Molecular Evolution of Protein Sequences and Codon Usage in Monkeypox Viruses.

The monkeypox virus (mpox virus, MPXV) epidemic in 2022 has posed a significant public health risk. Yet, the evolutionary principles of MPXV remain largely unknown. Here, we examined the evolutionary patterns of protein sequences and codon usage in MPXV. We first demonstrated the signal of positive selection in OPG027, specifically in the Clade I lineage of MPXV. Subsequently, we discovered accelerated protein sequence evolution over time in the variants responsible for the 2022 outbreak. Furthermore, we showed strong epistasis between amino acid substitutions located in different genes. The codon adaptation index (CAI) analysis revealed that MPXV genes tended to use more non-preferred codons compared to human genes, and the CAI decreased over time and diverged between clades, with Clade I > IIa and IIb-A > IIb-B. While the decrease in fatality rate among the three groups aligned with the CAI pattern, it remains unclear whether this correlation was coincidental or if the deoptimization of codon usage in MPXV led to a reduction in fatality rates. This study sheds new light on the mechanisms that govern the evolution of MPXV in human populations.

Clinical and molecular epidemiology of enterovirus D68 from 2013 to 2020 in Shanghai.

Enterovirus D68 (EV-D68) is an emerging pathogen that has caused outbreaks of severe respiratory disease worldwide, especially in children. We aim to investigate the prevalence and genetic characteristics of EV-D68 in children from Shanghai. Nasopharyngeal swab or bronchoalveolar lavage fluid samples collected from children hospitalized with community-acquired pneumonia were screened for EV-D68. Nine of 3997 samples were EV-D68-positive. Seven of nine positive samples were sequenced and submitted to GenBank. Based on partial polyprotein gene (3D) or complete sequence analysis, we found the seven strains belong to different clades and subclades, including three D1 (detected in 2013 and 2014), one D2 (2013), one D3 (2019), and two B3 (2014 and 2018). Overall, we show different clades and subclades of EV-D68 spread with low positive rates (0.2%) among children in Shanghai between 2013 and 2020. Amino acid mutations were found in the epitopes of the VP1 BC and DE loops and C-terminus; similarity analysis provided evidence for recombination as an important mechanism of genomic diversification. Both single nucleotide mutations and recombination play a role in evolution of EV-D68. Genetic instability within these clinical strains may indicate large outbreaks could occur following cumulative mutations.

Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing.

The monkeypox virus (MPXV) has triggered a current outbreak globally. Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control. It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads, as it is one of the DNA viruses with the largest genome and the most AT-biased, and has a significant number of tandem repeats. Here we evaluated the performance of metagenomic and amplicon sequencing techniques, and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland. We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens. Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes. Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences. Besides, several intra-host single nucleotide variations were identified in the first imported mpox case. This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens. The findings of this study will significantly enhance the surveillance of MPXV.

Discovery and identification of a novel canine coronavirus causing a diarrhea outbreak in Vulpes.

Cross-species transmission of viruses from wildlife animal reservoirs, such as bats, poses a threat to human and domestic animal health. Previous studies have shown that domestic animals have important roles as intermediate hosts, enabling the transmission of genetically diverse coronaviruses from natural hosts to humans. Here, we report the identification and characterization of a novel canine coronavirus (VuCCoV), which caused an epidemic of acute diarrhea in Vulpes (foxes) in Shenyang, China. The epidemic started on November 8, 2019, and caused more than 39,600 deaths by January 1, 2022. Full-length viral genomic sequences were obtained from 15 foxes with diarrhea at the early stage of this outbreak. The VuCCoV genome shared more than 90% nucleotide identity with canine coronavirus (CCoV) for three of the four structural genes, with the S gene showing a larger amount of divergence. In addition, 67% (10/15) of the VuCCoV genomes contained an open reading frame (ORF3) gene, which was previously only detected in CCoV-I genomes. Notably, VuCCoV had only two to three amino acid differences at the partial RNA-dependent RNA polymerase (RdRp) level to bat CoV, suggesting a close genetic relationship. Therefore, these novel VuCCoV genomes represent a previously unsampled lineage of CCoVs. We also show that the VuCCoV spike protein binds to canine and fox aminopeptidase N (APN), which may allow this protein to serve as an entry receptor. In addition, cell lines were identified that are sensitive to VuCCoV using a pseudovirus system. These data highlight the importance of identifying the diversity and distribution of coronaviruses in domestic animals, which could mitigate future outbreaks that could threaten livestock, public health, and economic growth.

Detecting SARS-CoV-2 BA.2, BA.4, and BA.5 Variants Utilizing a Robust RT-RPA-CRISPR/Cas12a-Based Method - China, 2023.

Introduction: Since 2019, numerous variants of concern for severe acute respiratory syndrome virus 2 (SARS-CoV-2) have emerged, leading to significant outbreaks. The development of novel, highly accurate, and rapid detection techniques for these new SARS-CoV-2 variants remains a primary focus in the ongoing efforts to control and prevent the coronavirus disease 2019 (COVID-19) pandemic. Methods: Reverse transcription-recombinase polymerase amplification combined with the clustered regularly interspaced short palindromic repeats-associated protein 12a (CRISPR/Cas12a) system was used to validate the detection of the Omicron BA.2, BA.4, and BA.5 variants of SARS-CoV-2. Results: Our results demonstrate that the CRISPR/Cas12a assay is capable of effectively detecting the SARS-CoV-2 BA.2, BA.4, and BA.5 variants with a limit of detection of 10, 1, and 10 copies/µL, respectively. Importantly, our assay successfully differentiated the three SARS-CoV-2 Omicron strains from one another. Additionally, we evaluated 46 SARS-CoV-2 positive clinical samples consisting of BA.2 (n=20), BA.4 (n=6), and BA.5 (n=20) variants, and the sensitivity of our assay ranged from 90% to 100%, while the specificity was 100%. Discussion: This research presents a swift and reliable CRISPR-based method that may be employed to track the emergence of novel SARS-CoV-2 variants.

Isolation and Characterization of Monkeypox Virus from the First Case of Monkeypox - Chongqing Municipality, China, 2022.

Introduction: The first imported case of monkeypox (MPX) from the mainland of China was reported in September 2022. Herein, the study reports the isolation and characterization of MPX virus (MPXV) in this case. Methods: Clinical specimens including skin blister fluid, oropharyngeal and nasopharyngeal swabs, and blood were collected and inoculated onto Vero cells. The isolated virus was identified as MPXV using quantitative polymerase chain reaction (qPCR), cytopathic effects (CPEs), immunofluorescence assay (IFA) and transmission electron microscopy (TEM). Plaque assays were employed to quantify infectious plaque-forming units (PFUs). The plaque reduction neutralization test (PRNT) was developed to determine the neutralizing antibody (nAb) against MPXV. Results: MPXV replication was confirmed with qPCR. Typical CPEs were observed 48 h post-incubation. The isolated virus was named MPXV-B.1-China-C-Tan-CQ01. IFA showed that MPXV reacted with serum of MPX case. Orthopoxvirus morphology was observed using TEM. The virus titer increased to >106 PFU/mL after three passages. The serum PRNT 50% neutralization titer (NT50) was 35 for the MPX patient 6 days after symptom onset. Discussion: The study successfully isolated the first MPXV strain in the mainland of China, MPXV-B.1-China-C-Tan-CQ01. Infectious titration and PRNT methods have been developed. The study provides key resources and technical platforms for further research as well as anti-viral drug and vaccine development against MPX.

Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa, IIb, and I clades and the B.1 lineage.

An ongoing multi-country outbreak of monkeypox was reported in May 2022 with several deaths, affecting 107 countries of all six World Health Organization (WHO) regions. The WHO has declared the current monkeypox outbreak to be a Public Health Emergency of International Concern. It is, thus, necessary to rapidly and accurately detect and distinguish different monkeypox virus (MPXV) clades. We designed primers and probes based on the alignment of 138 complete genomes of poxviruses. In Panel 1, we mixed one pair of primers and three probes to detect and differentiate the MPXV Western Africa (IIa, IIb clade) and Congo Basin (I clade) and other orthopoxviruses. In Panel 2, we mixed one pair of primers and two probes to detect the 2022 MPXV (B.1 lineage and its descendant lineages). In addition, we tested the specificity and sensitivity of the assay using real-time PCR. In Panel 1, the assay reproducibly identified various concentrations of two plasmids of the monkeypox virus, whereas other orthopoxviruses did not cross-react. In Panel 2, the probe annealed well to MPXV B.1 and showed the expected linearity. These two multiple real-time assays are inclusive and highly specific for identifying different clades of MPXV.

Mycoplasma pneumoniae and Adenovirus Coinfection Cause Pediatric Severe Community-Acquired Pneumonia.

Consolidation is one complication of pediatric severe community-acquired pneumonia (SCAP) that can respond poorly to conservative medical treatment. We investigated the pathogens that cause pediatric SCAP including cases with persistent consolidation that need bronchoscopy intervention. Alveolar lavage fluid (ALF) samples collected from cases admitted to Children's Hospital of Fudan University with SCAP during January 2019 to March in 2019 were retrospectively tested by the RespiFinder 2SMART multiplex PCR (multi-PCR) assay targeting 22 respiratory pathogens. A total of 90 cases and 91 samples were enrolled; 80.0% (72/90) of the cases had pulmonary consolidation and/or atelectasis. All samples were positive with targeted pathogens tested by multi-PCR, and 92.3% (84/91) of the samples were co-detected with pathogens. Mycoplasma pneumoniae (MP) and adenovirus (ADV) as the two dominant pathogens, with the positive rates of 96.7% (88/91) and 79.1% (72/91), respectively. Most of the samples were positive with MP and ADV simultaneously. As a control, 78.0% (71/91) of the samples were positive by conventional tests (CT), in which MP had the detection rate of 63.9% (55/86) by a traditional real-time PCR assay, while ADV were positive in 13.1% (12/91) of the samples by a direct immunofluorescence assay (DFA). In cases with persistent pulmonary consolidation, the positive rates of pathogens by multi-PCR and CT were 100% (72/72) and 81.9% (59/72), respectively. There were no significant differences of MP or ADV positive rates between cases with and without pulmonary consolidation. MP and ADV most prevalent in pediatric SCAP cases required fiberscope intervention, and presented with coinfections dominantly. IMPORTANCE Pathogens that cause pediatric severe community-acquired pneumonia (SCAP) requiring bronchoscopy intervention are understudied. Through this study, we explore the etiology of SCAP form alveolar lavage fluid (ALF) samples by the RespiFinder 2SMART multi-PCR assay. It is observed that high mixed detection rates of Mycoplasma pneumoniae and adenovirus in ALF samples collected from hospitalized SCAP children experienced bronchoscopy intervention. Eighty percent of the cases had pulmonary consolidation and/or atelectasis. The presence of possible coinfection of these two pathogens might contribute to poor clinical anti-infection response. The results of this study might be helpful for the selection of clinical strategies for the empirical treatment of such pediatric SCAP cases.

Epidemiology of Viruses Causing Pediatric Community Acquired Pneumonia in Shanghai During 2010-2020: What Happened Before and After the COVID-19 Outbreak?

INTRODUCTION: Since the global outbreak of COVID-19, there has been a significant reduction in pediatric outpatient and emergency visits for infectious diseases. The purpose of this study was to analyze the changes in respiratory viruses in children with community-acquired pneumonia (CAP) in Shanghai in the past 10 years, especially in the first year after COVID-19. METHODS: We conducted a retrospective, observational study; the results for eight common respiratory viruses (respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1-3 (PIV), adenovirus (ADV) and human metapneumovirus) tested by direct fluorescent antibody assays in hospitalized CAP cases in Children's Hospital of Fudan University during 2010-2020 were analyzed. RESULTS: Of the 5544 hospitalized CAP patients included in this study, 20.2% (1125/5544) were positive for the eight respiratory viruses. The top three pathogens were RSV, PIV3 and ADV, detected from 9.8% (543/5544), 5.3% (294/5544) and 2.0% (111/5544) of the samples, respectively. RSV had the highest positive rates among children < 2 years old. In 2020, the detection rate of all viruses showed a sharp decline from February to August compared with the previous 9 years. When the Shanghai community reopened in August 2020, the detection rate of eight viruses rebounded significantly in September. CONCLUSIONS: These eight respiratory viruses, especially RSV and PIV, were important pathogens of CAP in Shanghai children in the past 10 years. The COVID-19 pandemic had a significant impact on the detection rates for eight respiratory viruses in children with CAP in Shanghai.

Discovery and Nanosized Preparations of (S,R)-Tylophorine Malate as Novel anti-SARS-CoV-2 Agents.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has stimulated the search for effective drugs for its prevention and treatment. Natural products are an important source for new drug discovery. Here, we report that, NK007(S,R), a tylophorine malate, displays high antiviral activity against SARS-CoV-2 with an EC50 0.03 µM in vitro, which is substantially lower than that of remdesivir (EC50: 0.8 µM in vitro), the only authorized drug to date. The histopathological research revealed that NK007(S,R) (5 mg/kg/dose) displayed a protection effect in lung injury induced by SARS-CoV-2, which is better than remdesivir (25 mg/kg/dose). We also prepared two nanosized preparations of NK007(S,R), which also showed good efficacy (EC50: NP-NK007, 0.007 µM in vitro; LP-NK007, 0.014 µM in vitro). Our findings suggest that tylophora alkaloids, isolated from the traditional Chinese medicine Cynanchum komarovii AL, offer a new skeleton for the development of anticoronavirus drug candidate.

Real-time reverse transcription-polymerase chain reaction assay panel for the detection of severe acute respiratory syndrome coronavirus 2 and its variants.

BACKGROUND: With the ongoing worldwide coronavirus disease 2019 (COVID-19) pandemic, an increasing number of viral variants are being identified, which poses a challenge for nucleic acid-based diagnostic tests. Rapid tests, such as real-time reverse transcription-polymerase chain reaction (rRT-PCR), play an important role in monitoring COVID-19 infection and controlling its spread. However, the changes in the genotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARS-CoV-2 detection over time. METHODS: We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2. We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern (VOCs). In addition, we also developed two rRT-PCR assays (S484K and S501Y) targeting the spike gene, which when combined with the open reading frames (ORF)1ab assay, respectively, to form duplex rRT-PCR assays, were able to detect SARS-CoV-2 VOCs (lineages B.1.351 and B.1.1.7). RESULTS: Using a SARS-CoV-2 stock with predetermined genomic copies as a standard, the detection limit of both assays targeting RdRp and N was five copies/reaction. Furthermore, no cross-reactions with six others human CoVs (229E, OC43, NL63, HKU1, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus) were observed using these assays. In addition, the S484K and S501Y assays were combined with the ORF1ab assay, respectively. CONCLUSIONS: Four rRT-PCR assays (RdRp, N, S484K, and S501Y) were used to detect SARS-CoV-2 variants, and these assays were shown to be effective in screening for multiple virus strains.

Etiology of Severe Pneumonia in Children in Alveolar Lavage Fluid Using a High-Throughput Gene Targeted Amplicon Sequencing Assay.

Objective: To evaluate the diagnostic value of a high-throughput gene targeted amplicon sequencing (TAS) assay for detecting pathogenic microorganisms in alveolar lavage fluid (ALF) from children with severe community-acquired pneumonia (SCAP). Methods: A retrospective study was performed on 48 frozen ALF samples from 47 severe pneumonia cases admitted to Children's Hospital of Fudan University from January 1, 2019, to March 31, 2019. All samples were tested by a multiplex PCR (Multi-PCR) assay and a TAS assay. The results of the TAS panels were parallel compared with Multi-PCR and Conventional Tests (CT) including culture, direct fluorescent antibody method (DFA), and singleplex polymerase chain reaction (PCR). Results: The proportion of pathogens detection by CT was 81.2% (39/48). The 8 common respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), influenza A virus (FLUA), influenza B virus (FLUB), parainfluenza virus 1-3 (PIV1-3), and human Metapneumovirus (hMPV) were found in 31.2% (15/48) of the 48 samples by DFA. With the criteria of CT results used as "Golden Standard" for determing of TAS results, the proportion of pathogens detection by TAS was 70.8% (34/48). The difference of proportion of pathogens detection between TAS and CT was not statistically significant (p = 0.232). The sensitivity and specificity of TAS for pathogens detection based on CT were 87.1% (95% CI, 71.77-95.18%) and 100.0% (95% CI, 62.88-100%), the positive predictive value (PPV) and negative predictive value (NPV) were 100.0% (95% CI, 87.35-100%) and 64.2% (95% CI, 35.62-86.02%), respectively. While Multi-PCR results were used as "Golden Standard," the total pathogens detection rate of TAS was 83.3% (40/48), which had a significant difference with that of Multi-PCR (p = 0.003). The sensitivity and PPV of TAS compared with Multi-PCR were 83.3% (95% CI, 69.23-92.03%) and 100.0% (95% CI, 89.08-100%), respectively. High rates of co-infection were proved by CT, Multi-PCR, and TAS. Mycoplasma pneumoniae (MP) and ADV were the two most frequently detected pathogens in all three assays. Conclusion: Compared with the CT and Multi-PCR methods, this TAS assay had a good performance in detecting bacteriological and viral pathogens from ALF. More research is needed to establish interpretation criteria based on TAS reads or analysis platforms.

Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro.

COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CLpro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CLpro activity in vitro with IC50's of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC50's of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CLpro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CLpro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.

Mutations, Recombination and Insertion in the Evolution of 2019-nCoV.

Background: The 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. Methods: We 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. Results: Systematic 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. Conclusions: Our 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 Summary: Novel 2019-nCoV sequences revealed the evolution and specificity of betacoronavirus with possible mechanisms of enhanced infectivity.

In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) first broke out in 2019 and subsequently spread worldwide. Chloroquine has been sporadically used in treating SARS-CoV-2 infection. Hydroxychloroquine shares the same mechanism of action as chloroquine, but its more tolerable safety profile makes it the preferred drug to treat malaria and autoimmune conditions. We propose that the immunomodulatory effect of hydroxychloroquine also may be useful in controlling the cytokine storm that occurs late phase in critically ill patients with SARS-CoV-2. Currently, there is no evidence to support the use of hydroxychloroquine in SARS-CoV-2 infection. METHODS: The pharmacological activity of chloroquine and hydroxychloroquine was tested using SARS-CoV-2-infected Vero cells. Physiologically based pharmacokinetic (PBPK) models were implemented for both drugs separately by integrating their in vitro data. Using the PBPK models, hydroxychloroquine concentrations in lung fluid were simulated under 5 different dosing regimens to explore the most effective regimen while considering the drug's safety profile. RESULTS: Hydroxychloroquine (EC50 = 0.72 µM) was found to be more potent than chloroquine (EC50 = 5.47 µM) in vitro. Based on PBPK models results, a loading dose of 400 mg twice daily of hydroxychloroquine sulfate given orally, followed by a maintenance dose of 200 mg given twice daily for 4 days is recommended for SARS-CoV-2 infection, as it reached 3 times the potency of chloroquine phosphate when given 500 mg twice daily 5 days in advance. CONCLUSIONS: Hydroxychloroquine was found to be more potent than chloroquine to inhibit SARS-CoV-2 in vitro.