Metatranscriptomic data mining together with microfluidic card uncovered the potential pathogens and seasonal RNA viral ecology in a drinking water source.

AIMS: Despite metatranscriptomics becoming an emerging tool for pathogen surveillance, very little is known about the feasibility of this approach for understanding the fate of human-derived pathogens in drinking water sources. METHODS AND RESULTS: We conducted multiplexed microfluidic cards and metatranscriptomic sequencing of the drinking water source in a border city of North Korea in four seasons. Microfluidic card detected norovirus, hepatitis B virus (HBV), enterovirus, and Vibrio cholerae in the water. Phylogenetic analyses showed that environmental-derived sequences from norovirus GII.17, genotype C of HBV, and coxsackievirus A6 (CA6) were genetically related to the local clinical isolates. Meanwhile, metatranscriptomic assembly suggested that several bacterial pathogens, including Acinetobacter johnsonii and V. cholerae might be prevalent in the studied region. Metatranscriptomic analysis recovered 349 species-level groups with substantial viral diversity without detection of norovirus, HBV, and CA6. Seasonally distinct virus communities were also found. Specifically, 126, 73, 126, and 457 types of viruses were identified in spring, summer, autumn, and winter, respectively. The viromes were dominated by the Pisuviricota phylum, including members from Marnaviridae, Dicistroviridae, Luteoviridae, Potyviridae, Picornaviridae, Astroviridae, and Picobirnaviridae families. Further phylogenetic analyses of RNA (Ribonucleic Acid)-dependent RNA polymerase (RdRp) sequences showed a diverse set of picorna-like viruses associated with shellfish, of which several novel picorna-like viruses were also identified. Additionally, potential animal pathogens, including infectious bronchitis virus, Bat dicibavirus, Bat nodavirus, Bat picornavirus 2, infectious bursal disease virus, and Macrobrachium rosenbergii nodavirus were also identified. CONCLUSIONS: Our data illustrate the divergence between microfluidic cards and metatranscriptomics, highlighting that the combination of both methods facilitates the source tracking of human viruses in challenging settings without sufficient clinical surveillance.

Identifying Japanese Encephalitis Virus Using Metatranscriptomic Sequencing, Xinjiang, China.

The treat of infectious disease epidemics has increased the critical need for continuous broad-ranging surveillance of pathogens with outbreak potential. Using metatranscriptomic sequencing of blood samples, we identified several cases of Japanese encephalitis virus infection from Xinjiang Uyghur Autonomous Region, China. This discovery highlights the risk for known viral diseases even in nonendemic areas.

Multi-Omics Analysis Reveals the Nitrogen Removal Mechanism Induced by Electron Flow during the Start-up of the Anammox-Centered Process.

Significant progress in understanding the key enzymes or species of anammox has been made; however, the nitrogen removal mechanism in complex coupling systems centered on anammox remains limited. In this study, by the combination of metagenomics-metatranscriptomics analyses, the nitrogen removal in the anammox-centered coupling system that entails partial denitrification (PD) and hydrolytic acidification (HA, A-PDHA) was elucidated to be the nitrogen transformation driven by the electron generation-transport-consumption process. The results showed that a total nitrogen (TN) removal efficiency of >98%, with a TN effluence of <1 mg/L and a TN removal contribution via anammox of >98%, was achieved after 59 days under famine operation and alkaline conditions during the start-up process. Further investigation confirmed that famine operation promoted the activity of genes responsible for electron generation in anammox, and increased the abundance or expression of genes related to electron consumption. Alkaline conditions enhanced the electron generation for PD by upregulating the activity of glyceraldehyde 3-phosphate dehydrogenase and strengthened electron transfer by increasing the gene encoding quinone pool. Altogether, these variations in the electron flow led to efficient nitrogen removal. These results improve our understanding of the nitrogen removal mechanism and application of the anammox-centered coupling systems in treating nitrogen wastewater.

Response mechanism of microbial community during anaerobic biotransformation of marine toxin domoic acid.

Domoic acid (DA) is a potent neurotoxin produced by toxigenic Pseudo-nitzschia blooms and quickly transfers to the benthic anaerobic environment by marine snow particles. DA anaerobic biotransformation is driven by microbial interactions, in which trace amounts of DA can cause physiological stress in marine microorganisms. However, the underlying response mechanisms of microbial community to DA stress remain unclear. In this study, we utilized an anaerobic marine DA-degrading consortium GLY (using glycine as co-substrate) to systematically investigate the global response mechanisms of microbial community during DA anaerobic biotransformation.16S rRNA gene sequencing and metatranscriptomic analyses were applied to measure microbial community structure, function and metabolic responses. Results showed that DA stress markedly changed the composition of main species, with increased levels of Firmicutes and decreased levels of Proteobacteria, Cyanobacteria, Bacteroidetes and Actinobacteria. Several genera of tolerated bacteria (Bacillus and Solibacillus) were increased, while, Stenotrophomonas, Sphingomonas and Acinetobacter were decreased. Metatranscriptomic analyses indicated that DA stimulated the expression of quorum sensing, extracellular polymeric substance (EPS) production, sporulation, membrane transporters, bacterial chemotaxis, flagellar assembly and ribosome protection in community, promoting bacterial adaptation ability under DA stress. Moreover, amino acid metabolism, carbohydrate metabolism and lipid metabolism were modulated during DA anaerobic biotransformation to reduce metabolic burden, increase metabolic demands for EPS production and DA degradation. This study provides the new insights into response of microbial community to DA stress and its potential impact on benthic microorganisms in marine environments.

Characterisation of a Novel Insect-Specific Virus Discovered in Rice Thrips, Haplothrips aculeatus.

Insects constitute the largest proportion of animals on Earth and act as significant reservoirs and vectors in disease transmission. Rice thrips (Haplothrips aculeatus, family Phlaeothripidae) are one of the most common pests in agriculture. In this study, the full genome sequence of a novel Ollusvirus, provisionally named "Rice thrips ollusvirus 1" (RTOV1), was elucidated using transcriptome sequencing and the rapid amplification of cDNA ends (RACE). A homology search and phylogenetic tree analysis revealed that the newly identified virus is a member of the family Aliusviridae (order Jingchuvirales). The genome of RTOV1 contains four predicted open reading frames (ORFs), including a polymerase protein (L, 7590 nt), a glycoprotein (G, 4206 nt), a nucleocapsid protein (N, 2415 nt) and a small protein of unknown function (291 nt). All of the ORFs are encoded by the complementary genome, suggesting that the virus is a negative-stranded RNA virus. Phylogenetic analysis using polymerase sequences suggested that RTOV1 was closely related to ollusvirus 1. Deep small RNA sequencing analysis reveals a significant accumulation of small RNAs derived from RTOV1, indicating that the virus replicated in the insect. According to our understanding, this is the first report of an Ollusvirus identified in a member of the insect family Phlaeothripidae. The characterisation and discovery of RTOV1 is a significant contribution to the understanding of Ollusvirus diversity in insects.

An atlas of the blood virome in healthy individuals.

Emerging evidence indicates that gut virome plays a role in human health and disease, however, much less is known about the viral communities in blood. Here we conducted a direct metatranscriptomic sequencing of virus-like-particles in blood from 1200 healthy individuals, without prior amplification to avoid potential amplification bias and with a strictly bioinformatic and manual check for candidate viral reads to reduce false-positive matches. We identified 55 different viruses from 36 viral families, including 24 human DNA, RNA and retroviruses in 70% of the studied pools. The study showed that anelloviruses are widely distributed and dominate the blood virome in healthy individuals. Human herpesviruses and pegivirus-1 are commonly prevalent in asymptomatic humans. We identified the prevalence of RNA viruses often causing acute infection, like HEV, HPIV, RSV and HCoV-HKU1, revealing of a transmissible risk of asymptomatic infection. Several viruses possible related to transfusion safety were identified, including human Merkel cell polyomavirus, papillomavirus, parvovirus B19 and herpesvirus 8 in addition to HBV. In addition, phages in Caudovirales and Microviridae, were commonly found in pools of samples with a very low abundance; a few sequences for invertebrate, plant and giant viruses were found in some of individuals; however, the remaining 31 viruses mostly reflect extensive contamination from commercial reagents and the work environments. In conclusion, this study is the first comprehensive investigation of blood virome in healthy individuals by metatranscriptomic sequencing of VLP in China. Further investigation of potential false positives representing a major challenge for the identification of novel viruses in mNGS, will offer a systemic idea and means to reveal true viral infections of human.

Skin infectome of patients with a tick bite history.

Introduction: Ticks are the most important obligate blood-feeding vectors of human pathogens. With the advance of high-throughput sequencing, more and more bacterial community and virome in tick has been reported, which seems to pose a great threat to people. Methods: A total of 14 skin specimens collected from tick-bite patients with mild to severe symptoms were analyzed through meta-transcriptomic sequencings. Results: Four bacteria genera were both detected in the skins and ticks, including Pseudomonas, Acinetobacter, Corynebacterium and Propionibacterium, and three tick-associated viruses, Jingmen tick virus (JMTV), Bole tick virus 4 (BLTV4) and Deer tick mononegavirales-like virus (DTMV) were identified in the skin samples. Except of known pathogens such as pathogenic rickettsia, Coxiella burnetii and JMTV, we suggest Roseomonas cervicalis and BLTV4 as potential new agents amplified in the skins and then disseminated into the blood. As early as 1 day after a tick-bite, these pathogens can transmit to skins and at most four ones can co-infect in skins. Discussion: Advances in sequencing technologies have revealed that the diversity of tick microbiome and virome goes far beyond our previous understanding. This report not only identifies three new potential pathogens in humans but also shows that the skin barrier is vital in preventing horizontal transmissions of tick-associated bacteria or virus communities to the host. It is the first research on patients' skin infectome after a tick bite and demonstrates that more attention should be paid to the cutaneous response to prevent tick-borne illness.

Effect of chlorine disinfectant influx on biological sewage treatment process under the COVID-19 pandemic: Performance, mechanisms and implications.

Since the onset of the COVID-19 Pandemic, large amounts of chlorine-containing disinfectants have been used to interrupt the spread of SARS-CoV-2 and residual chlorine eventually entered the hospital or municipal sewage treatment facilities. However, little is known about the effect of chlorine influx on the biological sewage treatment process. Here we investigated the effect of chlorine on the microbiome and the mechanism of microbial chlorine resistance in the activated sludge of the aerobic treatment process, using metagenomic and metatranscriptomic sequencing. We found that chlorine could negatively impact the aerobic treatment performance regarding nitrogen/COD removal with a dose-dependent effect, and the dual effects of chlorine dose and interaction time differentiated the microbial community in activated sludge. The decline of nitrogen/COD removal was attributed to the compressed activity of functional microorganisms, such as the ammonia oxidation bacteria, under chlorinated conditions, and the damage cannot be recovered in a short term. In addition, some microorganisms could survive in chlorinated conditions by up-regulating the chlorine resistance genes (CRGs) expression (approximately 1.5 times) and stimulating new CRGs expression. In particular, species Acinetobacter johnsonii could resist high concentrations of chlorine through various mechanisms, especially the overexpression of efflux pump function encoded by qac genes play a key role. Based on these results, considering the persistence of the epidemic and extensive use of chlorine disinfectants, it cannot be ignored that large amounts of residual chlorine are entering the biological treatment facility, and strictly de-chlorination measures or microbial chlorine resistance regulations before entering should be implemented.

Metatranscriptomic analysis revealed Prevotella as a potential biomarker of oropharyngeal microbiomes in SARS-CoV-2 infection.

Background and objectives: Disease severity and prognosis of coronavirus disease 2019 (COVID-19) disease with other viral infections can be affected by the oropharyngeal microbiome. However, limited research had been carried out to uncover how these diseases are differentially affected by the oropharyngeal microbiome of the patient. Here, we aimed to explore the characteristics of the oropharyngeal microbiota of COVID-19 patients and compare them with those of patients with similar symptoms. Methods: COVID-19 was diagnosed in patients through the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Characterization of the oropharyngeal microbiome was performed by metatranscriptomic sequencing analyses of oropharyngeal swab specimens from 144 COVID-19 patients, 100 patients infected with other viruses, and 40 healthy volunteers. Results: The oropharyngeal microbiome diversity in patients with SARS-CoV-2 infection was different from that of patients with other infections. Prevotella and Aspergillus could play a role in the differentiation between patients with SARS-CoV-2 infection and patients with other infections. Prevotella could also influence the prognosis of COVID-19 through a mechanism that potentially involved the sphingolipid metabolism regulation pathway. Conclusion: The oropharyngeal microbiome characterization was different between SARS-CoV-2 infection and infections caused by other viruses. Prevotella could act as a biomarker for COVID-19 diagnosis and of host immune response evaluation in SARS-CoV-2 infection. In addition, the cross-talk among Prevotella, SARS-CoV-2, and sphingolipid metabolism pathways could provide a basis for the precise diagnosis, prevention, control, and treatment of COVID-19.

Aerobic starvation treatment of activated sludge enhances the degradation efficiency of refractory organic compounds.

Many refractory organic compounds (ROCs) in wastewater are toxic to human and aquatic organisms. Here, we reported an aerobic starvation approach to improve the degradation efficiencies of ROCs in activated sludge systems. The highest degradation rates of bisphenol AF (BPAF) (11.4 mg/g VSS · h) and gabapentin (GBP) (8.9 mg/g VSS · h) were achieved on the second day of the starvation process. While, the degradation rate of bisphenol A (BPA) on the 43rd day reached the maximum value of 0.8 mg/g VSS ·h, which was significantly higher than that of the seeding sludge (0.01 mg/g VSS · h). To investigate the mechanisms of this finding, we applied magnetic-nanoparticle mediated isolation, 16S rRNA gene sequencing, metagenomic sequencing and metatranscriptomic sequencing to analyze the microbial community structures and functions during the starvation process. The results showed that the increase of the BPA degradation ability was caused by the increase of the relative abundance of BPA degrading bacteria (Sphingomonas, Achromobacter, etc.), while, the enhancement of BPAF and GBP degradation was attributed to the increase of the expression of ROC degrading genes. Overall, these results improve our understanding of the microbial ecology of starved activated sludge and provide useful information for the future development of ROC removal technologies.

The Characterization of Three Novel Insect-Specific Viruses Discovered in the Bean Bug, Riptortus pedestris.

Insect-specific virus (ISV) is one of the most promising agents for the biological control of insects, which is abundantly distributed in hematophagous insects. However, few ISVs have been reported in Riptortus pedestris (Fabricius), one of the major pests threatening soybeans and causing great losses in yield and quality. In this work, field Riptortus pedestris was collected from six soybean-producing regions in China, and their virome was analyzed with the metatranscriptomic approach. Altogether, seven new insect RNA viruses were identified, three of which had complete RNA-dependent RNA polymerase (RdRp) and nearly full-length genome sequences, which were named Riptortus pedestris alphadrosrha-like virus 1 (RpALv1), Riptortus pedestris alphadrosrha-like virus 2 (RpALv2) and Riptortus pedestris almendra-like virus (RiALv). The three identified novel ISVs belonged to the family Rhabdoviridae, and phylogenetic tree analysis indicated that they were clustered into new distinct clades. Interestingly, the analysis of virus-derived small-interfering RNAs (vsiRNAs) indicated that only RiALv-derived siRNAs exhibited 22 nt length preference, whereas no clear 21 or 22 nt peaks were observed for RpALv1 and RpALv2, suggesting the complexity of siRNA-based antiviral immunity in R. pedestris. In conclusion, this study contributes to a better understanding of the microenvironment in R. pedestris and provides viral information for the development of potential soybean insect-specific biocontrol agents.

Characterization of Two Novel Insect-Specific Viruses Discovered in the Green Leafhopper, Cicadella viridis.

Insect-specific viruses (ISV) are one of the most promising agents for the biological control of insects. The green leafhopper, Cicadella viridis (Linnaeus), is an significant pest in agriculture, and causes economic losses to fruit trees, willows, and field crops. As a representative species of the large family Cicadellidae, ISVs in C. viridis have never been studied, to date. In this study, the full genome sequences of two ISVs, named Cicadella viridis iflavirus1 (CvIfV1), and Cicadella viridis nido-like virus 1 (CvNiLV1), were revealed using a metatranscriptomic approach. A homology search and phylogenetic analysis indicated that CvIfV1 is a new member in the family Iflaviridae (genus Iflavirus) with a typical iflavirus genome organization, whereas CvNiLV1 belongs to the unclassified clade/family of the order Nidovirales. In addition, analysis of virus-derived small interfering RNAs (vsiRNAs) was performed to investigate the antiviral RNA interference (RNAi) response of C. viridis. The vsiRNAs exhibit typical patterns produced by host siRNA-mediated antiviral immunity, including a preference of 21-nt vsiRNAs derived equally from the sense and antisense genomic strands, and a strong A/U bias in the 5'-terminus of the viral genomes. Our study provides valuable information for ISVs in leafhoppers for the first time, which might prove useful in the control of C. viridis in future.

Untangling an AGS Outbreak Caused by the Recombinant GII.12[P16] Norovirus With Nanopore Sequencing.

For a rapidly spreading virus such as NoV (norovirus), pathogen identification, genotype classification, and transmission tracing are urgent for epidemic control. Here, we applied the Nanopore metatranscriptomic sequencing to determine the causative pathogen of a community AGS (Acute gastroenteritis) outbreak. The results were also confirmed by RT-PCR. The NGS (Next Generation Sequencing) library was constructed within 8 hours and sequence analyses were carried out in real-time. NoV positive reads were detected in 13 of 17 collected samples, including two water samples from sewage treatment tank and cistern. A nearly complete viral genome and other genome fragments could be generated from metatranscriptomic sequencing of 13 samples. The NoV sequences from water samples and cases are identical suggesting the potential source of the outbreak. The sequencing results also indicated the outbreak was likely caused by an emerging recombinant GII.12[P16] virus, which was only identified in the United States and Canada in 2017-2018. This is the first report of this emerging variant in mainland China, following the large outbreaks caused by the recombinant GII.17[P17] and GII.2[P16] in 2014 and 2016, respectively. Closely monitoring of the prevalence of this recombinant strain is required. Our data also highlighted the importance of real-time sequencing in emerging pathogens' surveillance.

Metatranscriptomic Analysis Reveals Disordered Alterations in Oropharyngeal Microbiome during the Infection and Clearance Processes of SARS-CoV-2: A Warning for Secondary Infections.

This study was conducted to investigate oropharyngeal microbiota alterations during the progression of coronavirus disease 2019 (COVID-19) by analyzing these alterations during the infection and clearance processes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The diagnosis of COVID-19 was confirmed by using positive SARS-CoV-2 quantitative reverse transcription polymerase chain reaction (RT-qPCR). The alterations in abundance, diversity, and potential function of the oropharyngeal microbiome were identified using metatranscriptomic sequencing analyses of oropharyngeal swab specimens from 47 patients with COVID-19 (within a week after diagnosis and within two months after recovery from COVID-19) and 40 healthy individuals. As a result, in the infection process of SARS-CoV-2, compared to the healthy individuals, the relative abundances of Prevotella, Aspergillus, and Epstein-Barr virus were elevated; the alpha diversity was decreased; the beta diversity was disordered; the relative abundance of Gram-negative bacteria was increased; and the relative abundance of Gram-positive bacteria was decreased. After the clearance of SARS-CoV-2, compared to the healthy individuals and patients with COVID-19, the above disordered alterations persisted in the patients who had recovered from COVID-19 and did not return to the normal level observed in the healthy individuals. Additionally, the expressions of several antibiotic resistance genes (especially multi-drug resistance, glycopeptide, and tetracycline) in the patients with COVID-19 were higher than those in the healthy individuals. After SARS-CoV-2 was cleared, the expressions of these genes in the patients who had recovered from COVID-19 were lower than those in the patients with COVID-19, and they were different from those in the healthy individuals. In conclusion, our findings provide evidence that potential secondary infections with oropharyngeal bacteria, fungi, and viruses in patients who have recovered from COVID-19 should not be ignored; this evidence also highlights the clinical significance of the oropharyngeal microbiome in the early prevention of potential secondary infections of COVID-19 and suggests that it is imperative to choose appropriate antibiotics for subsequent bacterial secondary infection in patients with COVID-19.

Compositional and Functional Changes in Microbial Communities of Composts Due to the Composting-Related Factors and the Presence of Listeria monocytogenes.

Listeria monocytogenes is a leading foodborne pathogen that can contaminate fresh produce in farm environment, resulting in deadly outbreaks. Composts contain a diversity of microorganisms, and some of them may be compost-adapted competitive exclusion microorganisms against L. monocytogenes. To understand interactions between compost microflora and the pathogen, both dairy- and poultry-wastes based composts (n = 12) were inoculated with L. monocytogenes, and then analyzed by next-generation sequencing approaches along with culturing methods. DNA extraction and enumeration of L. monocytogenes were performed at 0 and 72 h post-incubation at room temperature. The major bacterial phyla were identified as Firmicutes (23%), Proteobacteria (23%), Actinobacteria (19%), Chloroflexi (13%), Bacteroidetes (12%), Gemmatimonadetes (2%), and Acidobacteria (2%). The top three indicator genera enriched in different compost types were identified by LEfSe with LDA score > 2. The interactions between L. monocytogenes and indigenous microflora were limited as no significant changes in the dominant microbial members in compost ecosystem, but some discriminatory species such as Bacillus, Geobacillus, and Brevibacterium were identified by Random Forest analysis. Besides, changes in metabolic pathways and the increased abundance of bacteriocins category in the compost samples containing L. monocytogenes after 72 h postinoculation were revealed by metatranscriptomic sequencing. Taken together, the compost-related factors such as compost types, composting stages, and the collection farms are major drivers that affect compost microbial compositions, and the analysis of compost metagenome implied that interactions between L. monocytogenes and compost microflora may include competition for nutrients and the presence of antimicrobials. IMPORTANCE Listeria monocytogenes has been recognized as the etiological agent causing foodborne disease outbreaks, with fresh produce as vulnerable for contamination at even preharvest stage. Owing to the richness in microbial community, compost may mediate suppression of pathogens. In this study, the impact of compost-related factors and L. monocytogenes intrusion on dynamic changes in compost microbiome was investigated by next generation sequencing techniques. The compost-related factors such as compost types, composting stages, and the collection farms are major drivers that affect compost microbiome. The interactions between L. monocytogenes and compost microflora may include the competition for nutrients and the presence of antimicrobials produced by native compost microorganisms as potential competitive exclusion microorganisms. Findings from this study are important for the composting industry to understand the composition and functionality of microbial community in their products and help developing organic fertilizers fortified with anti-L. monocytogenes competitive exclusion microorganisms.

The deletion of Schizosaccharomyces pombe decreased the production of flavor-related metabolites during traditional Baijiu fermentation.

The microbiota in traditional solid-state fermentation is a complex microbiota that plays a key role in the production of feed, fuel, food and pharmaceutical products. The function of microbiota is an important factor dictating the quantity and quality of products. Core functional species play key metabolic roles in the microbiota, and their disappearance could result in the abnormal fermentation process. In this work, we combined Baijiu production and laboratory experiments to explore the keystone microbes and their metabolites. We found the deletion of core functional microbe resulted in the loss of multiple metabolites involved many alcohols and acids. In the traditional Baijiu production, the absence or appearance of Schizosaccharomyces pombe caused the content divergence in 227 flavor-related metabolites, especially in ethanol, butanol and pentanoic acid between abnormal and normal group (each content > 1 mg/kg and the content ratio of normal/abnormal group > 2). Schi. pombe increased the expression level of related genes involving alcohol dehydrogenase (ADH), acyl-CoA oxidase (ACOX) and trans-2-enoyl-CoA reductase (TER). Moreover, in the verification experiment of laboratory, the absence or appearance of Schizosaccharomyces pombe C-11 caused the content divergence in 136 flavor-related metabolites, especially in ethanol, butanol and pentanoic acid between Sp- and Sp+ group (each content > 1 mg/kg and the content ratio of Sp+/Sp- group > 2). Our results identified specific member that were essential for the function of fermentation microbiota. This study also suggests species deletions from fermentation microbiota and synthetic consortium could be a useful approach to illustrate relevant microbe-metabolites association and defining metabolic roles in the traditional solid-state fermentation.

Multiple approaches for massively parallel sequencing of SARS-CoV-2 genomes directly from clinical samples.

BACKGROUND: COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. METHODS: We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. RESULTS: We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations. CONCLUSIONS: This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.

Heightened Innate Immune Responses in the Respiratory Tract of COVID-19 Patients.

The outbreaks of 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 infection have posed a severe threat to global public health. It is unclear how the human immune system responds to this infection. Here, we used metatranscriptomic sequencing to profile immune signatures in the bronchoalveolar lavage fluid of eight COVID-19 cases. The expression of proinflammatory genes, especially chemokines, was markedly elevated in COVID-19 cases compared to community-acquired pneumonia patients and healthy controls, suggesting that SARS-CoV-2 infection causes hypercytokinemia. Compared to SARS-CoV, which is thought to induce inadequate interferon (IFN) responses, SARS-CoV-2 robustly triggered expression of numerous IFN-stimulated genes (ISGs). These ISGs exhibit immunopathogenic potential, with overrepresentation of genes involved in inflammation. The transcriptome data was also used to estimate immune cell populations, revealing increases in activated dendritic cells and neutrophils. Collectively, these host responses to SARS-CoV-2 infection could further our understanding of disease pathogenesis and point toward antiviral strategies.

Schizosaccharomyces pombe can Reduce Acetic Acid Produced by Baijiu Spontaneous Fermentation Microbiota.

The spontaneous fermentation of alcoholic beverage is a bioprocess donated by microbiota with complex stress environments. Among various microbes, non-Saccharomyces yeasts have high stress tolerance and significantly affect the taste and quality of products in process. Although many researchers have focused on the influence of acid stress, the mechanism of non-Saccharomyces yeasts to tolerant stress remains unclear in microbiota. To bridge the gap, we constructed in situ and in vitro studies to explore the reduction pathway of acetic acid in non-Saccharomyces yeasts. In this study, we found Schizosaccharomyces pombe has special capacities to resist 10 g/L acetic acid in laboratory cultures and decrease the average concentration of acetic acid from 9.62 to 6.55 g/kg fermented grains in Chinese Maotai-flavor liquor (Baijiu) production. Moreover, Schi. pombe promoted metabolic level of mevalonate pathway (high expressions of gene ACCAT1, HMGCS1, and HMGCR1) to degrade a high concentration of acetic acid. Meanwhile, Schi. pombe also improved the concentration of mevalonic acid that is the precursor of terpenes to enhance the taste and quality of Baijiu. Overall, the synchronicity of reduction and generation in Schi. pombe advances the current knowledge to guide more suitable strategies for mechanism studies of non-Saccharomyces yeasts in fermented industries of alcoholic beverages.