ResearchPad - short-communications https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Persistent viral RNA positivity during recovery period of a patient with SARS‐CoV‐2 infection]]> https://www.researchpad.co/article/elastic_article_16740 As an emerging infectious disease, the clinical course and virological course of SARS‐CoV‐2 infection remain to be further investigated. In this case report, we described a case of SARS‐CoV‐2 infection with clinical course more than two months. This patient had recovered from the pneumonia after treatment. The viral RNA of throat swabs became negative and the viral specific antibodies were produced during recovery period. However, the viral RNA reappeared and additionally persisted in throat swabs for more than 40 days. In addition, the viral RNA was detected in multiple types of specimens with extremely high titers in the saliva. In conclusion, these findings indicate that SARS‐CoV‐2 can cause a long clinical course. The coexistence of viral RNA and viral specific antibodies may imply an immune evasion of SARAS‐CoV‐2 from host's immune system.

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<![CDATA[Diagnostic accuracy of an automated chemiluminescent immunoassay for anti‐SARS‐CoV‐2 IgM and IgG antibodies: an Italian experience]]> https://www.researchpad.co/article/elastic_article_16721 A pandemic of coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been spreading throughout the world. Though molecular diagnostic tests are the gold standard for COVID‐19, serological testing is emerging as a potential surveillance tool, in addition to its complementary role in COVID‐19 diagnostics. Indubitably quantitative serological testing provides greater advantages than qualitative tests but today there is still little known about serological diagnostics and what the most appropriate role quantitative tests might play. Sixty‐one COVID‐19 patients and 64 patients from a control group were tested by iFlash1800 CLIA analyzer for anti‐SARS CoV‐2 antibodies IgM and IgG. All COVID‐19 patients were hospitalized in San Giovanni di Dio Hospital (Florence, Italy) and had a positive oro/nasopharyngeal swab reverse‐transcription polymerase chain reaction result. The highest sensitivity with a very good specificity performance was reached at a cutoff value of 10.0 AU/mL for IgM and of 7.1 for IgG antibodies, hence near to the manufacturer's cutoff values of 10 AU/mL for both isotypes. The receiver operating characteristic curves showed area under the curve values of 0.918 and 0.980 for anti‐SARS CoV‐2 antibodies IgM and IgG, respectively. iFlash1800 CLIA analyzer has shown highly accurate results for the anti‐SARS‐CoV‐2 antibodies profile and can be considered an excellent tool for COVID‐19 diagnostics.

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<![CDATA[Asymptomatic COVID‐19 infection in late pregnancy indicated no vertical transmission]]> https://www.researchpad.co/article/elastic_article_16689 We show a timely case report regarding the current COVID‐19 outbreak.Pregnant woman with asymptomatic COVID‐19 infection was prensented in detail.

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<![CDATA[A pilot study of the antiviral activity of anionic and cationic polyamidoamine dendrimers against the Middle East respiratory syndrome coronavirus]]> https://www.researchpad.co/article/elastic_article_16613 MERS CoV is an emerging viral disease with fatal consequences.Anti‐MERS CoV studies are very limited.The anti‐MERS CoV activity of several generations polyvalent charge dendrimers was investigated.PAMAM dendrimers bears intrinsic anti‐MESR CoV activity.Polyanionic carboxylate PAMAM dendrimers were the most effective agents.

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<![CDATA[SARS‐CoV‐2 can be detected in urine, blood, anal swabs, and oropharyngeal swabs specimens]]> https://www.researchpad.co/article/elastic_article_16594 SARS‐CoV‐2 can be detected in multiple system specimens but without necessarily corresponding dysfunction. Further study is needed to determine the virus ability to destroy different organs.

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<![CDATA[A Statistical Modeling of the Course of COVID-19 (SARS-CoV-2) Outbreak: A Comparative Analysis]]> https://www.researchpad.co/article/elastic_article_11269 This study aims to provide both a model by using cumulative cases and cumulative death toll for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) outbreak in four countries, China, Italy, South Korea, and Turkey, starting from the first diagnosis and to compare associated indicators. The most successful estimation was obtained from the cubic model with natural logarithm for China, Italy, South Korea, and Turkey. The success of the models was around 99%. However, differences began to emerge in China, Italy, and South Korea after the second week. Although the highest number of new cases per 1 million people in China was 9.8 on February 28, 2020; it was 108.4 on March 21, 2020, in Italy; and this was 16.6 on March 5, 2020, in South Korea. On the other hand, the number of new cases was 24.6 per 1 million people on March 27, 2020, in Turkey. The log-cubic model proposed in this study has been set forth to obtain successful results for aforementioned countries, as well as to estimate the course of the COVID-19 outbreak. Other factors such as climacteric factors and genetic differences, which may have an impact on viral spreading and transmission, would also have strengthened the model prediction capacity.

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<![CDATA[Mapping of Medical Microbiology Content in a Clinical Presentation Curriculum]]> https://www.researchpad.co/article/Nde6441d4-8f31-49fb-b330-13b06b8b6a2f

Clinically important microbes, and the pathogenesis, symptoms and diagnosis of their corresponding infectious diseases were integrated into clinical schemes within a clinical presentation curriculum. Decisions on microbe placement considered a variety of factors, including spaced reinforcement of major pathogens. We report here the map of our integrated medical microbiology curriculum.

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<![CDATA[Novel mutations in ADAMTS13 CUB domains cause abnormal pre‐mRNA splicing and defective secretion of ADAMTS13]]> https://www.researchpad.co/article/N76380691-b15a-42b2-851d-e9092647a2e7

Abstract

Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss‐of‐function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice‐site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre‐mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra‐large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.

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<![CDATA[Genomic variance of the 2019‐nCoV coronavirus]]> https://www.researchpad.co/article/N70fed03f-cc8a-4c9c-af70-25e422542a60

Abstract

There is a rising global concern for the recently emerged novel coronavirus (2019‐nCoV). Full genomic sequences have been released by the worldwide scientific community in the last few weeks to understand the evolutionary origin and molecular characteristics of this virus. Taking advantage of all the genomic information currently available, we constructed a phylogenetic tree including also representatives of other coronaviridae, such as Bat coronavirus (BCoV) and severe acute respiratory syndrome. We confirm high sequence similarity (>99%) between all sequenced 2019‐nCoVs genomes available, with the closest BCoV sequence sharing 96.2% sequence identity, confirming the notion of a zoonotic origin of 2019‐nCoV. Despite the low heterogeneity of the 2019‐nCoV genomes, we could identify at least two hypervariable genomic hotspots, one of which is responsible for a Serine/Leucine variation in the viral ORF8‐encoded protein. Finally, we perform a full proteomic comparison with other coronaviridae, identifying key aminoacidic differences to be considered for antiviral strategies deriving from previous anti‐coronavirus approaches.

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<![CDATA[Knowledge and practices regarding Middle East Respiratory Syndrome Coronavirus among camel handlers in a Slaughterhouse, Kenya, 2015]]> https://www.researchpad.co/article/Nb9b0b1c9-d994-4a30-9bbd-b90d7d2dfab2

Abstract

Dromedary camels are implicated as reservoirs for the zoonotic transmission of Middle East Respiratory Syndrome coronavirus (MERS‐CoV) with the respiratory route thought to be the main mode of transmission. Knowledge and practices regarding MERS among herders, traders and slaughterhouse workers were assessed at Athi‐River slaughterhouse, Kenya. Questionnaires were administered, and a check list was used to collect information on hygiene practices among slaughterhouse workers. Of 22 persons, all washed hands after handling camels, 82% wore gumboots, and 65% wore overalls/dustcoats. None of the workers wore gloves or facemasks during slaughter processes. Fourteen percent reported drinking raw camel milk; 90% were aware of zoonotic diseases with most reporting common ways of transmission as: eating improperly cooked meat (90%), drinking raw milk (68%) and slaughter processes (50%). Sixteen (73%) were unaware of MERS‐CoV. Use of personal protective clothing to prevent direct contact with discharges and aerosols was lacking. Although few people working with camels were interviewed, those met at this centralized slaughterhouse lacked knowledge about MERS‐CoV but were aware of zoonotic diseases and their transmission. These findings highlight need to disseminate information about MERS‐CoV and enhance hygiene and biosafety practices among camel slaughterhouse workers to reduce opportunities for potential virus transmission.

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<![CDATA[Human coronaviruses in severe acute respiratory infection (SARI) cases in southwest India]]> https://www.researchpad.co/article/N5f0cf4a4-6ed2-48a8-b386-e6978a36a903

Acute viral respiratory infections (AVRI) are a leading cause of morbidity and mortality among all age groups globally. Except for Influenza virus and Respiratory Syncytial virus, mostly viral aetiology of AVRI remains undiagnosed. Lately, human coronaviruses (HCoVs) have emerged as an important aetiology of AVRI. A laboratory based retrospective cross sectional study was conducted in which respiratory samples (throat swabs) of patients (n = 864), with Influenza negative SARI, of all age groups between Jan 2011–Dec 2012 were tested for HCoVs including MERS‐CoV using Conventional and real time PCR assays. The prevalence of HCoV among SARI cases was 1.04% (9/864) [95% CI: 0.36–1.72]. Of these four (44.44%) were identified as HCoV OC43, three (33.33%) as HCoV NL63 and two (22.22%) as HCoV 229E. No HCoV HKU1 was detected. The samples were also negative for SARS‐CoV and MERS‐CoV. The results of this study documents low prevalence of human coronaviruses in SARI cases in south western India and the absence of highly pathogenic human coronaviruses. As the study included only SARI cases the prevalence reported could be an under estimate when it is extrapolated to community. J. Med. Virol. 88:163–165, 2016. © 2015 Wiley Periodicals, Inc.

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<![CDATA[Neotropical Bats from Costa Rica harbour Diverse Coronaviruses]]> https://www.researchpad.co/article/Nf753267c-673e-4498-a398-88e30ec7f59f

Summary

Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host–species barrier. For analysing coronavirus diversity in a bat species‐rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA‐dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavirus‐derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the α‐CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogenetically distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dissimilar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected.

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<![CDATA[Coronaviruses in guano from Pteropus medius bats in Peradeniya, Sri Lanka]]> https://www.researchpad.co/article/N90a30312-aaba-4238-9317-8e1ffbbc50e6

Summary

Bats are a unique group of mammals well suited to be hosts for emerging viruses. With current rates of deforestation and urbanization, redistribution of bat habitats to urban and suburban areas may bring bats into closer contact with livestock and humans. Common flying fox, Pteropus medius (previously known as Pteropus giganteus), forms large communal roosts on treetops, often in close proximity to human habitation in Sri Lanka. This report describes the detection of coronavirus RNA in P. medius bat guano collected in Peradeniya, Sri Lanka. These viruses had >97% nucleotide identity with coronaviruses detected in Cynopterus sphinx, Scotophilus heathii and S. kuhlii bats in Thailand. Pteropus medius is widespread in Asia and appears to excrete group D coronaviruses, which are hitherto confined to bats; however, these findings may have public health implications in the future.

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<![CDATA[Characterization oftrans- andcis-cleavage activity of the SARS coronavirus 3CLproprotease: basis for the in vitro screening of anti-SARS drugs]]> https://www.researchpad.co/article/N4bbdd442-2264-4f47-a2cc-cf3a1d710fe4

Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS‐CoV, was identified as the etiological agent of the disease. SARS‐CoV 3C‐like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS‐CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans‐cleavage and the cell‐based cis‐cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate‐binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln‐P1 in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell‐based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS‐CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti‐SARS drugs.

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<![CDATA[Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus]]> https://www.researchpad.co/article/N7b9bdcf3-c180-47ba-9601-11952a52b78f

The open reading frame 3 of the severe acute respiratory syndrome coronavirus (SARS‐CoV) genome encodes a predicted protein 3a, consisting of 274 amino acids, that lacks any significant similarities to any known protein. We generated specific antibodies against SARS protein 3a by using a synthetic peptide (P2) corresponding to amino acids 261–274 of the putative protein. Anti‐P2 antibodies and the sera from SARS patients could specifically detect the recombinant SARS protein 3a expressed in Escherichia coli and in Vero E6 cells. Expression of SARS protein 3a was detected at 8–12 h after infection and reached a higher level after ∼24 h in SARS‐CoV‐infected Vero E6 cells. Protein 3a was also detected in the alveolar lining pneumocytes and some intra‐alveolar cells of a SARS‐CoV‐infected patient's lung specimen. Recombinant protein 3a expressed in Vero E6 cells and protein 3a in the SARS‐CoV‐infected cells was distributed over the cytoplasm in a fine punctate pattern with partly concentrated staining in the Golgi apparatus. Our study demonstrates that SARS‐CoV indeed expresses a novel protein 3a, which is present only in SARS‐CoV and not in other known CoVs.

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<![CDATA[Tyrosine dephosphorylation of STAT3 in SARS coronavirus-infected Vero E6 cells]]> https://www.researchpad.co/article/Nd9baf976-21dc-43c2-96ef-01193fe3d532

Severe acute respiratory syndrome (SARS) has become a global public health emergency. p38 mitogen‐activated protein kinase (MAPK) and its downstream targets are activated in SARS coronavirus (SARS‐CoV)‐infected Vero E6 cells and activation of p38 MAPK enhances the cytopathic effects of SARS‐CoV infection. In addition, weak activation of Akt cannot prevent SARS‐CoV infection‐induced apoptosis in Vero E6 cells. In the present study, we demonstrated that signal transducer and activator of transcription (STAT) 3, which is constitutively phosphorylated at tyrosine (Tyr)‐705 and slightly phosphorylated at serine (Ser)‐727 in Vero E6 cells, was dephosphorylated at Tyr‐705 on SARS‐CoV infection. In addition to phosphorylation of p38 MAPK in virus‐infected cells, other MAPKs, i.e., extracellular signal‐regulated kinase (ERK) 1/2 and c‐Jun N‐terminal kinase (JNK), were phosphorylated. Although inhibitors of ERK1/2 and JNK (PD98059 and SP600125) had no effect on phosphorylation status of STAT3, inhibitors of p38 MAPK (SB203580 and SB202190) partially inhibited dephosphorylation of STAT3 at Tyr‐705. Tyr‐705‐phosphorylated STAT3 was localized mainly in the nucleus in mock infected cells, whereas STAT3 disappeared from the nucleus in virus‐infected cells. As STAT3 acts as an activator of transcription in the nucleus, these results suggest that STAT3 lacks its activity on transcription in SARS‐CoV‐infected Vero E6 cells.

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<![CDATA[Evolutional insights on uncharacterized SARS coronavirus genes]]> https://www.researchpad.co/article/Ndf56b0f1-87ea-4c72-8f22-f2c22dc232ab

The complete genome of the severe acute respiratory syndrome coronavirus (SARS‐CoV) and many of its variants has been determined by several laboratories. The genome contains fourteen predicted open reading frames (ORFs). However, a function had been clearly assigned for only six of these ORFs, in the viral replication, transcription and structural constituents. The others are herein referred to as uncharacterized ORFs (UC‐ORFs). Here, we try to provide a relational insight on those UC‐ORFs, suggesting that a number of them are remotely related to structural proteins of coronaviruses and other viruses infecting mammalian hosts. Surprisingly, several of the UC‐ORFs exhibit considerable similarity with other SARS‐CoV ORFs. These observations may provide clues on the evolution and genome dynamics of the SARS‐CoV.

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<![CDATA[Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system]]> https://www.researchpad.co/article/N012f17fd-bed5-45ee-bdf7-77fe1322e6a5

Virus‐like particles (VLPs) produced by recombinant expression of the major viral structural proteins could be an attractive method for severe acute respiratory syndrome (SARS) control. In this study, using the baculovirus system, we generated recombinant viruses that expressed S, E, M and N structural proteins of SARS‐CoV either individually or simultaneously. The expression level, size and authenticity of each recombinant SARS‐CoV protein were determined. In addition, immunofluorescence and FACS analysis confirmed the cell surface expression of the S protein. Co‐infections of insect cells with two recombinant viruses demonstrated that M and E could assemble readily to form smooth surfaced VLPs. On the other hand, simultaneous high level expression of S, E and M by a single recombinant virus allowed the very efficient assembly and release of VLPs. These data demonstrate that the VLPs are morphological mimics of virion particles. The high level expression of VLPs with correct S protein conformation by a single recombinant baculovirus offers a potential candidate vaccine for SARS.

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<![CDATA[Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis]]> https://www.researchpad.co/article/N5bee19c0-c885-4037-ac3b-0913b32af246

The function of Semliki Forest Virus nsP2 protease was investigated by site‐directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature‐sensitive virus strains, rendered the enzyme temperature‐sensitive in vitro as well. Five conserved residues were required for the proteolytic activity of Pro39. Changes affecting Cys478, His548, and Trp549 resulted in complete inactivation of the enzyme, whereas the replacements N600D and N605D significantly impaired its activity. The importance of Trp549 for the proteolytic cleavage specificity is discussed and a new structural motif involved in substrate recognition by cysteine proteases is proposed.

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<![CDATA[Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells]]> https://www.researchpad.co/article/N0fcea84b-edf4-4e77-823d-c664827967b2

Coronavirus envelope (E) protein is a small integral membrane protein with multi‐functions in virion assembly, morphogenesis and virus–host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS‐CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N‐ and C‐termini of the SARS‐CoV E protein are exposed to the cytoplasmic side of the membranes (NcytoCcyto). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N‐terminus exposed luminally and the C‐terminus exposed cytoplasmically (Nexo(lum)Ccyto). Intriguingly, a minor proportion of the SARS‐CoV E protein was found to be modified by N‐linked glycosylation on Asn 66 and inserted into the membranes once with the C‐terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS‐CoV E protein may provide a useful clue to the pathogenesis of SARS‐CoV.

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