Dysbiosis of oropharyngeal microbiome and antibiotic resistance in hospitalized COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is ongoing and multiple studies have elucidated its pathogenesis, however, the related- microbiome imbalance caused by SARS-CoV-2 is still not clear. In this study, we have comprehensively compared the microbiome composition and associated function alterations in the oropharyngeal swabs of healthy controls and coronavirus disease 2019 (COVID-19) patients with moderate or severe symptoms by metatranscriptomic sequencing. We did observe a reduced microbiome alpha-diversity but significant enrichment of opportunistic microorganisms in patients with COVID-19 compared with healthy controls, and the microbial homeostasis was rebuilt following the recovery of COVID-19 patients. Correspondingly, less functional genes in multiple biological processes and weakened metabolic pathways such as carbohydrate metabolism, energy metabolism were also observed in COVID-19 patients. We only found higher relative abundance of limited genera such as Lachnoanaerobaculum between severe patients and moderate patients while no worthy-noting microbiome diversity and function alteration were observed. Finally, we noticed that the co-occurrence of antibiotic resistance and virulence was closely related to the microbiome alteration caused by SRAS-CoV-2. Overall, our findings demonstrate that microbial dysbiosis may enhance the pathogenesis of SARS-CoV-2 and the antibiotics treatment should be critically considered.

[Western area surge for controlling Ebola hemorrhagic fever outbreak in Sierra Leone and evaluation of its effect].

OBJECTIVE: To investigate the Western Area Surge (WAS) program in the Ebola outbreak of Sierra Leone, and to analyze its implementing effect. METHODS: The subject of this study was 3,813 laboratory confirmed Ebola hemorrhagic fever (EHF) cases reported in Sierra Leone from November 19, 2014 through January 27, 2015, a period before and after the implementation of the WAS program. To analyze and make conclusions according to the working experience of China Mobile Laboratory Reponses Team in the fight of Ebola outbreak, using WHO published EHF case definition to make diagnosis and compare the number of bed numbers, confirmed EHF cases, samples tested, and positive rates before and after implementation of WAS program. RESULTS: From the implementation of WAS program on 17th December 2014 to half a month later, the total numbers of Ebola holding and treatment centers increased from 640 to 960, six additional laboratories were established. On January, 2015, another two laboratories from America and The Netherlands were established. The numbers of samples tested one month before and after WAS program were 7,891 and 9,783, respectively, with an increase of 24.0 percent, while the positive rate of Ebola virus decreased from 22.2% (1,752/7,891) to 11.0% (1,077/9,783). The positive rate of blood samples decreased from 39.6% (248/626) in the month before WAS program to 27.4% (131/478) (χ2=17.93, P<0.001) in the mother after WAS program, the positive rate of blood samples 22.7% (103/454) to 10% (62/609) (χ2=31.03, P<0.001), accordingly. After 3 weeks of WAS program, in addition to Western Area, another four hotspots in Sierra Leone had also reported a significant decrease of the numbers of confirmed EVD cases. Forty-two days after implementation of WAS program, the daily number of laboratory confirmed EHF cases decreased from 63 to 10. CONCLUSION: WAS program played a vital role in controlling the EHF outbreak rapidly in Sierra Leone. It could also provide guidance for the control similar large infectious diseases outbreak in the future.

Economical, green synthesis of fluorescent carbon nanoparticles and their use as probes for sensitive and selective detection of mercury(II) ions.

The present article reports on a simple, economical, and green preparative strategy toward water-soluble, fluorescent carbon nanoparticles (CPs) with a quantum yield of approximately 6.9% by hydrothermal process using low cost wastes of pomelo peel as a carbon source for the first time. We further explore the use of such CPs as probes for a fluorescent Hg(2+) detection application, which is based on Hg(2+)-induced fluorescence quenching of CPs. This sensing system exhibits excellent sensitivity and selectivity toward Hg(2+), and a detection limit as low as 0.23 nM is achieved. The practical use of this system for Hg(2+) determination in lake water samples is also demonstrated successfully.

Development of an ELISA-array for simultaneous detection of five encephalitis viruses.

Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.

Synthesis of Ag nanoparticle-decorated 2,4,6-tris(2-pyridyl)-1,3,5-triazine nanobelts and their application for H2O2 and glucose detection.

The present paper reports on the first preparation of 2,4,6-tris(2-pyridyl)-1,3,5-triazine nanobelts (TPTNBs) by adjusting the pH value of the solution and the subsequent synthesis of Ag nanoparticle (AgNP)-decorated TPTNBs (AgNP-TPTNBs) by mixing an aqueous AgNO(3) solution with preformed TPTNBs without use of any external reducing agent. It is found that the resultant AgNP-TPTNBs exhibit notable catalytic performance for H(2)O(2) reduction. A glucose biosensor was fabricated by immobilizing glucose oxidase (GOD) onto a AgNP-TPTNBs-modified glassy carbon electrode (GCE) for glucose detection. The constructed glucose sensor has a wide linear response range from 3 mM to 20 mM (r: 0.999) with a detection limit of 190 µM. It is further shown that this glucose biosensor can be used for glucose detection in human blood serum.

Novel application of CoFe layered double hydroxide nanoplates for colorimetric detection of H(2)O(2) and glucose.

The present communication demonstrates the proof of concept of using CoFe layered double hydroxide (CoFe-LDHs) nanoplates as an effective peroxidase mimetic to catalyze the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine in the presence of H(2)O(2) to produce a blue solution. We further demonstrate successfully CoFe-LDHs nanoplate-based colorimetric assay to detect H(2)O(2) and glucose.

2,4,6-Tris (2-pyridyl)-1,3,5-triazine nanobelts as an effective fluorescent sensing platform for DNA detection.

In the present study, we report on the use of 2,4,6-tris (2-pyridyl)-1,3,5-triazine nanobelts (TPTNBs) as an effective fluorescent sensing platform for DNA detection for the first time. The general concept used in this approach is based on adsorption of fluorescently labeled single-stranded DNA (ssDNA) probe by TPTNB, due to the strong pi-pi stacking between unpaired DNA bases and TPTNB. As a result, the fluorophor is brought into close proximity of TPTNB, leading to fluorescence quenching. Upon presence of the target ssDNA, specific hybridization with the target takes place to form a double-stranded DNA (dsDNA). The resultant dsDNA cannot be adsorbed by TPTNB due to its rigid conformation and the absence of unpaired DNA bases. Thus, the fluorophor is seperated from TPTNB accompanied by fluorescence recovery. The present system shows a detection limit as low as 3 nM and has a high selectivity down to single-base mismatch.

Carbon nanocapsules as an effective sensing platform for fluorescence-enhanced nucleic acid detection.

In this communication, we demonstrate the proof of concept that carbon nanocapsules (CNCs) can be used as an effective fluorescent sensing platform for nucleic acid detection with selectivity down to single-base mismatch. The detection is accomplished by two steps: (1) CNC adsorbs and quenches the fluorescence of the dye-labeled single-stranded DNA (ssDNA) probe; (2) in the presence of the target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the CNC surface, leading to recovery of the dye fluorescence.

Anchoring gold nanoparticles on graphene nanosheets functionalized with cationic polyelectrolyte: a novel catalyst for 4-nitrophenol reduction.

In this paper, a stable aqueous dispersion of graphene nanosheets (GNs) has been prepared by chemical reduction of graphene oxide (GO) with hydrazine hydrate in the presence of poly [(2-ethyldimethylammonioethyl methacrylate ethyl sulfate)-co-(1-vinylpyrrolidone)] (PQ11). Taking advantages of the fact that PQ11 is a positively charged polymer exhibiting reducing ability, we further demonstrated the subsequent decoration of GN with gold nanoparticals (AuNPs) by in-situ chemical reduction of HAuCl4. It was found that such nanocomposites exhibit good catalytic activity toward 4-nitrophenol (4-NP) reduction and the GN supports also enhance the catalytic activity via a synergistic effect.

Synthesis of Pt nanoparticles decorated 1,5-diaminoanthraquinone nanofibers and their application toward catalytic reduction of 4-nitrophenol.

The present communication reports on the rapid preparation of 1,5-diaminoanthraquinone nanofibers (DAAQNFs) on a large scale by a reprecipitation method and their subsequent decoration with small platinum nanoparticles (PtNPs) using tannic acid (TA) as a reducing agent. It suggests the resultant PtNP/DAAQNF composites exhibit a good catalytic activity toward reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) by NaBH4. It also suggests that the composites exhibit higher catalytic activity than the PtNPs due to that the DAAQNF support may play an active part in the catalysis, yielding a synergistic effect. The possible mechanism involved has also been discussed.

Genetic analysis of murine hepatitis virus non-structural protein 16.

MHV-Wüts18 is an RNA-negative, temperature-sensitive mutant of mouse coronavirus, strain murine hepatitis virus (MHV)-A59. We have previously identified the putative causal mutation of MHV-Wüts18 as a C to U transition at codon 2446 in ORF1b, which results in a substitution of proline 12 with serine in non-structural protein 16. Here, we have used a vaccinia virus-based reverse genetic system to produce a recombinant virus, inf-MHV-Wüts18((AGC)) that encodes nsp16 serine 12 with AGC rather than UCU; a difference that facilitates the isolation of second-site revertants. Sequence analysis of nine inf-MHV-Wüts18((AGC)) revertant viruses suggests that their phenotype is most probably due to the intra-molecular substitution of amino acids in nsp16. However, the revertant viruses displayed different plaque sizes and whole genome sequencing of two revertants showed that they were isogenic apart from a mutation in nsp13. These results are discussed in the context of a model of coronavirus MHV nsp16 structure.

PB1-mediated virulence attenuation of H5N1 influenza virus in mice is associated with PB2.

H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passages in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned isolates from the lungs of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and large-plaque isolates differed in several amino acids. In order to assess their effects on pathogenicity in mice, two amino acid changes common to attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M), were inserted into a wild-type recombinant virus construct. The PB2 (I63T) or PB1 (T677M) mutations alone did not alter the phenotype of H5N1 virus, whereas recombinant virus with both mutations was less pathogenic than the wild-type recombinant virus. Furthermore, the PB1 (T677M) mutation showed a lower replication efficiency, although it had higher polymerase activity. The recombinant virus with the PB2 (63T) mutation replicated as well as the wild-type recombinant virus. These results suggest that the C terminus of PB1 of H5N1 influenza virus mediates virulence attenuation of H5N1 influenza virus in mice, associating with the N terminus of PB2. However, the role of the N terminus of PB2 in virulence attenuation in mice remains unclear.

Virulence of H5N1 virus in mice attenuates after in vitro serial passages.

The virulence of A/Vietnam/1194/2004 (VN1194) in mice attenuated after serial passages in MDCK cells and chicken embryos, because the enriched large-plaque variants of the virus had significantly reduced virulence. In contrast, the small-plaque variants of the virus and the variants isolated from the brain of mice that were infected with the parental virus VN1194 had much higher virulence in mice. The virulence attenuation of serially propagated virus may be caused by the reduced neurotropism in mice. Our whole genome sequence analysis revealed substitutions of a total of two amino acids in PB1, three in PB2, two in PA common for virulence attenuated variants, all or part of which may be correlated with the virulence attenuation and reduced neurotropism of the serially propagated VN1194 in mice. Our study indicates that serial passages of VN1194 in vitro lead to adaptation and selection of variants that have markedly decreased virulence and neurotropism, which emphasizes the importance of direct analysis of original or less propagated virus samples.

Iron-substituted SBA-15 microparticles: a peroxidase-like catalyst for H2O2 detection.

In this communication, we demonstrate our recent finding that iron-substituted SBA-15 (Fe-SBA-15) microparticles possess intrinsic peroxidase-like activity and can catalyze the oxidation of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) by H(2)O(2) to develop a blue color in aqueous solution, leading to a simple approach towards colorimetric detection of H(2)O(2) with a linear detection range from 0.4 µM to 15 µM (r = 0.997) and a detection limit of 0.2 µM.

Coordination polymer nanobelts for nucleic acid detection.

Herein, coordination polymer nanobelts (CPNBs) were prepared rapidly and on a large scale, by directly mixing aqueous AgNO(3) solution and an ethanol solution of 4, 4'-bipyridine at room temperature. The application of such CPNBs as a fluorescent sensing platform for nucleic acid detection was further explored. CPNB is a π-rich structure, the strong π-π stacking interactions between unpaired DNA bases and CPNB leads to adsorption of fluorescently labeled single-stranded DNA (ssDNA) accompanied by 66% fluorescence quenching. However, the presence of target ssDNA will hybridize with the probe. The resultant helix cannot be adsorbed by CPNB due to its rigid conformation and the absence of unpaired DNA bases. Thus, a significant fluorescence enhancement, 73% fluorescence recovery, was observed in DNA detection as long as the target exists. The present system has excellent sensitivity; a substantial fluorescence enhancement was observed when the concentration of the target was as low as 5 nM. It also exhibits outstanding discrimination ability down to a single-base mismatch.

Organ-specific attenuation of murine hepatitis virus strain A59 by replacement of catalytic residues in the putative viral cyclic phosphodiesterase ns2.

The Murine hepatitis virus (MHV) strain A59 ns2 protein is a 30-kDa nonstructural protein that is expressed from a subgenomic mRNA in the cytoplasm of virus-infected cells. Its homologs are also encoded in other closely related group 2a coronaviruses and more distantly related toroviruses. Together, these proteins comprise a subset of a large superfamily of 2H phosphoesterase proteins that are distinguished by a pair of conserved His-x-Thr/Ser motifs encompassing catalytically important residues. We have used a vaccinia virus-based reverse genetic system to produce recombinant viruses encoding ns2 proteins with single-amino-acid substitutions in, or adjacent to, these conserved motifs, namely, inf-ns2 H46A, inf-ns2 S48A, inf-ns2-S120A, and inf-ns2-H126R. All of the mutant viruses replicate in mouse 17 clone 1 fibroblast cells and mouse embryonic cells to the same extent as the parental wild-type recombinant virus, inf-MHV-A59. However, compared to inf-MHV-A59, the inf-ns2 H46A and inf-ns2-H126R mutants are highly attenuated for replication in mouse liver following intrahepatic inoculation. Interestingly, none of the mutant viruses were attenuated for replication in mouse brain following intracranial inoculation. These results show that the ns2 protein of MHV-A59 has an important role in virus pathogenicity and that a substitution of the histidine residues of the MHV-A59 ns2 His-x-Thr/Ser motifs is critical for virus virulence in the liver but not in the brain. This novel phenotype suggests a strategy to investigate the function of the MHV-A59 ns2 protein involving the search for organ-specific proteins or RNAs that react differentially to wild-type and mutant ns2 proteins.

Monoclonal antibody induced with inactived EV71-Hn2 virus protects mice against lethal EV71-Hn2 virus infection.

BACKGROUND: Enterovirus 71 (EV71) is a viral pathogen that belongs to the Picornaviridae family, EV71-infected children can develop severe neurological complications leading to rapid clinical deterioration and death. RESULTS: In this study, several monoclonal antibodies (MAbs) were produced by immunizing mice with the inactived EV71 Henan (Hn2) virus strain. The isolated MAbs were characterised by in vitro neutralizing analysis and peptide ELISA. ELISA assay showed that the neutralizing monoclonal antibody 4E8 specifically reacted with synthetic peptides which contain amino acid 240-250 and 250-260 of EV71 VP1. The in vivo protection assay showed that 4E8 can protect two-day-old BALB/c mice against the lethal challenge of EV71 virus. CONCLUSION: The MAb 4E8 could be a promising candidate to be humanized and used for treatment of EV71 infection.

A duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses.

In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.

A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus.

A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

Subtype identification of the novel A H1N1 and other human influenza A viruses using an oligonucleotide microarray.

A novel strain of influenza A (H1N1) virus was isolated in Mexico and the US in March and April 2009. This novel virus spread to many countries and regions in a few months, and WHO raised the level of pandemic alert from phase 5 to phase 6 on June 11, 2009. The accurate identification of H1N1 virus and other human seasonal influenza A viruses is very important for further treatment and control of their infections. In this study, we developed an oligonucleotide microarray to subtype human H1N1, H3N2 and H5N1 influenza viruses, which could distinguish the novel H1N1 from human seasonal H1N1 influenza viruses and swine H1N1 influenza viruses. The microarray utilizes a panel of primers for multiplex PCR amplification of the hemagglutinin (HA), neuraminidase (NA) and matrix (MP) genes of human influenza A viruses. The 59-mer oligonucleotides were designed to distinguish different subtypes of human influenza A viruses. With this microarray, we accurately identified and correctly subtyped the reference virus strains. Moreover, we confirmed 4 out of 39 clinical throat swab specimens from suspected cases of novel H1N1.