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1.
Virol J ; 19(1): 70, 2022 04 20.
Article in English | MEDLINE | ID: covidwho-1862136

ABSTRACT

BACKGROUND: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples. METHODS: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system. RESULTS: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits. CONCLUSION: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.


Subject(s)
Central Nervous System Viral Diseases , Enterovirus Infections , Enterovirus , Herpesvirus 1, Human , Parechovirus , Picornaviridae Infections , Enterovirus/genetics , Enterovirus Infections/diagnosis , Herpesvirus 1, Human/genetics , Herpesvirus 2, Human/genetics , Humans , Parechovirus/genetics , Real-Time Polymerase Chain Reaction/methods
2.
PLoS One ; 17(3): e0264855, 2022.
Article in English | MEDLINE | ID: covidwho-1736511

ABSTRACT

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).


Subject(s)
Bacteria/genetics , COVID-19/diagnosis , High-Throughput Nucleotide Sequencing/methods , RNA Viruses/genetics , Respiratory Tract Infections/diagnosis , SARS-CoV-2/genetics , Bacteria/isolation & purification , COVID-19/virology , Coronavirus/genetics , Coronavirus/isolation & purification , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/isolation & purification , Humans , Multiplex Polymerase Chain Reaction , Nanopores , Orthomyxoviridae/genetics , Orthomyxoviridae/isolation & purification , RNA Viruses/isolation & purification , RNA, Viral/chemistry , RNA, Viral/metabolism , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , SARS-CoV-2/isolation & purification
3.
Mem Inst Oswaldo Cruz ; 116: e210176, 2022.
Article in English | MEDLINE | ID: covidwho-1725021

ABSTRACT

BACKGROUND: During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES: We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS: Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS: Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS: We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.


Subject(s)
COVID-19 , Coinfection , Herpesvirus 1, Human , Herpesvirus 1, Human/genetics , Humans , Real-Time Polymerase Chain Reaction , SARS-CoV-2
4.
Ocul Immunol Inflamm ; 29(6): 1238-1240, 2021 Aug 18.
Article in English | MEDLINE | ID: covidwho-1462164

ABSTRACT

PURPOSE: To report two cases of herpes simplex virus keratitis reactivation following Pfizer-BioNTech COVID-19 (BNT162b2) mRNA vaccination. METHODS: Two patients (one male, age 42 years, and one female, age 29 years) who are known to have herpetic keratitis presented to our emergency room in a time frame between 4 days and 4 weeks of receiving the vaccine. One patient presented with necrotizing stromal keratitis; the other presented with endotheliitis and epithelial keratitis. PCR for herpes simplex virus (HSV) was obtained from the two patients, and all cases received systemic acyclovir. RESULTS: PCR for HSV came positive in both cases. Patients responded well to the provided treatment. CONCLUSION: Ocular herpetic infection may be activated by COVID-19 (BNT162b2) mRNA vaccine. Treating physician should be alert to such associations, and patients should be followed closely. No direct causality has been proven, but further reporting and investigating similar conditions is recommended.


Subject(s)
/adverse effects , COVID-19/prevention & control , Keratitis, Herpetic/etiology , Latent Infection/etiology , SARS-CoV-2 , Vaccination/adverse effects , Acyclovir/therapeutic use , Adult , Antiviral Agents/therapeutic use , Female , Herpesvirus 1, Human/genetics , Humans , Keratitis, Herpetic/diagnosis , Keratitis, Herpetic/drug therapy , Latent Infection/diagnosis , Latent Infection/drug therapy , Male , Polymerase Chain Reaction
5.
Virology ; 559: 165-172, 2021 07.
Article in English | MEDLINE | ID: covidwho-1198236

ABSTRACT

SARS coronavirus 1 (SARS-CoV-1) causes a respiratory infection that can lead to acute respiratory distress characterized by inflammation and high levels of cytokines in the lung tissue. In this study we constructed a herpes simplex virus 1 replication-defective mutant vector expressing SARS-CoV-1 spike protein as a potential vaccine vector and to probe the effects of spike protein on host cells. The spike protein expressed from this vector is functional in that it localizes to the surface of infected cells and induces fusion of ACE2-expressing cells. In immunized mice, the recombinant vector induced antibodies that bind to spike protein in an ELISA assay and that show neutralizing activity. The spike protein expressed from this vector can induce the expression of cytokines in an ACE2-independent, MyD88-dependent process. These results argue that the SARS-CoV-1 spike protein intrinsically activates signaling pathways that induce cytokines and contribute directly to the inflammatory process of SARS.


Subject(s)
Antibodies, Neutralizing/immunology , Herpesvirus 1, Human/genetics , Immunity, Innate , SARS Virus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibodies, Viral/immunology , Cell Fusion , Cell Line , Cytokines/immunology , Genetic Vectors , Humans , Mice , SARS Virus/genetics , Signal Transduction , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vaccines, Virus-Like Particle/immunology , Viral Vaccines/immunology
6.
Front Immunol ; 12: 676828, 2021.
Article in English | MEDLINE | ID: covidwho-1320577

ABSTRACT

In coronavirus disease 2019 (COVID-19), ulcerative lesions have been episodically reported in various segments of the gastrointestinal (GI) tract, including the oral cavity, oropharynx, esophagus, stomach and bowel. In this report, we describe an autopsy case of a COVID-19 patient who showed two undiagnosed ulcers at the level of the anterior and posterior walls of the hypopharynx. Molecular testing of viruses involved in pharyngeal ulcers demonstrated the presence of severe acute respiratory syndrome - coronavirus type 2 (SARS-CoV-2) RNA, together with herpes simplex virus 1 DNA. Histopathologic analysis demonstrated full-thickness lympho-monocytic infiltration (mainly composed of CD68-positive cells), with hemorrhagic foci and necrosis of both the mucosal layer and deep skeletal muscle fibers. Fibrin and platelet microthrombi were also found. Cytological signs of HSV-1 induced damage were not found. Cells expressing SARS-CoV-2 spike subunit 1 were immunohistochemically identified in the inflammatory infiltrations. Immunohistochemistry for HSV1 showed general negativity for inflammatory infiltration, although in the presence of some positive cells. Thus, histopathological, immunohistochemical and molecular findings supported a direct role by SARS-CoV-2 in producing local ulcerative damage, although a possible contributory role by HSV-1 reactivation cannot be excluded. From a clinical perspective, this autopsy report of two undiagnosed lesions put the question if ulcers along the GI tract could be more common (but frequently neglected) in COVID-19 patients.


Subject(s)
COVID-19/complications , Hypopharynx/pathology , SARS-CoV-2/isolation & purification , Ulcer/pathology , Aged , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Autopsy , Blood Platelets/metabolism , Blood Platelets/pathology , COVID-19/mortality , COVID-19/pathology , COVID-19/physiopathology , Gastrointestinal Tract/pathology , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/isolation & purification , Humans , Hypopharynx/virology , Immunohistochemistry , Inflammation/immunology , Inflammation/metabolism , Inflammation/virology , Lymphocytes/metabolism , Monocytes/metabolism , Mucous Membrane/pathology , Muscle, Skeletal/pathology , Necrosis/pathology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism , Thrombosis/pathology , Ulcer/virology
7.
PLoS One ; 16(2): e0247396, 2021.
Article in English | MEDLINE | ID: covidwho-1090539

ABSTRACT

Among various delivery systems for vaccine and drug delivery, cell-penetrating peptides (CPPs) have been known as a potent delivery system because of their capability to penetrate cell membranes and deliver some types of cargoes into cells. Several CPPs were found in the proteome of viruses such as Tat originated from human immunodeficiency virus-1 (HIV-1), and VP22 derived from herpes simplex virus-1 (HSV-1). In the current study, a wide-range of CPPs was identified in the proteome of SARS-CoV-2, a new member of coronaviruses family, using in silico analyses. These CPPs may play a main role for high penetration of virus into cells and infection of host. At first, we submitted the proteome of SARS-CoV-2 to CellPPD web server that resulted in a huge number of CPPs with ten residues in length. Afterward, we submitted the predicted CPPs to C2Pred web server for evaluation of the probability of each peptide. Then, the uptake efficiency of each peptide was investigated using CPPred-RF and MLCPP web servers. Next, the physicochemical properties of the predicted CPPs including net charge, theoretical isoelectric point (pI), amphipathicity, molecular weight, and water solubility were calculated using protparam and pepcalc tools. In addition, the probability of membrane binding potential and cellular localization of each CPP were estimated by Boman index using APD3 web server, D factor, and TMHMM web server. On the other hand, the immunogenicity, toxicity, allergenicity, hemolytic potency, and half-life of CPPs were predicted using various web servers. Finally, the tertiary structure and the helical wheel projection of some CPPs were predicted by PEP-FOLD3 and Heliquest web servers, respectively. These CPPs were divided into: a) CPP containing tumor homing motif (RGD) and/or tumor penetrating motif (RXXR); b) CPP with the highest Boman index; c) CPP with high half-life (~100 hour) in mammalian cells, and d) CPP with +5.00 net charge. Based on the results, we found a large number of novel CPPs with various features. Some of these CPPs possess tumor-specific motifs which can be evaluated in cancer therapy. Furthermore, the novel and potent CPPs derived from SARS-CoV-2 may be used alone or conjugated to some sequences such as nuclear localization sequence (NLS) for vaccine and drug delivery.


Subject(s)
COVID-19 Vaccines/chemistry , Cell-Penetrating Peptides/chemistry , Computational Biology , Computer Simulation , Drug Delivery Systems , Proteome , SARS-CoV-2/chemistry , Animals , COVID-19 , COVID-19 Vaccines/genetics , COVID-19 Vaccines/metabolism , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/metabolism , HIV-1/chemistry , HIV-1/genetics , Herpesvirus 1, Human/chemistry , Herpesvirus 1, Human/genetics , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Viral Structural Proteins/chemistry , Viral Structural Proteins/genetics , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/genetics
8.
Acta Ophthalmol ; 98(8): 859-863, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-260153

ABSTRACT

BACKGROUND: The current outbreak of COVID-19 has spread rapidly all over the world. Respiratory droplets and contaction with infected patients are the two major transmission routes. However, the value of tear virus nucleic acid is still not clear. We dynamic detected the SARS-CoV-2 in eye sample of one COVID-19 patient with obstruction of common lacrimal ducts. METHODS: Besides the routine examination, nasopharyngeal and eye swab were continuously measured by polymerase chain reaction assay and next-generation sequencing (NGS). Gene detection was performed for drug use guidance, and flow cytometry was performed to analyse the lymphocyte subsets. RESULTS: Nasopharyngeal swabs were positive for 22 days, but eye swabs were still continuously positive for 2 weeks after nasopharyngeal swabs turned negative. The low level of lymphocyte and the high level IL-6 lasted for almost 4 weeks, then became near normal. Next-generation sequencing (NGS) confirmed the existing of SARS-CoV-2, HSV1 and HHV6B virus nucleic acid. The gene detection for drug use guidance showed the genetic locus ABCB1 (3435T>C) rs1045642 belonged to type CC and it mean the efficiency of lopinavir-ritonavir would be significantly decreased. The flow cytometry of lymphocyte subsets showed PD-1+  CD95+ cells was accounting for 94.8% in CD3+  CD8+ T subset and for 94.8% in CD3+  TCRγδ+ T subset. CONCLUSIONS: As obstruction of common lacrimal duct, positively detection in one eye for 2 weeks more after nasopharyngeal swab became negative. More eye swabs should be collected from COVID-19 patients, especially from those immunocompromised, those with eye symptoms and those had a history of ocular diseases.


Subject(s)
COVID-19/diagnosis , Conjunctiva/virology , Eye Infections, Viral/diagnosis , Herpesvirus 1, Human/isolation & purification , Herpesvirus 6, Human/isolation & purification , Lacrimal Duct Obstruction/diagnosis , SARS-CoV-2/isolation & purification , Tears/virology , Aged , Anti-Bacterial Agents/therapeutic use , COVID-19/drug therapy , COVID-19/virology , Conjunctivitis, Viral/diagnosis , Drug Therapy, Combination , Eye Infections, Viral/drug therapy , Eye Infections, Viral/virology , Flow Cytometry , HIV Protease Inhibitors/therapeutic use , Herpes Simplex/diagnosis , Herpes Simplex/drug therapy , Herpes Simplex/virology , Herpesvirus 1, Human/genetics , Herpesvirus 6, Human/genetics , High-Throughput Nucleotide Sequencing , Humans , Lacrimal Duct Obstruction/drug therapy , Lacrimal Duct Obstruction/virology , Lopinavir/therapeutic use , Male , Medicine, Chinese Traditional , Moxifloxacin/therapeutic use , Nasopharynx/virology , Nucleic Acids/genetics , Polymerase Chain Reaction , RNA, Viral/genetics , Ritonavir/therapeutic use , Roseolovirus Infections/diagnosis , Roseolovirus Infections/drug therapy , Roseolovirus Infections/virology , SARS-CoV-2/genetics
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