Genetic characteristics of classical astroviruses in Shenzhen, China, 2016-2019.

Human astrovirus (HAstV) is a single-stranded, positive-sense RNA virus and is the leading cause of viral gastroenteritis. However, despite its prevalence, astroviruses still remain one of the least studied enteroviruses. In this study, we sequenced 11 classical astrovirus strains from clinical samples collected in Shenzhen, China from 2016 to 2019, analyzed their genetic characteristics, and deposited them into GenBank. We conducted phylogenetic analysis using IQ-TREE software, with references to astrovirus sequences worldwide. The phylogeographic analysis was performed using the Bayesian Evolutionary Analysis Sampling Trees program, through Bayesian Markov Chain Monte Carlo sampling. We also conducted recombination analysis with the Recombination Detection Program. The newly sequenced strains were categorized as HAstV genotype 1, which is the predominant genotype in Shenzhen. Phylogeographic reconstruction indicated that HAstV-1 may have migrated from the United States to China, followed by frequent transmission between China and Japan. The recombination analysis revealed recombination events within and across genotypes, and identified a recombination-prone region that produced relatively uniform recombination breakpoints and fragment lengths. The genetic analysis of HAstV strains in Shenzhen addresses the current lack of astrovirus data in the region of Shenzhen and provides key insights to the evolution and transmission of astroviruses worldwide. These findings highlight the importance of improving surveillance of astroviruses.

CRISPR/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform.

Respiratory viruses have long been a major cause of a global pandemic, emphasizing the urgent need for high-sensitivity diagnostic tools. Typical PCR technology can only determine the type of virus in the sample, which is unable to detect different variants of the same virus without costly and time-consuming gene sequencing. Here, we introduce a simple, fully enclosed, and highly integrated microfluidic system based on CRISPR/Cas12a and isothermal amplification techniques (LOC-CRISPR) that can specifically identify multiple common respiratory viruses and their variants. The LOC-CRISPR chip integrates viral nucleic acid extraction, recombinant polymerase amplification, and CRISPR/Cas12a cleavage reaction-based detection, contamination-free detection. In addition, the LOC-CRISPR chip was designed for multiplexed detection (two-sample input and ten-result outputs), which can not only detect the presence of SARS-CoV-2, H1N1, H3N2, IVB and HRSV but also differentiate the BA.1, BA.2, and BA.5 variants of SARS-COV-2. For clinical validation, the LOC-CRISPR chip was used to analyze 50 nasopharyngeal swab samples (44 positive and 6 negative) and achieved excellent sensitivity (97.8%) and specificity (100%). This innovative LOC-CRISPR system has the ability to quickly, sensitively, and accurately detect multiple target nucleic acid sequences with single-base mutations, which will further improve the rapid identification and traceability of respiratory viruses infectious diseases.

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.

Metabolic alterations upon SARS-CoV-2 infection and potential therapeutic targets against coronavirus infection.

The coronavirus disease 2019 (COVID-19) caused by coronavirus SARS-CoV-2 infection has become a global pandemic due to the high viral transmissibility and pathogenesis, bringing enormous burden to our society. Most patients infected by SARS-CoV-2 are asymptomatic or have mild symptoms. Although only a small proportion of patients progressed to severe COVID-19 with symptoms including acute respiratory distress syndrome (ARDS), disseminated coagulopathy, and cardiovascular disorders, severe COVID-19 is accompanied by high mortality rates with near 7 million deaths. Nowadays, effective therapeutic patterns for severe COVID-19 are still lacking. It has been extensively reported that host metabolism plays essential roles in various physiological processes during virus infection. Many viruses manipulate host metabolism to avoid immunity, facilitate their own replication, or to initiate pathological response. Targeting the interaction between SARS-CoV-2 and host metabolism holds promise for developing therapeutic strategies. In this review, we summarize and discuss recent studies dedicated to uncovering the role of host metabolism during the life cycle of SARS-CoV-2 in aspects of entry, replication, assembly, and pathogenesis with an emphasis on glucose metabolism and lipid metabolism. Microbiota and long COVID-19 are also discussed. Ultimately, we recapitulate metabolism-modulating drugs repurposed for COVID-19 including statins, ASM inhibitors, NSAIDs, Montelukast, omega-3 fatty acids, 2-DG, and metformin.

CRISPR-Cas13a-powered electrochemical biosensor for the detection of the L452R mutation in clinical samples of SARS-CoV-2 variants.

Since the end of 2019, a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has deprived numerous lives worldwide, called COVID-19. Up to date, omicron is the latest variant of concern, and BA.5 is replacing the BA.2 variant to become the main subtype rampaging worldwide. These subtypes harbor an L452R mutation, which increases their transmissibility among vaccinated people. Current methods for identifying SARS-CoV-2 variants are mainly based on polymerase chain reaction (PCR) followed by gene sequencing, making time-consuming processes and expensive instrumentation indispensable. In this study, we developed a rapid and ultrasensitive electrochemical biosensor to achieve the goals of high sensitivity, the ability of distinguishing the variants, and the direct detection of RNAs from viruses simultaneously. We used electrodes made of MXene-AuNP (gold nanoparticle) composites for improved sensitivity and the CRISPR/Cas13a system for high specificity in detecting the single-base L452R mutation in RNAs and clinical samples. Our biosensor will be an excellent supplement to the RT-qPCR method enabling the early diagnosis and quick distinguishment of SARS-CoV-2 Omicron BA.5 and BA.2 variants and more potential variants that might arise in the future.

Temporal dynamics of SARS-CoV-2 genome mutations that occurred in vivo on an aircraft.

We analyzed variations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome during a flight-related cluster outbreak of coronavirus disease 2019 (COVID-19) in Shenzhen, China, to explore the characteristics of SARS-CoV-2 transmission and intra-host single nucleotide variations (iSNVs) in a confined space. Thirty-three patients with COVID-19 were sampled, and 14 were resampled 3-31 days later. All 47 nasopharyngeal swabs were deep-sequenced. iSNVs and similarities in the consensus genome sequence were analyzed. Three SARS-CoV-2 variants of concern, Delta (n = 31), Beta (n = 1), and C.1.2 (n = 1), were detected among the 33 patients. The viral genome sequences from 30 Delta-positive patients had similar SNVs; 14 of these patients provided two successive samples. Overall, the 47 sequenced genomes contained 164 iSNVs. Of the 14 paired (successive) samples, the second samples (T2) contained more iSNVs (median: 3; 95% confidence interval [95% CI]: 2.77-10.22) than did the first samples (T1; median: 2; 95% CI: 1.63-3.74; Wilcoxon test, P = 0.021). 38 iSNVs were detected in T1 samples, and only seven were also detectable in T2 samples. Notably, T2 samples from two of the 14 paired samples had additional mutations than the T1 samples. The iSNVs of the SARS-CoV-2 genome exhibited rapid dynamic changes during a flight-related cluster outbreak event. Intra-host diversity increased gradually with time, and new site mutations occurred in vivo without a population transmission bottleneck. Therefore, we could not determine the generational relationship from the mutation site changes alone.

Relationship between blood amyloid A and resting magnetic resonance functional brain connections in patients with obstructive sleep apnea-hypopnea syndrome.

OBJECTIVE: The aim of this study was to analyze the relationship between serum amyloid A (SAA) concentrations in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS) and their magnetic resonance imaging (MRI) of resting brain function. METHODS: Male patients with OSAHS were enrolled from January to June 2019 in Suzhou Ninth People's Hospital Affiliated to Soochow University, and nineteen healthy male volunteers were selected as the normal control group. The patients with OSAHS were divided into mild, moderate, and severe groups according to their apnea-hypopnea index (AHI). Early in the morning after the polysomnography (PSG), blood samples were collected and serum levels of serum amyloid A (SAA) were measured by enzyme-linked immunosorbent assay. All subjects were scored by the Auditory Verbal Learning Test (AVLT) scale. Resting brain function images of healthy male volunteers and patients in the severe group were collected by 3.0 T magnetic resonance scanner. SPSS25.0 software was used for statistical analysis. RESULTS: The SAA of the OSAHS group (n = 43) were higher than those of control group (n = 19). The scores of AVLT-immediate and AVLT-delay in the severe OSAHS group were lower than those in the control group (P < 0.05), and it was negatively correlated with SAA. In the severe OSAHS group, the rest state Function Connection (rsFC) in temporal lobe, marginal lobe, and frontal lobe was lower than that in the control group (P < 0.05) and was significantly negatively correlated with SAA. The rsFC in bilateral parietal lobes was higher than that in the control group (P < 0.05), was significantly positively correlated with SAA, and was negatively correlated with AVLT-delay. CONCLUSIONS: The significant increase in SAA concentration in patients with OSAHS correlated with brain rsFC intensity, providing a reference role for the diagnosis, treatment, and prognosis of cognitive dysfunction in patients with OSAHS.

Preliminary study on brain resting-state networks and cognitive impairments of patients with obstructive sleep apnea-hypopnea syndrome.

BACKGROUND: To investigate functional changes in brain resting-state networks (RSNs) in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS) and their correlations with sleep breathing disorders and neurocognitive performance. METHODS: In this study, 18 OSAHS patients and 18 matched healthy controls underwent neurocognitive assessment and magnetic resonance imaging (MRI). Group-level independent component analysis (ICA) and statistical analyses were used to explore between-group differences in RSNs and the relationship between functional changes in RSNs, sleep breathing disorders and neurocognitive performance. RESULTS: The OSAHS patients performed worse on neuropsychological tests than the healthy controls. Eight RSNs were identified, and between-group analyses showed that OSAHS patients displayed significantly decreased functional connectivity in the bilateral posterior cingulate gyri (PCC) within the default mode network (DMN), the right middle frontal gyrus (MFG) within the dorsal attention network (DAN), and the left superior temporal gyrus (STG) within the ventral attention network (VAN), and increased functional connectivity in the right superior frontal gyrus (SFG) within the salience network (SN). Further correlation analyses revealed that the average ICA z-scores in the bilateral PCC were correlated with sleep breathing disorders. CONCLUSIONS: Our findings demonstrate that the DMN, SN, DAN, and VAN are impaired during the resting state and are associated with decreased functionally distinct aspects of cognition in patients with OSAHS. Moreover, the intermittent hypoxia and sleep fragmentation caused by OSAHS are likely to be the main influencing factors.

A CRISPR/Cas12a-empowered surface plasmon resonance platform for rapid and specific diagnosis of the Omicron variant of SARS-CoV-2.

The outbreak of the COVID-19 pandemic was partially due to the challenge of identifying asymptomatic and presymptomatic carriers of the virus, and thus highlights a strong motivation for diagnostics with high sensitivity that can be rapidly deployed. On the other hand, several concerning SARS-CoV-2 variants, including Omicron, are required to be identified as soon as the samples are identified as 'positive'. Unfortunately, a traditional PCR test does not allow their specific identification. Herein, for the first time, we have developed MOPCS (Methodologies of Photonic CRISPR Sensing), which combines an optical sensing technology-surface plasmon resonance (SPR) with the 'gene scissors' clustered regularly interspaced short palindromic repeat (CRISPR) technique to achieve both high sensitivity and specificity when it comes to measurement of viral variants. MOPCS is a low-cost, CRISPR/Cas12a-system-empowered SPR gene-detecting platform that can analyze viral RNA, without the need for amplification, within 38 min from sample input to results output, and achieve a limit of detection of 15 fM. MOPCS achieves a highly sensitive analysis of SARS-CoV-2, and mutations appear in variants B.1.617.2 (Delta), B.1.1.529 (Omicron) and BA.1 (a subtype of Omicron). This platform was also used to analyze some recently collected patient samples from a local outbreak in China, identified by the Centers for Disease Control and Prevention. This innovative CRISPR-empowered SPR platform will further contribute to the fast, sensitive and accurate detection of target nucleic acid sequences with single-base mutations.

CRISPR-Cas12a-Empowered Electrochemical Biosensor for Rapid and Ultrasensitive Detection of SARS-CoV-2 Delta Variant.

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The gold standard method for the diagnosis of SARS-CoV-2 depends on quantitative reverse transcription-polymerase chain reaction till now, which is time-consuming and requires expensive instrumentation, and the confirmation of variants relies on further sequencing techniques. Herein, we first proposed a robust technique-methodology of electrochemical CRISPR sensing with the advantages of rapid, highly sensitivity and specificity for the detection of SARS-CoV-2 variant. To enhance the sensing capability, gold electrodes are uniformly decorated with electro-deposited gold nanoparticles. Using DNA template identical to SARS-CoV-2 Delta spike gene sequence as model, our biosensor exhibits excellent analytical detection limit (50 fM) and high linearity (R2 = 0.987) over six orders of magnitude dynamic range from 100 fM to 10 nM without any nucleic-acid-amplification assays. The detection can be completed within 1 h with high stability and specificity which benefits from the CRISPR-Cas system. Furthermore, based on the wireless micro-electrochemical platform, the proposed biosensor reveals promising application ability in point-of-care testing.

Possible recombination between two variants of concern in a COVID-19 patient.

We identified an individual who was coinfected with two SARS-CoV-2 variants of concern, the Beta and Delta variants. The ratio of the relative abundance between the two variants was maintained at 1:9 (Beta:Delta) in 14 days. Furthermore, possible evidence of recombinations in the Orf1ab and Spike genes was found.

Ten emerging SARS-CoV-2 spike variants exhibit variable infectivity, animal tropism, and antibody neutralization.

Emerging mutations in SARS-CoV-2 cause several waves of COVID-19 pandemic. Here we investigate the infectivity and antigenicity of ten emerging SARS-CoV-2 variants-B.1.1.298, B.1.1.7(Alpha), B.1.351(Beta), P.1(Gamma), P.2(Zeta), B.1.429(Epsilon), B.1.525(Eta), B.1.526-1(Iota), B.1.526-2(Iota), B.1.1.318-and seven corresponding single amino acid mutations in the receptor-binding domain using SARS-CoV-2 pseudovirus. The results indicate that the pseudovirus of most of the SARS-CoV-2 variants (except B.1.1.298) display slightly increased infectivity in human and monkey cell lines, especially B.1.351, B.1.525 and B.1.526 in Calu-3 cells. The K417N/T, N501Y, or E484K-carrying variants exhibit significantly increased abilities to infect mouse ACE2-overexpressing cells. The activities of furin, TMPRSS2, and cathepsin L are increased against most of the variants. RBD amino acid mutations comprising K417T/N, L452R, Y453F, S477N, E484K, and N501Y cause significant immune escape from 11 of 13 monoclonal antibodies. However, the resistance to neutralization by convalescent serum or vaccines elicited serum is mainly caused by the E484K mutation. The convalescent serum from B.1.1.7- and B.1.351-infected patients neutralized the variants themselves better than other SARS-CoV-2 variants. Our study provides insights regarding therapeutic antibodies and vaccines, and highlights the importance of E484K mutation.

Norovirus GII.2[P16] strain in Shenzhen, China: a retrospective study.

BACKGROUND: Norovirus (NoV) is the main cause of non-bacterial acute gastroenteritis (AGE) outbreaks worldwide. From September 2015 through August 2018, 203 NoV outbreaks involving 2500 cases were reported to the Shenzhen Center for Disease Control and Prevention. METHODS: Faecal specimens for 203 outbreaks were collected and epidemiological data were obtained through the AGE outbreak surveillance system in Shenzhen. Genotypes were determined by sequencing analysis. To gain a better understanding of the evolutionary characteristics of NoV in Shenzhen, molecular evolution and mutations were evaluated based on time-scale evolutionary phylogeny and amino acid mutations. RESULTS: A total of nine districts reported NoV outbreaks and the reported NoV outbreaks peaked from November to March. Among the 203 NoV outbreaks, 150 were sequenced successfully. Most of these outbreaks were associated with the NoV GII.2[P16] strain (45.3%, 92/203) and occurred in school settings (91.6%, 186/203). The evolutionary rates of the RdRp region and the VP1 sequence were 2.1 × 10-3 (95% HPD interval, 1.7 × 10-3-2.5 × 10-3) substitutions/site/year and 2.7 × 10-3 (95% HPD interval, 2.4 × 10-3-3.1 × 10-3) substitutions/site/year, respectively. The common ancestors of the GII.2[P16] strain from Shenzhen and GII.4 Sydney 2012[P16] diverged from 2011 to 2012. The common ancestors of the GII.2[P16] strain from Shenzhen and previous GII.2[P16] (2010-2012) diverged from 2003 to 2004. The results of amino acid mutations showed 6 amino acid substitutions (*77E, R750K, P845Q, H1310Y, K1546Q, T1549A) were found only in GII.4 Sydney 2012[P16] and the GII.2[P16] recombinant strain. CONCLUSIONS: This study illustrates the molecular epidemiological patterns in Shenzhen, China, from September 2015 to August 2018 and provides evidence that the epidemic trend of GII.2[P16] recombinant strain had weakened and the non-structural proteins of the recombinant strain might have played a more significant role than VP1.

Molecular Epidemiological Characteristics of Gastroenteritis Outbreaks Caused by Norovirus GII.4 Sydney [P31] Strains - China, October 2016-December 2020.

INTRODUCTION: Human noroviruses are the leading cause of acute viral gastroenteritis (AGE) worldwide in all age groups. GII.4 strains have been the predominant genotype circulating globally over the last 2 decades and since 2012. GII.4 Sydney viruses have emerged and caused the majority of AGE outbreaks worldwide. METHODS: Data from norovirus outbreaks from the laboratory-based surveillance of norovirus outbreaks in China (CaliciNet China) between October 2016-December 2020 were analyzed. RESULTS: During October 2016-December 2020, 1,954 norovirus outbreaks were reported, and positive fecal samples from 1,352 (69.19%) outbreaks were genotyped. GII.4 Sydney [P31] viruses accounted for 2.1% (October 2016-August 2017), 5.5% (September 2017-August 2018), 3.3% (September 2018-August 2018), 26.6% (September 2019-August 2020), and and 1.1% (September 2020-December 2020) of GII outbreaks, respectively. Compared to reference strains of GII.4 Sydney [P31] from 2012 to 2013, 7 amino acid mutations in epitopes[A (297, 372 and 373), B (333), E (414), and H (309 and 310)] and 1 in human histo-blood group antigens binding site at site II 372 were found by analyzing 9 GII.4 Sydney [P31] complete genomic sequences. CONCLUSIONS: This report identified the genomic variation of GII.4 Sydney [P31] from CaliciNet China. Continued surveillance with prompt genotyping and genetic analysis is necessary to monitor the emergence of novel GII.4 variants.