Identification of novel bat coronaviruses sheds light on the evolutionary origins of SARS-CoV-2 and related viruses.

Despite the discovery of animal coronaviruses related to SARS-CoV-2, the evolutionary origins of this virus are elusive. We describe a meta-transcriptomic study of 411 bat samples collected from a small geographical region in Yunnan province, China, between May 2019 and November 2020. We identified 24 full-length coronavirus genomes, including four novel SARS-CoV-2-related and three SARS-CoV-related viruses. Rhinolophus pusillus virus RpYN06 was the closest relative of SARS-CoV-2 in most of the genome, although it possessed a more divergent spike gene. The other three SARS-CoV-2-related coronaviruses carried a genetically distinct spike gene that could weakly bind to the hACE2 receptor in vitro. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species, with the largest contiguous hotspots extending from South Laos and Vietnam to southern China. Our study highlights the remarkable diversity of bat coronaviruses at the local scale, including close relatives of both SARS-CoV-2 and SARS-CoV.

Surveillance of SARS-CoV-2 at the Huanan Seafood Market.

SARS-CoV-2, the causative agent of COVID-19, emerged in December 2019. Its origins remain uncertain. It has been reported that a number of the early human cases had a history of contact with the Huanan Seafood Market. Here we present the results of surveillance for SARS-CoV-2 within the market. From January 1st 2020, after closure of the market, 923 samples were collected from the environment. From 18th January, 457 samples were collected from 18 species of animals, comprising of unsold contents of refrigerators and freezers, swabs from stray animals, and the contents of a fish tank. Using RT-qPCR, SARS-CoV-2 was detected in 73 environmental samples, but none of the animal samples. Three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.99% to 100% with the human isolate HCoV-19/Wuhan/IVDC-HB-01/2019. SARS-CoV-2 lineage A (8782T and 28144C) was found in an environmental sample. RNA-seq analysis of SARS-CoV-2 positive and negative environmental samples showed an abundance of different vertebrate genera at the market. In summary, this study provides information about the distribution and prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stages of the COVID-19 outbreak.

The emergence, genomic diversity and global spread of SARS-CoV-2.

Since the first cases of COVID-19 were documented in Wuhan, China in 2019, the world has witnessed a devastating global pandemic, with more than 238 million cases, nearly 5 million fatalities and the daily number of people infected increasing rapidly. Here we describe the currently available data on the emergence of the SARS-CoV-2 virus, the causative agent of COVID-19, outline the early viral spread in Wuhan and its transmission patterns in China and across the rest of the world, and highlight how genomic surveillance, together with other data such as those on human mobility, has helped to trace the spread and genetic variation of the virus and has also comprised a key element for the control of the pandemic. We pay particular attention to characterizing and describing the international spread of the major variants of concern of SARS-CoV-2 that were first identified in late 2020 and demonstrate that virus evolution has entered a new phase. More broadly, we highlight our currently limited understanding of coronavirus diversity in nature, the rapid spread of the virus and its variants in such an increasingly connected world, the reduced protection of vaccines, and the urgent need for coordinated global surveillance using genomic techniques. In summary, we provide important information for the prevention and control of both the ongoing COVID-19 pandemic and any new diseases that will inevitably emerge in the human population in future generations.

Isolation and characterization of a novel rodent hepevirus in long-tailed dwarf hamsters (Cricetulus longicaudatus) in China.

Hepeviruses have been identified in a broad range of animal hosts, including mammals, birds, and fish. In this study, rodents (n=91) from seven different species and ten pikas (Ochotona curzoniae) were collected in Qinghai Province, China. Using transcriptomic sequencing and confirmatory molecular testing, hepeviruses were detected in 27 of 45 (60 %) long-tailed dwarf hamsters (Cricetulus longicaudatus) and were undetected in other rodents and pika. The complete genome sequences from 14 representative strains were subsequently obtained, and phylogenetic analyses suggested that they represent a novel species within the genus Rocahepevirus, which we tentatively designated as Cl-2018QH. The virus was successfully isolated in human hepatoma (Huh-7) and murine fibroblast (17 Cl-1) cell lines, though both exhibited limited replication as assayed by detection of negative-sense RNA intermediates. A129 immunodeficient mice were inoculated with Cl-2018QH and the virus was consistently detected in multiple organs, despite relatively low viral loads. In summary, this study has described a novel rodent hepevirus, which enhances our knowledge of the genetic diversity of rodent hepeviruses and highlights its potential for cross-species transmission.

Combination of IL-33 with PD-1 blockade augment mILC2s-mediated anti-tumor immunity.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) represent one of the main tissue-specific innate lymphoid cell populations, which are key drivers of cytokine secretion in their occupational niche. However, the precise involvement of ILC2s in cancer immunity and their potential impact on immunotherapeutic approaches remain poorly understood. METHODS: The proportion of ILC2s originating from various tissue sources were quantified through flow cytometry, along with the determination of CD4+ T cell and CD8+ T cell percentages. Flow cytometry was also employed to assess IFN-γ production and programmed cell death protein-1 (PD-1) expression in T cells. Immunohistochemistry was utilized to detect IL-33 expression in tumor tissues, while immunofluorescence was employed to confirm the infiltration of ILC2s in both murine and human tumor tissues. RESULTS: In this study, we provide evidence that intra-tumoral ILC2s in lung adenocarcinoma (LUAD) exist in a quiescent state. However, the activation of intra-tumoral ILC2s is induced by IL-33 specifically in a natural ILC2s (nILC2, ST2+KLRG1-) phenotype. Considering the pivotal role of PD-1 in cancer immunotherapy and its immunoregulatory functions, we investigated the synergistic effects of IL-33 and anti-PD-1 and found that their combination enhances anti-tumor immunity and improves the efficacy of immunotherapy. Moreover, this combination leads to the upregulation of activated mature ILC2s (mILC2, ST2+KLRG1+) phenotype, thereby highlighting the activated ILC2s as a novel enhancer of the immunoregulatory properties of anti-PD-1. CONCLUSIONS: Collectively, these findings underscore the significance of ILC2s and their contribution to the anti-tumor response in the context of cancer immunotherapy. Consequently, the simultaneous targeting of ILC2s and T cells represents a potentially promising and widely applicable strategy for immunotherapeutic interventions.

Ubiquitin E3 ligase SPOP is a host negative regulator of enterovirus 71-encoded 2A protease.

IMPORTANCE: EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2Apro, which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response.

Hypoxia promotes metastasis by relieving miR-598-3p-restricted glycolysis in gastric cancer.

The activation of glycolysis, particularly in the context of reprogrammed energy metabolism, is increasingly recognized as a significant characteristic of cancer. However, the precise mechanisms by which glycolysis is promoted in metastatic gastric cancer cells under normal oxygen conditions remain poorly understood. MicroRNAs (miRNAs) play a crucial role in the development of malignant phenotypes in gastric cancer. Nevertheless, our understanding of the specific involvement of miRNAs in hypoxia-induced metabolic shifting and the subsequent metastatic processes is limited. Hypoxia-induced downregulation of miR-598-3p mechanistically leads to the upregulation of RMP and IGF1r, thereby promoting glycolysis. Either overexpression of miR-598-3p or R406 treatment effectively suppresses the metastasis of gastric cancer cells both in vitro and in vivo. Collectively, the depletion of miR-598-3p alters glucose metabolism from oxidative phosphorylation to glycolysis, thereby exacerbating the malignancy of gastric cancer cells. The present findings indicate a potential target for the development of therapeutics against gastric cancers with increased miR-598-3p expression.

High salt activates p97 to reduce host antiviral immunity by restricting Viperin induction.

High-salt diets have recently been implicated in hypertension, cardiovascular disease, and autoimmune disease. However, whether and how dietary salt affects host antiviral response remain elusive. Here, we report that high salt induces an instant reduction in host antiviral immunity, although this effect is compromised during a long-term high-salt diet. Further studies reveal that high salt stimulates the acetylation at Lys663 of p97, which promotes the recruitment of ubiquitinated proteins for proteasome-dependent degradation. p97-mediated degradation of the deubiquitinase USP33 results in a deficiency of Viperin protein expression during viral infection, which substantially attenuates host antiviral ability. Importantly, switching to a low-salt diet during viral infection significantly enhances Viperin expression and improves host antiviral ability. These findings uncover dietary salt-induced regulation of ubiquitinated cellular proteins and host antiviral immunity, and could offer insight into the daily consumption of salt-containing diets during virus epidemics.

Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin.

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.

Fog Density Analysis Based on the Alignment of an Airport Video and Visibility Data.

The density of fog is directly related to visibility and is one of the decision-making criteria for airport flight management and highway traffic management. Estimating fog density based on images and videos has been a popular research topic in recent years. However, the fog density estimated results based on images should be further evaluated and analyzed by combining weather information from other sensors. The data obtained by different sensors often need to be aligned in terms of time because of the difference in acquisition methods. In this paper, we propose a video and a visibility data alignment method based on temporal consistency for data alignment. After data alignment, the fog density estimation results based on images and videos can be analyzed, and the incorrect estimation results can be efficiently detected and corrected. The experimental results show that the new method effectively combines videos and visibility for fog density estimation.

Novel Avian Influenza Virus (H5N1) Clade 2.3.4.4b Reassortants in Migratory Birds, China.

Two novel reassortant highly pathogenic avian influenza viruses (H5N1) clade 2.3.4.4b.2 were identified in dead migratory birds in China in November 2021. The viruses probably evolved among wild birds through different flyways connecting Europe and Asia. Their low antigenic reaction to vaccine antiserum indicates high risks to poultry and to public health.

A Novel Coronavirus from Patients with Pneumonia in China, 2019.

In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.).

Pathogenicity and Structural Basis of Zika Variants with Glycan Loop Deletions in the Envelope Protein.

The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.

Bioinformatics resources for SARS-CoV-2 discovery and surveillance.

In early January 2020, the novel coronavirus (SARS-CoV-2) responsible for a pneumonia outbreak in Wuhan, China, was identified using next-generation sequencing (NGS) and readily available bioinformatics pipelines. In addition to virus discovery, these NGS technologies and bioinformatics resources are currently being employed for ongoing genomic surveillance of SARS-CoV-2 worldwide, tracking its spread, evolution and patterns of variation on a global scale. In this review, we summarize the bioinformatics resources used for the discovery and surveillance of SARS-CoV-2. We also discuss the advantages and disadvantages of these bioinformatics resources and highlight areas where additional technical developments are urgently needed. Solutions to these problems will be beneficial not only to the prevention and control of the current COVID-19 pandemic but also to infectious disease outbreaks of the future.

Assessing the role of live poultry trade in community-structured transmission of avian influenza in China.

The live poultry trade is thought to play an important role in the spread and maintenance of highly pathogenic avian influenza A viruses (HP AIVs) in Asia. Despite an abundance of small-scale observational studies, the role of the poultry trade in disseminating AIV over large geographic areas is still unclear, especially for developing countries with complex poultry production systems. Here we combine virus genomes and reconstructed poultry transportation data to measure and compare the spatial spread in China of three key subtypes of AIV: H5N1, H7N9, and H5N6. Although it is difficult to disentangle the contribution of confounding factors, such as bird migration and spatial distance, we find evidence that the dissemination of these subtypes among domestic poultry is geographically continuous and likely associated with the intensity of the live poultry trade in China. Using two independent data sources and network analysis methods, we report a regional-scale community structure in China that might explain the spread of AIV subtypes in the country. The identification of this structure has the potential to inform more targeted strategies for the prevention and control of AIV in China.

Temporal Proteomic and Phosphoproteomic Analysis of EV-A71-Infected Human Cells.

Human enterovirus A71 (EV-A71), a member of the Picornaviridae family, is one of the main etiological viruses that lead to hand, foot, and mouth disease (HFMD). We utilized a multiplex tandem mass tag-based quantitative proteomic technique to monitor the alternation of the whole cell proteome and phosphoproteome of human rhabdomyosarcoma cells over the course of EV-A71 infection. We successfully quantified more than 7000 host proteins and 17,000 phosphosites, of which 80 proteins and nearly 1700 phosphosites were significantly regulated upon viral infection. We found that Myc proto-oncogene protein level decreased significantly, benefiting EV-A71 replication. Multiple signaling pathways were regulated in phosphorylation events that converge for protein translation, cell cycle control, and cell survival. Numerous host factors targeted by virus proteins are phosphoproteins. These factors are involved in host translational initiation, unfolded protein response, endoplasmic reticulum stress, and stress granule formation, and their phosphorylation may play key roles in the virus life cycle. Notably, we identified three conserved phosphorylation sites on viral polyproteins that have not been previously reported. Our study provides valuable resources for a systematic understanding of the interaction between the host cells and the EV-A71 at the protein and the post-translational level.

One-Year Sustained Cellular and Humoral Immunities in Coronavirus Disease 2019 (COVID-19) Convalescents.

BACKGROUND: The longitudinal antigen-specific immunity in COVID-19 convalescents is crucial for long-term protection upon individual re-exposure to SARS-CoV-2, and even more pivotal for ultimately achieving population-level immunity. We conducted this cohort study to better understand the features of immune memory in individuals with different disease severities at 1 year post-disease onset. METHODS: We conducted a systematic antigen-specific immune evaluation in 101 COVID-19 convalescents, who had asymptomatic, mild, moderate, or severe disease, through 2 visits at months 6 and 12 after disease onset. The SARS-CoV-2-specific antibodies, comprising neutralizing antibody (NAb), immunoglobulin (Ig) G, and IgM, were assessed by mutually corroborated assays (ie, neutralization, enzyme-linked immunosorbent assay [ELISA], and microparticle chemiluminescence immunoassay [MCLIA]). Meanwhile, T-cell memory against SARS-CoV-2 spike, membrane, and nucleocapsid proteins was tested through enzyme-linked immunospot assay (ELISpot), intracellular cytokine staining, and tetramer staining-based flow cytometry, respectively. RESULTS: SARS-CoV-2-specific IgG antibodies, and NAb, can persist among >95% of COVID-19 convalescents from 6 to 12 months after disease onset. At least 19/71 (26%) of COVID-19 convalescents (double positive in ELISA and MCLIA) had detectable circulating IgM antibody against SARS-CoV-2 at 12 months post-disease onset. Notably, numbers of convalescents with positive SARS-CoV-2-specific T-cell responses (≥1 of the SARS-CoV-2 antigen S1, S2, M, and N proteins) were 71/76 (93%) and 67/73 (92%) at 6 and 12 months, respectively. Furthermore, both antibody and T-cell memory levels in the convalescents were positively associated with disease severity. CONCLUSIONS: SARS-CoV-2-specific cellular and humoral immunities are durable at least until 1 year after disease onset.

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding.

BACKGROUND: In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. METHODS: We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus. FINDINGS: The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues. INTERPRETATION: 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. FUNDING: National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone.

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

Long noncoding RNAs: Novel regulators of virus-host interactions.

Long noncoding RNAs (lncRNAs) represent a key class of cellular regulators, involved in the modulation and control of multiple biological processes. Distinct classes of lncRNAs are now known to be induced by host cytokines following viral infections. Current evidence demonstrates that lncRNAs play essential roles at the host-pathogen interface regulating viral infections by either innate immune responses at various levels including activation of pathogen recognition receptors or by epigenetic, transcriptional, and posttranscriptional effects. We review the newly described mechanisms underlying the interactions between lncRNAs, cytokines, and metabolites differentially expressed following viral infections; we highlight the regulatory networks of host antiviral responses and emphasize the need for interdisciplinary research between lncRNA biology and immunology to deepen understanding of viral pathogenesis.