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.

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.

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.

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.

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.

The first complete genome sequence and pathogenicity characterization of fowl adenovirus serotype 2 with inclusion body hepatitis and hydropericardium in China.

Since 2015, fowl adenovirus (FAdV) has been frequently reported worldwide, causing serious economic losses to the poultry industry. In this study, a FAdV-2, namely GX01, was isolated from liver samples of chickens with hepatitis and hydropericardium in Guangxi Province, China. The complete genome sequence of GX01 was determined about 43,663 base pairs (bp) with 53% G+C content. To our knowledge, this is the first FAdV-2 complete genome in China. There was a deleting fragment in ORF25 gene. Phylogenetic analysis based on the hexon loop-1 gene showed that GX01 is most closely related to FAdV-2 strain 685. Pathogenicity experiment of GX01 in 3-day-old and 10-day-old specific-pathogen-free chickens showed that although no mortality was observed within 21 days post infection (dpi), strain GX01 significantly inhibited weight gain of infected chickens. Moreover, FAdV-2 was still detectable in the anal swabs of infected chickens at 21 dpi. Necropsy analysis showed that the main lesions were observed in liver, heart, and spleen. Of note, hepatitis and hydropericardium were observed in the infected chickens. In addition, massive necrosis of lymphocyte was observed in spleen of infected 3-days-old chickens. We concluded that FAdV-2 strain GX01 is capable of causing hepatitis and hydropericardium, which will make serious impact on the growth of chickens. Our research lays a foundation to investigate the molecular epidemiology and etiology of FAdV.

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.

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 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.

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.

Identification of coronaviruses in farmed wild animals reveals their evolutionary origins in Guangdong, southern China.

Coronavirus infections cause diseases that range from mild to severe in mammals and birds. In this study, we detected coronavirus infections in 748 farmed wild animals of 23 species in Guangdong, southern China, by RT-PCR and metagenomic analysis. We identified four coronaviruses in these wild animals and analysed their evolutionary origins. Coronaviruses detected in Rhizomys sinensis were genetically grouped into canine and rodent coronaviruses, which were likely recombinants of canine and rodent coronaviruses. The coronavirus found in Phasianus colchicus was a recombinant pheasant coronavirus of turkey coronavirus and infectious bronchitis virus. The coronavirus in Paguma larvata had a high nucleotide identity (94.6-98.5 per cent) with a coronavirus of bottlenose dolphin (Tursiops truncates). These findings suggested that the wildlife coronaviruses may have experienced homologous recombination and/or crossed the species barrier, likely resulting in the emergence of new coronaviruses. It is necessary to reduce human-animal interactions by prohibiting the eating and raising of wild animals, which may contribute to preventing the emergence of the next coronavirus pandemic.

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.

Development of a Cleaved Probe-Based Loop-Mediated Isothermal Amplification Assay for Rapid Detection of African Swine Fever Virus.

African Swine Fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and lethal viral disease of pigs. However, commercial vaccines are not yet available, and neither are drugs to prevent or control ASF. Therefore, rapid, accurate on-site diagnosis is urgently needed for detection during the early stages of ASFV infection. Herein, a cleaved probe-based loop-mediated isothermal amplification (CP-LAMP) detection method was established. Based on the original primer sets, we targeted the ASFV 9GL gene sequence to design a probe harboring a ribonucleotide insertion. Ribonuclease H2 (RNase H2) enzyme activity can only be activated when the probe is perfectly complementary, resulting in hydrolytic release of a quencher moiety, and consequent signal amplification. The method displayed robust sensitivity, with copy number detection as low as 13 copies/µL within 40 min at constant temperature (62°C). Visualization of the fluorescence product was employed using a self-designed 3D-printed visualization function cassette, and the CP-LAMP method achieved specific identification and visual detection of ASFV. Moreover, coupling the dual function cassette and smartphone quantitation makes the CP-LAMP assay first user-friendly, cost-effective, portable, rapid, and accurate point-of-care testing (POCT) platform for ASFV.

First Identification and Genetic Characterization of a Novel Duck Astrovirus in Ducklings in China.

Four divergent groups of duck astroviruses (DAstVs) have been identified that infect domestic ducks. In March 2021, a fatal disease characterized by visceral urate deposition broke out in 5-day-old Beijing ducks on a commercial farm in Guangdong province, China. We identified a novel duck astrovirus from the ducklings suffering from gout disease. The complete genome sequence of this DAstV was obtained by virome sequencing and amplification. Phylogenetic analyses and pairwise comparisons demonstrated that this DAstV represented a novel group of avastrovirus. Thus, we designated this duck astrovirus as DAstV-5 JM strain. DAstV-5 JM shared genome sequence identities of 15-45% with other avastroviruses. Amino acid identities with proteins from other avastroviruses did not exceed 59% for ORF1a, 79% for ORF1b, and 60% for ORF2. The capsid region of JM shared genetic distances of 0.596 to 0.695 with the three official avastrovirus species. suggesting that JM could be classified as a novel genotype species in the Avastrovirus genus. Meanwhile, JM shares genetic distances of 0.402-0.662 with all the other known unassigned avastroviruses, revealing that it represents an additional unassigned avastrovirus. In summary, we determined that the DAstV-5 JM strain is a novel genotype species of avastrovirus.

Isolation and characterization of a new goose orthoreovirus causing liver and spleen focal necrosis in geese, China.

Since July 2020, an infectious disease characterized by liver and spleen white focal necrosis has been spreading widely through geese farms in many regions of China. A novel goose orthoreovirus (GRV), designated GRV-GD2020, was isolated from the liver and spleen of dead geese. Phylogenetic analysis and sequence comparison revealed that all the genes of GRV-GD2020 clustered with other waterfowl-origin orthoreovirus. However, the gene constellation of GRV-GD2020 was not similar to that of any particular strain. Instead, the genomic segments of GRV-GD2020 showed 27.5-97.3% similarities to that of other waterfowl-origin orthoreovirus isolates. Waterfowl-origin orthoreovirus infections characterized by liver and spleen focal necrosis had not emerged in recent years. The re-emergence of the disease may be related to the recombination of the genome segments of Muscovy duck reovirus (MDRV), GRV, and new-type duck orthoreovirus. In summary, we determined that the GRV-GD2020 strain, causing goose liver and spleen focal necrosis, is a new variant of waterfowl-origin orthoreovirus.

Isolation and partial genetic characterization of a new duck adenovirus in China.

A novel duck adenovirus, isolated from Jinding Ducks(Anas platyrhynchos domestica), was proposed to be duck adenovirus 4 (DAdV-4), extending the genus Aviadenovirus. In this study, we sequenced the central genome part from Iva2 gene to fiber gene of the DAdV-4 that is conserved in all adenovirus genera. Phylogenetic analysis and protease cleavage site analysis verified the classification of DAdV-4 in the genus Aviadenovirus. Nucleotide identity analysis showed low sequence identity between central genome part genes of DAdV-4 with that of other aviadenoviruses. The phylogenetic tree based on the full amino acid sequence of hexon and DNA polymerase showed that the DAdV-4 appeared on a relatively independent branch. Our analysis suggested that DAdV-4 is a distinct type and represents a novel species. Although DAdV-4 has not caused serious disease outbreaks among ducks yet, the virus should be considered as a potential threat to the poultry industry.

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.

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.

The truncated E protein of DTMUV provide protection in young ducks.

Duck Tembusu virus (DTMUV) is a major pathogen of duck industry in China. In the current study, we generated different constructs containing envelope (E) protein, pre-membrane-envelope (prM-E) protein, and C-terminally truncated E protein of the DTMUV. The constructed proteins could induce specific antibody responses in young ducks. When ducklings were immunized with the constructed proteins, they were 100% protected against DTMUV infection. Furthermore, the fluorescent signal of the truncated E protein was stronger than other constructed proteins, when Bac-to-Bac baculovirus expression system was applied. Our data demonstrated that the truncated E protein used in the current study could be applied as a potential vaccine candidate to control DTMUV infection in young ducks.