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Since the outbreak of SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, the viral genome has acquired numerous mutations with the potential to increase transmission. One year after its emergence, we now further analyze emergent SARS-CoV-2 genome sequences in an effort to understand the evolution of this virus.

Accessory proteins play important roles in the interaction between coronaviruses and their hosts. Accordingly, a comprehensive study of the compositional diversity and evolutionary patterns of accessory proteins is critical to understanding the host adaptation and epidemic variation of coronaviruses. Here, we developed a standardized genome annotation tool for coronavirus (CoroAnnoter) by combining open reading frame prediction, transcription regulatory sequence recognition and homologous alignment. Using CoroAnnoter, we annotated 39 representative coronavirus strains to form a compositional profile for all of the accessary proteins. Large variations were observed in the number of accessory proteins of 1–10 for different coronaviruses, with SARS-CoV-2 and SARS-CoV having the most (9 and 10, respectively). The variation between SARS-CoV and SARS-CoV-2 accessory proteins could be traced back to related coronaviruses in other hosts. The genomic distribution of accessory proteins had significant intra-genus conservation and inter-genus diversity and could be grouped into 1, 4, 2 and 1 types for alpha-, beta-, gamma-, and delta-coronaviruses, respectively. Evolutionary analysis suggested that accessory proteins are more conservative locating before the N-terminal of proteins E and M (E-M), while they are more diverse after these proteins. Furthermore, comparison of virus-host interaction networks of SARS-CoV-2 and SARS-CoV accessory proteins showed that they share multiple antiviral signaling pathways, those involved in the apoptotic process, viral life cycle and response to oxidative stress. In summary, our study provides a tool for coronavirus genome annotation and builds a comprehensive profile for coronavirus accessory proteins covering their composition, classification, evolutionary pattern and host interaction.

West Nile virus (WNV) is an important neurotropic flavivirus that is widely distributed globally. WNV strain XJ11129 was first isolated in Xinjiang, China, and its genetic and biological characteristics remain largely unknown. In this study, phylogenetic and sequence analyses revealed that XJ11129 belongs to lineage 1a and shares high genetic identity with the highly pathogenic strain NY99. Then, the full-length genomic cDNA of XJ11129 was amplified and assembled using a modified Gibson assembly (GA) method. The virus (named rXJ11129) was successfully rescued in days following this method. Compared with other wild-type WNV isolates, rXJ11129 exhibited virulence indistinguishable from that of the NY99 strain in vivo. In summary, the genomic and virulence phenotypes of rXJ11129 were characterized in vivo and in vitro, and these data will improve the understanding of the spread and pathogenesis of this reemerging virus. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-020-00246-x) contains supplementary material, which is available to authorized users.

Recently, patients co-infected by two SARS-CoV-2 lineages have been sporadically reported. Concerns are raised because previous studies have demonstrated co-infection may contribute to the recombination of RNA viruses and cause severe clinic symptoms. In this study, we have estimated the compositional lineage(s), tendentiousness, and frequency of co-infection events in population from a large-scale genomic analysis for SARS-CoV-2 patients. SARS-CoV-2 lineage(s) infected in each sample have been recognized from the assignment of within-host site variations into lineage-defined feature variations by introducing a hypergeometric distribution method. Of all the 29,993 samples, 53 (~0.18%) co-infection events have been identified. Apart from 52 co-infections with two SARS-CoV-2 lineages, one sample with co-infections of three SARS-CoV-2 lineages was firstly identified. As expected, the co-infection events mainly happened in the regions where have co-existed more than two dominant SARS-CoV-2 lineages. However, co-infection of two sub-lineages in Delta lineage were detected as well. Our results provide a useful reference framework for the high throughput detecting of SARS-CoV-2 co-infection events in the Next Generation Sequencing (NGS) data. Although low in average rate, the co-infection events showed an increasing tendency with the increased diversity of SARS-CoV-2. And considering the large base of SARS-CoV-2 infections globally, co-infected patients would be a nonnegligible population. Thus, more clinical research is urgently needed on these patients.

Neuroblastoma (NB) is a rare type of cancer but frequently occurred in children. However, it is still unclear whether circular RNAs (circRNAs) play key roles in NB tumorigenesis or progression. In this study, we identified 39,022 circRNAs across the 39 neuroblastoma and 2 normal cell lines. With the gene and circRNA expression data, we classified the NB cell lines, identified and characterized the functional circRNAs in the 3 NB classes. Specifically, 29 circRNAs were found to be dysregulated in the NB classes. Notably, 7 circRNAs located within MYCN-amplified regions were upregulated in cell lines with the high activities of MYC targets and MYCN amplification, and were highly correlated with expression of their parental gene, NBAS. Subsequently, we constructed ceRNA networks for the functional circRNAs. Specifically, hsa_circ_0005379 was identified as a critical regulator in the ceRNA networks because of targeting 13 genes, which formed a complex competing endogenous RNA (ceRNA) network. Moreover, hsa_circ_0002343, which was connected with few genes, might regulate the PI3K/Akt/mTOR signaling via RAC1. Furthermore, 3 genes, including NOTCH2, SERPINH1, and LAMC1, involved in epithelial mesenchymal transition (EMT) were observed to connect with hsa_circ_0001361, suggesting that this circRNA was closely associated with EMT. Consequently, 7 genes, such as DAD1, PPIA, NOTCH2, PGK1, BUB1, EIF2S1, and TCF7L2, were found to be closely associated with both event-free survival (EFS) and overall survival (OS). In conclusion, the present study identified functional circRNAs and predicted their functionality in neuroblastoma cell lines, which not only improved the understanding of circRNAs in neuroblastoma, but also provided the evidences for the related researchers.

Infection with live-attenuated vaccines always inevitably induces side effects that reduce their safety. This study suggests a concept of magnetic virus produced by genetically modifying viral surfaces with Fe3 O4 nanoparticles (NPs) to control their tropisms. An iron-affinity peptide is designed to be displayed on the viral surface protein (VP1) of human enterovirus type 71 (EV71), a typical nonenveloped picornavirus, as the model. The modified EV71 can self-bind with Fe3 O4 NPs under physiological conditions, resulting in novel EV71-Fe3 O4 hybrid materials. This rationally engineered EV71 with Fe3 O4 retains its original biological infectivity, but its tropism can be precisely controlled by magnetism. Both in vitro and in vivo experiments demonstrate that EV71-Fe3 O4 can infect only a desired area within the limit of the applied magnetic field, which effectively reduces its pathological damage. More importantly, this characteristic of EV71 can be inherited due to the gene-induced coassembly of viruses and NPs. This achievement provides a proof of concept in virus vaccine improvement by a combination of gene modification and material incorporation, leading to great potential for biomedical developments.

BackgroundThe 2019 novel coronavirus (2019-nCoV or SARS-CoV-2) has spread more rapidly than any other betacoronavirus including SARS-CoV and MERS-CoV. However, the mechanisms responsible for infection and molecular evolution of this virus remained unclear.MethodsWe collected and analyzed 120 genomic sequences of 2019-nCoV including 11 novel genomes from patients in China. Through comprehensive analysis of the available genome sequences of 2019-nCoV strains, we have tracked multiple inheritable SNPs and determined the evolution of 2019-nCoV relative to other coronaviruses.ResultsSystematic analysis of 120 genomic sequences of 2019-nCoV revealed co-circulation of two genetic subgroups with distinct SNPs markers, which can be used to trace the 2019-nCoV spreading pathways to different regions and countries. Although 2019-nCoV, human and bat SARS-CoV share high homologous in overall genome structures, they evolved into two distinct groups with different receptor entry specificities through potential recombination in the receptor binding regions. In addition, 2019-nCoV has a unique four amino acid insertion between S1 and S2 domains of the spike protein, which created a potential furin or TMPRSS2 cleavage site.ConclusionsOur studies provided comprehensive insights into the evolution and spread of the 2019-nCoV. Our results provided evidence suggesting that 2019-nCoV may increase its infectivity through the receptor binding domain recombination and a cleavage site insertion.One Sentence SummaryNovel 2019-nCoV sequences revealed the evolution and specificity of betacoronavirus with possible mechanisms of enhanced infectivity.

Although the circulation of avian influenza viruses in humans is limited, they can be transmitted from Aves (birds) to humans, representing a great challenge. Herein, we suggest that influenza viruses from Aves might exist in a mineralized state owing to the high calcium concentrations in the avian intestine. Using two typical influenza viruses as examples, we demonstrate that these viruses can self-mineralize in simulated avian intestinal fluid, resulting in egg-like virus-mineral structured composites. The mineralized viruses are more robust, with enhanced infectivity and thermostability. More importantly, the mineral exterior of mineralized viruses can alter their cell internalization, expanding the possible tropisms. The discovery of a mineralized state of influenza viruses highlights the integration of nanomaterials and viruses in the environment, which provides a new understanding of avian influenza infection and its control.

Conventional therapeutic monoclonal antibodies (mAbs) are invalid for intracellular viruses but by using in situ biomineralization treatment, they can be successfully delivered into cells to inhibit intracellular viral replication. This achievement significantly expands the applications of mAbs and provides a new intracellular strategy to control viral infections.

Hydroquinone (HQ) is produced commercially from benzene by multi-step Hock-type processes with equivalent amounts of acetone as side-product. We describe an efficient biocatalytic alternative using the cytochrome P450-BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi-rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one-pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450-BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti-inflammatory and anti-bacterial activities.

Respiratory Pathogen 13 Detection Kit (13× kit) is able to simultaneously detect 11 respiratory viruses, Mycoplasma pneumoniae (MP) and Chlamydia in a single reaction. Using 572 Nasopharyngeal aspirates collected from hospitalized children, the clinical performance of 13× kit for detecting 11 respiratory viruses was evaluated in comparison with a routinely used 2-tube multiplex reverse transcription PCR assay (2-tube assay) at provincial Centers for Disease Control and Prevention in China. The clinical performance of 13× kit for detecting MP and Chlamydia was evaluated by commercial real-time quantitative PCR (qPCR) kits or sequencing. For tested viruses, the assay concordance was 95.98% and the kappa coefficient was 0.89. All the MP and Chlamydia positive samples detected by 13× kit were confirmed as true positives. The utilization of the 13× kit in clinical settings will be helpful for doctors to assess clinical outcome according to virus type or multiple infections, and to limit the use of antibiotics., Highlights • A 13× kit allowed for simultaneous detection of 11 respiratory viruses, MP and Ch. • The detection rate of 13× kit was comparable to that of 2-tube assay (kappa >0.75). • A 13× kit has high sensitivity (97.41%) and specificity (91.89%). • MP and Chlamydia detected by 13× kit were verified by qPCR/sequencing as true positives.

Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.

Heat-lability is a key roadblock that strangles the widespread applications of many biological products. In nature, archaeal and extremophilic organisms utilize amorphous silica as a protective biomineral and exhibit considerable thermal tolerance. Here we present a bioinspired approach to generate thermostable virus by introducing an artificial hydrated silica exterior on individual virion. Similar to thermophiles, silicified viruses can survive longer at high temperature than their wild-type relatives. Virus inactivation assays showed that silica hydration exterior of the modified virus effectively prolonged infectivity of viruses by ∼ 10-fold at room temperature, achieving a similar result as that obtained by storing native ones at 4 °C. Mechanistic studies indicate that amorphous silica nanoclusters stabilize the inner virion structure by forming a layer that restricts molecular mobility, acting as physiochemical nanoanchors. Notably, we further evaluate the potential application of this biomimetic strategy in stabilizing clinically approved vaccine, and the silicified polio vaccine that can retain 90% potency after the storage at room temperature for 35 days was generated by this biosilicification approach and validated with in vivo experiments. This approach not only biomimetically connects inorganic material and living virus but also provides an innovative resolution to improve the thermal stability of biological agents using nanomaterials.