NDV) strain LaSota or the velogenic NDV strain GM, the mRNA and protein levels of IRF7 showed a significant increase, and part of the IRF7 protein was translocated from the cytoplasm to the nucleus. In order to further explore the effect of IRF7-mediated innate immune response on the replication of NDV in CEFs, the mRNA levels of IFN-α, IFN-ß and STAT1 were measured and the replication kinetics of NDV determined. The results showed that specific siRNA could inhibit the expression of IRF7 and limit the mRNA level of IFN-α, IFN-ß and STAT1 and, accordingly, the replication kinetics of both NDVs were enhanced after the inhibition of IRF7. In conclusion, IRF7 is an important nuclear transcription factor for the induction of Type I IFNs during the antiviral response, which can affect the replication of NDV and spread to CEFs in the early phase of viral infection.

Prevalence, genomic characteristics, and pathogenicity of fowl adenovirus 2 in Southern China.

In recent years, the occurrence of fowl adenovirus 2 (FAdV-2) has been on the rise in China, posing a significant threat to the poultry industry. This study aimed to investigate the epidemiology, phylogenetic relationship, genomic characteristics, and pathogenicity of FAdV-2. The epidemiological analysis revealed the detection of multiple FAdV serotypes, including FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-8a, FAdV-8b, and FAdV-11 serotypes. Among them, FAdV-2 exhibited the highest proportion, accounting for 21.05% (8/38). The complete genomes of these 8 FAdV-2 strains were sequenced. Genetic evolution analysis indicated that these FAdV-2 strains formed a separate branch within the FAdV-D group, sharing 94.60 to 97.90% nucleotide similarity with the reference FAdV-2 and FAdV-11 strains. Notably, the recombination analysis revealed that 5 out of the 8 FAdV-2 strains, exhibited recombination events between FAdV-2 and FAdV-11. The recombination regions involved Hexon, Fiber, ORF19 genes and 3' end. Furthermore, pathogenicity experiments demonstrated that recombinant FAdV-2 XX strain is capable of inducing mortality rate of 66.70% and causing more severe hepatitis hydropericardium syndrome (HHS) in 6-wk-old specific-pathogen-free chickens. These findings contribute to our understanding of the prevalence, genomic characteristics, and the pathogenicity of FAdV-2, providing foundations for FAdV-2 vaccine development.

Natural variant R246K in hemagglutinin increased zoonotic characteristics and renal inflammation in mice infected with H9N2 influenza virus.

Considered a potential pandemic candidate, the widespread among poultry of H9N2 avian influenza viruses across Asia and North Africa pose an increasing threat to poultry and human health. The massive epidemic of H9N2 viruses has expanded the host range; however, the molecular basis and characteristic underlying the transmission to poultry and mammals remains unclear. Our previous study has proved that some natural mutations in the HA gene enhanced the binding ability of the H9N2 virus to α-2,6 SA receptors. Here, we systematically analyzed the impact of these natural mutations on zoonotic characteristics and the pathogenicity of H9N2 AIVs in poultry and mammals. Our study demonstrated that mutation R246K increased the replication in human lung epithelial cells in vitro. Mutation R246K increased the virus shedding of oropharyngeal swabs during early-stage infection in chickens. Moreover, mutation R246K displayed stronger pH stability and pathogenicity in mice. The strong renal tropism and inflammatory response may accelerate the pathogenicity. In summary, we found that natural variation R246K in HA of prevalent H9N2 in China promoted the transmissibility in chicken and accelerate the pathogenicity in mice, posing a great concern for zoonotic and pandemic emergence.

Chicken-origin Cluster 3.2 Tembusu virus exhibits higher infectivity than duck-origin Cluster 2 Tembusu virus in chicks.

In 2020, a chicken-origin Cluster 3 Tembusu virus (TMUV) caused outbreaks of a disease characterized by egg-drop syndrome in laying hens in China. In the present study, a TMUV strain, TMUV-GX, was isolated from tissue samples of laying hens with egg drop syndrome in south China. Phylogenetic analysis grouped TMUV-GX into TMUV Cluster 3.2, which was distinct from the prevalent TMUV Cluster 2 in duck flocks. To study the infectivity and pathogenicity of TMUV-GX in chickens and ducks, 7 day-old specific pathogen-free (SPF) chicks and SPF ducklings were infected with the same dose of the TMUV-GX. As a comparison, the duck-origin Cluster 2 strain, TMUV-JM, infection groups were set up in chicks and ducklings. Compared with the low infectivity and pathogenicity of TMUV-JM in chicks, the chicken-origin TMUV-GX displayed high replication competence in multiple tissues and caused tissues histopathological damage. In addition, the replication competence of TMUV-GX in ducklings was comparable to that of TMUV-JM. Our study revealed chicken-origin Cluster 3.2 TMUV exhibits high infectivity in chicks and ducklings, and suggested that chicken-origin Cluster 3.2 TMUV possesses a biological basis for widespread infection of chickens and ducks.

Global analysis of gene expression profiles and gout symptoms in goslings infected with goose astrovirus.

While blocking inflammation is an effective way to ease the symptoms of gout disease in humans, the treatment and prevention of gout in goslings infected with goose astrovirus (GAstV), a recently emergent condition, remain unclear. In this study, we investigated the reprogramming of the host genes as a result of GAstV infection by combining analysis of the global transcriptome and metabolic network pathways in the kidneys of goslings infected with GAstV. We showed that as GAstV replication increased in vivo, the regulation of key enzymes in the host metabolism progressively increased, flowing metabolites into the purine/pyrimidine biosynthesis pathways. Furthermore, we found that GAstV: 1) inhibits the host oxidation-reduction response by inhibiting the expression of the catalase gene; 2) activates the Toll-like receptor 2 pathway to enhance the immune inflammatory response; and 3) activates the key enzyme in lactic acid synthesis to produce lactate accumulation which inhibits the host's antiviral response, so as to facilitate the replication of the virus itself. This study provided the first insight into the overall metabolic requirements of GAstV for replication in vivo by combining transcriptome with metabolic network pathway information.

Cryo-EM structure of severe fever with thrombocytopenia syndrome virus.

The severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne human-infecting bunyavirus, which utilizes two envelope glycoproteins, Gn and Gc, to enter host cells. However, the structure and organization of these glycoproteins on virion surface are not yet known. Here we describe the structure of SFTSV determined by single particle reconstruction, which allows mechanistic insights into bunyavirus assembly at near-atomic resolution. The SFTSV Gn and Gc proteins exist as heterodimers and further assemble into pentameric and hexameric peplomers, shielding the Gc fusion loops by both intra- and inter-heterodimer interactions. Individual peplomers are associated mainly through the ectodomains, in which the highly conserved glycans on N914 of Gc play a crucial role. This elaborate assembly stabilizes Gc in the metastable prefusion conformation and creates some cryptic epitopes that are only accessible in the intermediate states during virus entry. These findings provide an important basis for developing vaccines and therapeutic drugs.

Identification and genomic analysis of two duck-origin Tembusu virus strains in southern China.

Egg-laying duck flocks in Guangdong province, southern China, have been suffering a widely spreading infectious disease with abrupt egg drops and death since the winter of 2010. However, the causative pathogen was not known. We obtained two independent virus isolates named FS and JM from the diseased layer duck flocks and identified them as duck-origin Tembusu virus by PCR detection, sequencing the entire length of the open reading frames (ORFs). The two isolates FS and JM shared high sequence similarity to the isolates of duck-origin Tembusu virus that was first emerged in eastern China in April 2010. Blast analysis shows that the whole ORF sequences of FS and JM have the highest similarity (>99 %) to BYD-1(the first reported duck-origin Tembusu virus) and JS804 (the first reported goose-origin Tembusu virus), indicating that the full-length genomes were highly conserved in waterfowl-origin Tembusu viruses. The present study suggests that duck-origin Tembusu viruses have spread fast from eastern China to southern China, causing widely spreading infections. The high conservation of duck-origin Tembusu virus strains provides the genomic basis for choosing the strains for vaccine preparation for better protection against this new virus infection.

Influence of the interatomic repulsive hardness on the microstructure and dynamics of CuZr metallic glasses.

The influence of the interatomic repulsive hardness (RH) on the microstructure and dynamics of CuZr metallic glass is studied by the molecular dynamics simulation method. By constructing potential energy functions that characterize different RH, we calculated their atomic diffusion coefficients, thermal expansion coefficients, radial distribution functions, fivefold symmetry order, and other related structural properties during the quenching process. We found that the atomic diffusion coefficients and thermal expansion coefficients decrease with RH, and the variation of radial distribution functions with temperatures becomes slower in softer RH. The softening RH is also accompanied by the enhancement of the icosahedral order in the liquids. Our results explain the experimental conclusion of "soft atoms make strong glasses" when considering the relation of the repulsive potential and liquid dynamics fragility.

Molecular dynamics simulation of minor Zr addition on short and medium-range orders of Cu-Zr metallic glass.

The compositional dependence of the atomic structure and glass-forming ability (GFA) was systematically studied in a binary alloy series Cu100-xZrx (x = 0.5, 1.0, 2.0, 3.0, 5.0, 7.0, 10.0) by molecular dynamics simulations. Several structural analysis techniques are adopted to find a direct relationship between the atomic structures and GFA by minor Zr addition. The simulation results confirm that the difference among the critical cooling rates proves the enhancement of GFA. It is found that the Zr addition can enhance the icosahedra short-range order (SRO). From another side, in terms of MRO, the addition of Zr can enhance interpenetrating icosahedra connection which will give rise to the Bergman-icosahedra medium-range order, resulting in a more stable, more compact, and more complex structures, which is responsible for the enhanced GFA in CuZr alloys. Furthermore, the five-fold symmetry governs the formation of the amorphous state and may behave as a principal indication of the formation of the glass state during the cooling process. We also found a critical Zr content of 3%, below which the effect of Zr on the structures is not obvious. However, when the Zr content is higher than 3%, the Zr can rapidly change the structures of the liquid and glassy structure. These results are helpful for understanding the GFA of CuZr alloys.

A Review of Emerging Goose Astrovirus Causing Gout.

In recent years, an infection in geese caused by goose astrovirus (GAstV) has repeatedly occurred in coastal areas of China and rapidly spread to inland provinces. The infection is characterized by joint and visceral gout and is fatal. The disease has caused huge economic losses to China's goose industry. GAstV is a nonenveloped, single-stranded, positive-sense RNA virus. As it is a novel virus, there is no specific classification. Here, we review the current understanding of GAstV. The virus structure, isolation, diagnosis and detection, innate immune regulation, and transmission route are discussed. In addition, since GAstV can cause gout in goslings, the possible role of GAstV in gout formation and uric acid metabolism is discussed. We hope that this review will inform researchers to rapidly develop effective methods to prevent and treat this disease.