In vitro anti-viral activity of hexetidine (Bactidol®) oral mouthwash against human coronavirus OC43 and influenza A (H1N1) virus

Background@#Mouthwashes are used to decrease oral cavity microbial load due to their antiseptic properties. Hexetidine is a broad-spectrum antiseptic used for minor infections of mucous membranes, and, as a 0.1% mouthwash for local infections and oral hygiene.@*Objectives@#This study determined the anti-viral activity of the mouthwash hexetidine (Bactidol®), specifically in reducing viral concentration of Human Coronavirus OC43 (HCoV- OC43; ATCC®VR-1558™) and Influenza A virus (IAV H1N1; clinical strain) cultured in cell lines.@*Methodology@#In-vitro suspension assay (ASTM E-1052-11) was used to evaluate the virucidal property of hexetidine. Tissue Culture Infective Dose or TCID50/ml in 25%, 50%, and 100% hexetidine concentration at 15- and 30-seconds were determined. Vero E6 and MDCK cell lines were utilized for HCoV OC43 and IAV H1N1, respectively.@*Results@#Hexetidine-treated cell lines achieved >80% survival rate for MDCK and Vero E6. Hexetidine reduced the infectivity of HCoV-OC43 and IAV H1N1 at 25%, 50%, and 100% concentrations by more than 80% at 15- and 30-seconds exposure times.@*Conclusion and Recommendation@#This in vitro study showed that hexetidine, even at diluted concentrations, reduced the infectivity of HCoV-OC43 and Influenza A virus H1N1 when used for 15 and 30 seconds. The antiviral activity of hexetidine mouthwash against the other virulent members of the Coronavirus Family, SARSCoV- 2 can be explored using the methods used in this in vitro study.

Therapeutic Effect of Gegentang Granules on Mouse Model with hCoV-229E Pneumonia and Hanshi Yidu Xifei Syndrome / 中国实验方剂学杂志

ObjectiveTo determine the therapeutic effect of Gegentang granules on a disease-syndrome mouse model combining human coronavirus 229E （hCoV-229E） pneumonia with Hanshi Yidu Xifei syndrome in vivo. MethodMice were randomly divided into normal group， infection group， cold-dampness group， model group， chloroquine phosphate group （0.18 g·kg-1）， interferon-α2b （IFN-α2b） group （1.83×106 U·kg-1）， Gegentang granules high-dose and low-dose groups （6.6， 3.3 g·kg-1） with 10 mice in each group. Cold-dampness environment and hCoV-229E infection were used for modeling， and the general status and lung index of mice in each group were observed. The viral load in lung tissue was detected by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR）， the pathological changes in lung tissue were evaluated by hematoxylin-eosin （HE） staining， the levels of serum gastrointestinal hormones and inflammatory factors in lung tissue were detected by enzyme-linked immunosorbent assay （ELISA）， and the percentage of peripheral blood lymphocytes was detected by flow cytometry. ResultComparing with model group， Gegentang granules could significantly alleviate the physical signs of Hanshi Yidu Xifei syndrome， including listlessness， weakness of limbs， sticky stool， etc. Comparing with model group， Gegentang granules high-dose group significantly reduced lung index， histopathological score of interstitial lung and bronchus， and the level of serum motilin （P<0.05， P<0.01）， two doses of Gegentang granules could significantly increase the level of serum gastrin （P<0.05， P<0.01）， the percentage of CD4+， CD8+ T lymphocytes in peripheral blood （P<0.05， P<0.01）， and the level of tumor necrosis factor-α （TNF-α） in lung tissue was significantly decreased （P<0.01）， and the level of interleukin-6 （IL-6） showed decreasing tendency. ConclusionGegentang granules has therapeutic effect on model mice. It can improve the appearance and behavior characterization， regulate the level of gastrointestinal hormones， decrease lung index and histopathological score， and possibly play an immunomodulatory role by inhibiting the expression of inflammatory cytokines in lung tissue and restoring the percentage of peripheral blood lymphocytes.

Therapeutic Effect of Gegentang Granules on Mouse Model with hCoV-229E Pneumonia and Hanshi Yidu Xifei Syndrome / 中国实验方剂学杂志

ObjectiveTo determine the therapeutic effect of Gegentang granules on a disease-syndrome mouse model combining human coronavirus 229E （hCoV-229E） pneumonia with Hanshi Yidu Xifei syndrome in vivo. MethodMice were randomly divided into normal group， infection group， cold-dampness group， model group， chloroquine phosphate group （0.18 g·kg-1）， interferon-α2b （IFN-α2b） group （1.83×106 U·kg-1）， Gegentang granules high-dose and low-dose groups （6.6， 3.3 g·kg-1） with 10 mice in each group. Cold-dampness environment and hCoV-229E infection were used for modeling， and the general status and lung index of mice in each group were observed. The viral load in lung tissue was detected by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR）， the pathological changes in lung tissue were evaluated by hematoxylin-eosin （HE） staining， the levels of serum gastrointestinal hormones and inflammatory factors in lung tissue were detected by enzyme-linked immunosorbent assay （ELISA）， and the percentage of peripheral blood lymphocytes was detected by flow cytometry. ResultComparing with model group， Gegentang granules could significantly alleviate the physical signs of Hanshi Yidu Xifei syndrome， including listlessness， weakness of limbs， sticky stool， etc. Comparing with model group， Gegentang granules high-dose group significantly reduced lung index， histopathological score of interstitial lung and bronchus， and the level of serum motilin （P<0.05， P<0.01）， two doses of Gegentang granules could significantly increase the level of serum gastrin （P<0.05， P<0.01）， the percentage of CD4+， CD8+ T lymphocytes in peripheral blood （P<0.05， P<0.01）， and the level of tumor necrosis factor-α （TNF-α） in lung tissue was significantly decreased （P<0.01）， and the level of interleukin-6 （IL-6） showed decreasing tendency. ConclusionGegentang granules has therapeutic effect on model mice. It can improve the appearance and behavior characterization， regulate the level of gastrointestinal hormones， decrease lung index and histopathological score， and possibly play an immunomodulatory role by inhibiting the expression of inflammatory cytokines in lung tissue and restoring the percentage of peripheral blood lymphocytes.

Aspectos clínicos, epidemiológicos y cardiovasculares en niños convalecientes por COVID 19 en Villa Clara, Cuba / Clinical, epidemiological and cardiovascular aspects in children convalescents of COVID-19 in in Villa Clara province, Cuba

Introducción: La COVID-19, enfermedad causada por Coronavirus SARS-CoV 2, ha devenido en pandemia en 185 países. Cuba la ha enfrentado sin informar fallecidos en edad pediátrica Objetivo: Examinar aspectos clínicos, epidemiológicos y cardiovasculares en menores de 18 años en período de convalecencia por COVID 19. Métodos: estudio descriptivo transversal en 36 pacientes diagnosticados de marzo a mayo de 2020 en Villa Clara, Cuba y evaluados posterior al alta clínica y epidemiológica en consulta de cardiología del Hospital Universitario "José Luis Miranda". La muestra no probabilística, se conformó por los que asistieron a consulta. Se estudiaron variables: edad, sexo, peso, talla, antecedentes patológicos y epidemiológicos, período de hospitalización, examen físico, exámenes complementarios iniciales y en la convalecencia. Se utilizaron técnicas de estadística descriptiva. Resultados: Predominaron edades mayores de 10 años (60,0 por ciento), sexo femenino (55,5 por ciento), estado nutricional óptimo sin enfermedad crónica previa. En Santa Clara y Camajuaní se presentaron eventos de transmisión local con mayores tasas de incidencia. La mayoría de los niños se encontraban asintomáticos, detectados por historia epidemiológica de contactos, de confirmados y sospechosos (89,0 por ciento). Las alteraciones en fases iniciales de convalecencia fueron miocarditis y pericarditis (13,8 por ciento). Conclusiones: En este estudio se detectaron complicaciones cardiovasculares secundarias a infección viral por coronavirus SARS COV 2, con criterios epidemiológicos, clínicos, electrocardiográficos, radiológicos de tórax y ecocardiográficos(AU)

Introduction: COVID-19 is caused by SARS-CoV-2 coronavirus and it has become a pandemic in 185 countries. Cuba has faced this pandemic without having any deaths in the pediatric ages. Objective: Examine clinical, epidemiological and cardiovascular aspects in patients under 18 years' old who are convalescents of COVID-19. Methods: Descriptive cross-sectional study in 36 patients diagnosed with COVID-19 from March to May, 2020 in Villa Clara province, Cuba; whom were attended after the clinical and epidemiological discharge in the Cardiology consultation of "José Luis Miranda" University Hospital in July, 2020.The non-probabilistic sample was formed with the patients that went to the consultation. The variables studied were: age, sex, weight, size, personal and epidemiological pathologic background, period of hospitalization, physical exam, initial and during recovery's complementary tests. The results were expressed in absolute and relative values. Results: There was a predominance of patients older than 10 years (60.0 percent), female sex (55.5 percent), optimal nutritional state and no chronic diseases. Santa Clara and Camajuaní presented events of local transmission and showed higher incidence rates. Most of the patients there were asymptomatic, and were detected mainly due to the epidemiological record of confirmed and suspicious cases (89.0 percent). Complications in the initial phases of convalescents were myocarditis and pericarditis (13.8 percent). Conclusions: There were detected cardiovascular complications as a consequence of the viral infection by SARS-CoV-2 coronavirus, with epidemiological, clinical, electrocardiographic, thorax radiology and echocardiographic criteria(AU)

Papel de las pruebas rápidas (POCT) en el diagnóstico del SARS-COV-2, agente causal de COVID-19 / Role of Rapid Tests (POCT) in the Diagnosis of SARS-VOC-2, Causal Agent of COVID-19

Resumen El estándar de oro actual para la detección de SARS-CoV-2, agente causal de la pandemia de neumonía atípica (COVID-19) que apareció por primera vez en la ciudad de Wuhan (provincia de Hubei, China) en diciembre de 2019 (1), es la RT-qPCR. El protocolo estándar implica la transcripción inversa de ARN de SARS-CoV-2 en cadenas de ADN complementarias (ADNc), seguida de la amplificación de regiones específicas del ADNc. Este procedimiento demanda varias horas para ser completado y deriva en que la información final del estado de la infección pueda demorar hasta 24 horas. Ante la necesidad de disminuir el riesgo de una posible propagación viral dentro de la población originada por la rápida transmisión del SARS-CoV-2, se ha buscado prevenir el contagio, la propagación nosocomial y la transmisión comunitaria posterior, a través de la identificación rápida de casos sospechosos, y predecir las posteriores ondas infecciosas de recurrencia viral. Para esto, se vienen desarrollando métodos de laboratorio rápidos o point of care testing (POCT), que disminuyen el tiempo de diagnóstico y minimizan el riesgo de contagio por parte de los operadores.

Abstract The gold test to detect SARS-CoV-2, the etiologic agent that leads to the pandemic of atypical pneumonia (COVID 2019) that first appeared in Wuhan City, Hubei Province of China in December 2019 (1), is the RT-qPCR. The standard protocol involves reverse transcription of SARS-CoV-2 RNA into complementary DNA strands (cDNA), followed by the amplification of cDNA specific regions, a procedure that takes several hours to complete and which results in the final information from the infection status can take up to 24 hours. For this reason, and due to the need to reduce the risk of possible viral spread within the population caused by the fast transmission of SARS-CoV-2, in order to prevent nosocomial spread and subsequent community transmission through the quick identification of suspected cases, and to predict the further infectious waves of viral recurrence, rapid laboratory methods or Point of Care Testing (POCT) are being developed to reduce the diagnosis time and minimize the risk of contagion by the operators. These tests are discussed below.

El nuevo coronavirus que llegó de Oriente: análisis de la epidemia inicial en México / The new coronavirus that came from the East: analysis of the initial epidemic in Mexico

Resumen Introducción: A partir del 23 de marzo de 2020, en México se declaró la suspensión de actividades no esenciales en todo el país para mitigar la diseminación de la pandemia de COVID-19. Objetivo: Analizar los datos sobre los primeros 1510 casos de COVID-19 confirmados por laboratorio en México, describir la distribución geográfica de la enfermedad y su dinámica de transmisión. Método: Descripción de los primeros casos de COVID-19 con prueba positiva de RT-PCR en tiempo real, así como evaluación de las medidas epidemiológicas, incidencia acumulada, razón de contagios y tasas de mortalidad y letalidad durante el primer mes de la epidemia. Resultados: La edad promedio fue de 43 años y 58 % fue del sexo masculino; 44 % de los casos iniciales fue importado. La letalidad en la población durante el primer mes pasó de 1.08 a 3.97 por 100 casos; sin embargo, la tendencia es lineal y similar a la observada en Europa. Conclusiones: En México se está aplicando el distanciamiento social, pero aún se requieren estudios sobre la dinámica de la epidemia, la transmisión de persona a persona, la incidencia de infecciones subclínicas y la supervivencia de los enfermos.

Abstract Introduction As of March 23, 2020, suspension of non-essential activities was declared in Mexico throughout the country in order to mitigate the spread of the COVID-19 pandemic. Objective: To analyze data on the first 1510 laboratory-confirmed cases of COVID-19 in Mexico, and to describe the geographical distribution of the disease and its transmission dynamics. Method: Description of the first COVID-19 cases with real-time RT-PCR-positive test, as well as evaluation of epidemiological measures, cumulative incidence, rate of transmission, and mortality and lethality rates during the 1st month of the epidemic. Results: Average age was 43 years, and 58% were males; 44% of initial cases were imported. Lethality in the population during the 1st month went from 1.08 to 3.97 per 100 cases; however, the trend is linear and similar to that observed in Europe. Conclusions: In Mexico, social distancing is being applied, but studies are still required on the dynamics of the epidemic, person-to-person transmission, incidence of subclinical infections, and patient survival.

Research progress of intervention of Chinese herbal medicine and its active components on human coronavirus / 中国中药杂志

The outbreak caused by 2019 novel coronavirus(2019-nCoV) is still spreading, posing a great threat to the safety and health of general population. However, there have not been any effective drugs for treatment, with symptomatic treatment and prevention prevailing. The treatment plans of severe acute respiratory syndrome(SARS) and Middle East respiratory syndrome(MERS) are often used for reference in clinic. The advantages of traditional Chinese medicine(TCM) in treating SARS and MERS are that it can intervene and block the progression of disease in early stage, significantly reduce symptoms, shorten the treatment duration of patients, reduce complications and side effects caused by hormone therapy. The coronavirus disease 2019(COVID-19) belongs to the category of TCM epidemic diseases. Chinese patent medicines and prescriptions in medical observation and clinical treatment were recommended in the "pneumonia diagnosis and treatment plan for new coronavirus infection"(trial version fifth) of the National Health Commission of the People's Republic of China. Qingfei Paidu Decotion was recommended for the treatment of COVID-19 by the National Health Commission of the People's Republic of China and National Administration of Traditional Chinese Medicine. TCM shows good clinical efficacy and great potential in the treatment of COVID-19. Previous studies of TCM have shown broad-spectrum antiviral activity, providing a variety of sources for the discovery of new antiviral drugs. In this paper, we reviewed traditional Chinese medicines and its active ingredients in the hope of bringing novel inspirations to the drug screening and clinical treatment for COVID-19.

Intervening Effect of Promoting Lung and Resolving Phlegm Method on Mouse Model Combining Disease and Syndrome of Human Coronavirus Pneumonia with Cold-dampness Pestilence Attacking Lung / 中国实验方剂学杂志

Objective:To observe the therapeutic effect of Kesuting syrups and Keqing capsules, which have the function of promoting lung and resolving phlegm, on a mouse model combining disease and syndrome of human coronavirus pneumonia with cold-dampness pestilence attacking lung. Method:The therapeutic effects of Kesuting syrups (the doses of 22, 11 mL·kg-1) and Keqing capsules (the doses of 1.155, 0.577 5 g·kg-1) on this model were evaluated by the inflammatory changes of lung tissue, the expression of viral nucleic acid, the contents of inflammatory factors [interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α and interferon-γ (IFN-γ)], and the percentages of immune cells in peripheral blood (CD4+ T cells, CD8+ T cells and B cells). Result:Compared with the model group, high- and low-dose groups of Keqing capsules and Kesuting syrups could significantly reduce the inflammatory damage in the lung tissues of mice, Keqing capsules could significantly increase the percentages of CD4+ T cells, CD8+ T cells and B cells in peripheral blood, Keqing capsules and Kesuting syrups could reduce the expression levels of IL-6, IL-10, TNF-α and IFN-γ, inhibit the viral load in lung tissue, as well as improve the pathogenic manifestations of lung tissue. Conclusion:As the first-line drugs for novel coronavirus pneumonia, Keqing capsules and Kesuting syrups have significant therapeutic effect on the mouse model combining disease and syndrome of human coronavirus pneumonia with cold-dampness pestilence attacking lung, and the mechanism may be related to regulating immune function and reducing cytokine storm.

Study on therapeutic effect of Chaiyin Particles on combining disease with syndrome of human coronavirus pneumonia with pestilence attacking lung syndrome based on regulation of immune function / 中国中药杂志

According to the classification of traditional Chinese medicine syndromes of coronavirus disease 2019 by the national competent authority, this study determined that human coronavirus 229 E(HCoV-229 E) was infected in a mouse model of cold and dampness syndrome, so as to build the human coronavirus pneumonia with pestilence attacking lung syndrome model. The model can simulate the traditional Chinese medicine treatment of common disease syndromes in Coronavirus Disease 2019 Diagnosis and Treatment Program(the sixth edition for trial). Specific steps were as follows. ABALB/c mouse model of cold and dampness syndrome was established, based on which, HCoV-229 E virus was infected; then the experiment was divided into normal control group, infection control group, cold-dampness control group, cold-dampness infection group(the model group), high-dose Chaiyin Particles group(8.8 g·kg~(-1)·d~(-1)), and low-dose Chaiyin Particles group(4.4 g·kg~(-1)·d~(-1)). On the day of infection, Chaiyin Particles was given for three consecutive days. Lung tissues were collected the day after the last dose, and the lung index and inhibition rate were calculated. The nucleic acid of lung tissue was extracted, and the HCoV-229 E virus load was detected by Real-time fluorescent quantitative RT-PCR. Blood leukocytes were separated, and the percentage of T and B lymphocytes was detected by flow cytometry. Lung tissue protein was extracted, and IL-6, IL-10, TNF-α and IFN-γ contents were detected by ELISA. High and low-dose Chaiyin Particles significantly reduced the lung index(P<0.01) of mice of human coronavirus pneumonia with pestilence attacking the lung syndrome, and the inhibition rates were 61.02% and 55.45%, respectively. Compared with the model control group, high and low-dose Chaiyin Particles significantly increased cross blood CD4~+ T lymphocytes, CD8~+T lymphocytes and total B lymphocyte percentage(P<0.05, P<0.01), and reduced IL-10, TNF-α and IFN-γ levels in lungs(P<0.01). In vitro results showed that TC_(50), TC_0, IC_(50) and TI of Chaiyin Particles were 4.46 mg·mL~(-1), 3.13 mg·mL~(-1), 1.12 mg·mL~(-1) and 4. The control group of in vitro culture cells had no HCoV-229 E virus nucleic acid expression. The expression of HCoV-229 E virus nucleic acid in the virus control group was 1.48×10~7 copies/mL, and Chaiyin Particles significantly reduced HCoV-229 E expression at doses of 3.13 and 1.56 mg·mL~(-1), and the expression of HCoV-229 E nucleic acid was 9.47×10~5 and 9.47×10~6 copies/mL, respectively. Chaiyin Particles has a better effect on the mouse model with human coronavirus pneumonia with pestilence attacking the lung syndrome, and could play a role by enhancing immunity, and reducing inflammatory factor expression.

Research progress of the role of angiotensin-converting enzyme 2 (ACE2) in the highly pathogenic human coronavirus pneumonia / 中国胸心血管外科临床杂志

@#A novel coronavirus (SARS-CoV-2) that broke out at the end of 2019 is a newly discovered highly pathogenic human coronavirus and has some similarities with severe acute respiratory syndrome coronavirus (SARS-CoV). Angiotensin-converting enzyme 2 (ACE2) is the receptor for infected cells by SARS-CoV. SARS-CoV can invade cells by binding to ACE2 through the spike protein and SARS-CoV-2 may also infect cells through ACE2. Meanwhile, ACE2 also plays an important role in the course of pneumonia. Therefore the possible role of ACE2 in SARS and coronavirus disease 2019 (COVID-19) is worth discussing. This paper briefly summarized the role of ACE2 in SARS, and discussed the possible function of ACE2 in COVID-19 and potential risk of infection with other organs. At last, the function of ACE2 was explored for possible treatment strategies for SARS. It is hoped to provide ideas and theoretical support for clinical treatment of COVID-19.

Advance in human coronaviruses research of host interactions / 中华实用儿科临床杂志

2019-novel coronavirus (2019-nCoV) is a highly pathogenic human CoV that first emerged in Wuhan in 2019.2019-nCoV has a zoonotic origin and poses a major threat to public health.However, little is known about the viral factors contributing to the high virulence of 2019-nCoV.Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors.Human coronaviruses (HCoVs) are known respiratory pathogens associated with a range of respiratory infection.In the past 17 years, the onset of 2019-nCoV, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have thrust HCoVs into spotlight of the research community due to their high pathogenicity in humans.The recent study of HCoVs-host interactions has contributed extensively to our understanding of infection pathogenesis of 2019-nCoV.This review discuss various host physiopathologic mechanism, such as apoptosis, innate immunity, endoplasmic reticulum (ER) stress response, mitogen-activated protein kinase (MAPK) pathway and nuclear factor kappa B (NF-κB) pathway that may be modulated by HCoVs and provides evidence for the intensive investigate of 2019-nCoV infection.

Detection of four human coronaviruses in respiratory infections in children in Fuzhou, China / 中华实验和临床病毒学杂志

Objective@#In this study, we tested for the presence of four human coronaviruses (HCoVs) in children with respiratory tract disease in Fuzhou, Fujian, China.@*Methods@#Nasopharyngeal aspirates were collected from children with respiratory tract disease from Nov, 2007 to Jan, 2015. A total of 266 clinical samples were tested for HCoVs using reverse-transcription polymerase chain reaction (RT-PCR). The positive products were sequenced and compared with those in GenBank by BLAST. The positive samples were then tested for HCoV-HKU1 and HCoV-NL63 using RT-PCR method . We compared the 440 bp pol gene sequence of the 8 HCoV isolates in Fuzhou, China to other HCoV isolates documented in the GenBank database by using MEGA software version 6.06 and the neighbor-joining method .@*Results@#HCoVs were detected in 8 patients (3.0%) out of the 266 children. Two of 266 (0.38%) were positive for HCoV-HKU1; 1 of 266(0.38%)were positive for HCoV-NL63; 1 of 266 (0.38%) were positive for HCoV-229E; 4 of 266 (1.5%)were positive for HCoV-OC43. All of children who were positive for HCoV had respiratory illness. Two HCoV-HKU1 were found to co-infect with human parainfluenza virus type 3 (HPIV-3). The 8 HCoV strains in our study fell into four clusters. Two strains of HCoV-HKU1 were genotype A.@*Conclusions@#HCoV infections were probably associated with upper and lower respiratory illness in children. Additional studies are needed to investigate the potential roles of these HCoVs in diseases.

Molecular evolution of human coronavirus in Guangzhou from 2010 to 2012 / 中华实验和临床病毒学杂志

Objective@#To investigate the molecular evolution characteristics of human coronavirus (HCoV) subtypes in patients with fever and respiratory tract infection in Guangzhou from 2010 to 2012.@*Methods@#Partial fragments of NP, RdRp and S genes of HCoV-OC43, HCoV-229E and HCoV-NL63 positive samples were amplified by RT-PCR and sequencing. Bioinformatics software, including Bio-edit, Mega4.0 and Clustal1.83 were used for comparison and analysis of NP, RDRp and S gene sequences. Molecular evolutionary tree of different gene regions of HCoV-OC43, HCoV-229E and HCoV-NL63 were built.@*Results@#No remarkable variation or recombinant strain of HCoV-OC43, HCoV-229E and HCoV-NL63 was found in Guangzhou during 2010—2012. The HCoV-OC43 substrains were genetically closest to the strains found in Belgium and Hong Kong (GenBank accession number JN129834 and AY903460). HCoV-229E substrains were genetically closest to those found in Amsterdam (GenBank accession number JX503060) and HCoV-NL63 most genetically close to those in Amsterdam and Beijing (GenBank accession number JX104161 and DQ445911). The NP and RDRp genes of all subtypes were highly conserved, while S gene was more variable.@*Conclusions@#There were at least 3 substrains of HCoV-OC43, HCoV-229E and HCoV-NL63 epidemic in Guangzhou during 2010—2012, and no remarkable variation or recombinant viral strain was found. The NP and RDRp genes of all subtypes were highly conserved and can be used in virus detection, while S gene was more variable and suitable for phylogenetic and variation study.

Preliminary mechanism study of HCoV-OC43 escape from human dendritic cell immune elimination / 中国免疫学杂志

Objective:To study the possible immune escape mechanisms of HCoV-OC43 from human dendritic cells(DC).Methods:HCoV-OC43 was isolated from clinical specimen using BSC-1 cells and identified by Real-time PCR,and the cytopathic effect was observed by phase contrast microscope.DCs were induced in vivo using hu-GM-CSF and IL-4 cytokines,and after 7 days of differentiation,DCs were infected by HCoV-OC43.The morphology of HCoV-OC43 infected DC was observed by transmission electron microscope,and the cytokines related to DC functions were detected by Real-time PCR after infection.DC proportion and function related co-stimulatory molecules were analyzed by flow cytometry.Results:In vitro HCoV-OC43 infected human DC model was successfully built.HCoV-OC43 can infect DC and generate immune response of DC in vitro,but no virus nucleonic acid could be detected in culture supernatant.The DC expression of IFN-α,IFN-β,CCL3 and CCL5 were significant decreased when infected with HCoV-OC43,but the expression of costimulatory molecules including HLA-DR,CD1c and CD86 were not affected by HCoV-OC43 infection.Conclusion:Human DC could be infected by HCoV-OC43 and generate immune response,but could not produce progeny virus.HCoV-OC43 may escape from immune response by suppressing the expression of IFN-α and other inflammatory cytokines and chemokines in DC.

Clinical characteristics of human coronavirus in children with acute lower respiratory tract infection / 中华实用儿科临床杂志

Objective To describe the clinical characteristics of acute lower respiratory tract infection (ALRTI)caused by human coronavirus (HCoV)in children.Methods Three thousand five hundred and three hospi-talized children diagnosed with ALRTI in Beijing Children′s Hospital from March 2007 to February 201 3 were re-viewed.Nasopharyngeal aspirate(NPA)specimen was collected from each patient.Reverse transcription (RT)-poly-merase chain reaction(PCR)methods were applied to detect common respiratory viruses including respiratory syncytial virus (RSV),rhinovirus (RV),parainfluenza virus (PIV)type 1 -4,influenza virus type A and B (IFA,IFB),adeno-virus (AdV),enterovirus (EV),HCoV,human metapneumovirus (hMPV)and human bocavirus (HBoV).Serum anti-bodies of mycoplasma and sputum bacterial culture were also detected.Only HCoV positive patients were analyzed in this study.Results Eleven of 3 503 patients were proved as HCoV -positive in NPA specimens.Of the 1 1 children,8 cases were male and 3 cases were female (2.71 .0).The median age was 3 months.The clinical symptoms of HCoV infection included cough (1 1 /1 1 cases,1 00.0%),wheezing (1 0 /1 1 cases,90.9%),fever (6 /1 1 cases,54.5%)and poor appetite (7 /1 1 cases,63.6%).Wheezing (8 /1 1 cases,72.7%)and moist rale in inspiratory phase (5 /1 1 ca-ses,45.4%)could be heard.Most patient′s chest X -ray showed bronchopneumonia.Full blood count displayed that leukocyte was in the normal range.Conclusions Respiratory tract infection with HCoV -positive will be easier to spread to ALRTI,especially in infants less than 1 year old.The symptoms include fever,cough and wheezing,but poor appetite and diarrhea can also be detected.

Construction of recombinant infectious clones of HCoV-OC43 expressing green fluorescent protein and viral rescue / 中华微生物学和免疫学杂志

Objective To investigate the feasibility of using recombinant infectious clones of hu-man coronavirus OC43 (HCoV-OC43) as a vector for the expression of exogenous genes and to analyze the insertion sites. Methods Based upon pBAC-OC43FL, a full-length cDNA infectious clone of HCoV-OC43, three recombinant expression plasmids ( pBAC-OC43-GFPΔNS2, pBAC-OC43-GFPΔNS12. 9 and pBAC-OC43-N-GFP) were respectively constructed by replacing NS2 and NS12. 9 genes with the reporter gene en-coding the green fluorescent protein ( GFP ) and inserting the reporter gene after the N gene by using the overlapping-PCR and in vitro ligation. Reverse genetics techniques were used for viral rescue. All of the res-cued virus strains were characterized by immunofluorescence assay ( IFA) and Western blot ( WB) assay af-ter transfecting BHK-21 cells with the recombinant viruses. Results Two recombinant viruses, OC43-GFPΔNS2 and OC43-GFPΔNS12. 9, could be successfully rescued by transfection the BHK-21 cells with pBAC-OC43-GFPΔNS2 and pBAC-OC43-GFPΔNS12. 9 plasmids. The expressed GFP was observed in BHK-21 cells transfected with pBAC-OC43-GFPΔNS2 or pBAC-OC43-GFPΔNS12. 9 plasmids, but not in the cells transfected with the pBAC-OC43-N-GFP plasmid. An efficient and stable expression of GFP was observed in the pBAC-OC43-GFPΔNS2 plasmid-transfected cells. The 10th generation of OC43-GFPΔNS2 virus was ob-tained after repeated freezing and thawing. The expression of GFP and N protein were detected in cells infec-ted with the OC43-GFPΔNS2 virus after 10 passages. Conclusion The NS2 gene of HCoV-OC43 could be used as a promising insertion site of the pBAC-OC43 FL infectious clone for the expression of exogenous genes. This study might provide a platform for further researches on the replication of HCoV-OC43 and the development of human coronavirus-based vectors.

Characterization of complete genome sequences of human coronavirus NL63 strains derived from Chinese patients / 中华微生物学和免疫学杂志

Objective To sequence and analyze the complete genome of two human coronavirus NL63 (HCoV-NL63) strains collected from Beijing Children Hospital .Methods Eighteen pairs of primers were designed according to the gene sequences of HCoV-NL63 reference strain ( HCoV-NL63_Amsterdam 1) and used to amplify the target fragments covering the complete genome of HCoV-NL63 strains.Rapid ampli-fication of cDNA ends ( RACE) and RT-PCR assays were used to amplify the full length genome of HCoV-NL63 strains.Phylogenetic analysis was conducted by using Mega 5.0 software.Results The complete ge-nome sequences of the two HCoV-NL63 strains were 27 538 bp in length, showing a homology of 99.1%in nucleotide sequences .There were 15 consecutive bases deleted from 1a region.The systematic phylogenetic analysis demonstrated that four genotypes of NL 63 virus including A , B, C and D have been identified , and two domestic strains were belonged to the new genotype D .Conclusion The complete genome sequences of two domestic HCoV-NL63 isolates were identified for the first time .This study provided evidence for further investigation on molecular epidemiology of HCoV-NL63 in China .

Infections with human coronaviruses NL63 and OC43 among hospitalised and outpatient individuals in São Paulo, Brazil

The incidence and clinical features of human coronaviruses (HCoVs) among Brazilian patients with respiratory illness are not well known. We investigated the prevalence of HCoVs among Brazilian outpatients and hospitalised patients with respiratory illnesses during 2009 and 2010. To identify the HCoVs, pancoronavirus and species-specific reverse-transcriptase polymerase chain reaction assays were performed. Five of 394 samples were positive for HCoVs (1.2%): 1/182 (0.5%) outpatients and 4/212 (1.8%) hospitalised patients. The OC43 and NL63 HCoVs were identified. Two patients were admitted to the intensive care unit. Underlying chronic disease was reported in cases and one diabetic adult died. HCoVs can cause lower respiratory infections and hospitalisation. Patients with pre-existing conditions and respiratory infections should be evaluated for HCoV infections.

Association between Kawasaki disease and acute respiratory viral infections / 소아과

PURPOSE: The etiology of Kawasaki disease (KD) is still unknown. Recently, an association between human coronavirus NL63 (HCoV-NL63) and KD was implicated. Hence, we attempted to determine the association between KD and acute respiratory viral infections. METHODS: Nasopharyngeal aspirate samples were obtained from 54 patients diagnosed with KD at the Seoul National University (SNU) Children's Hospital and SNU-Bundang Hospital between October 2003 and September 2006. Viral diagnoses of 11 respiratory viruses were made using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR): respiratory syncytial virus (RSV), adenovirus, rhinovirus (RV), parainfluenza viruses (PIVs) 1 and 3, influenza viruses (IFVs) A and B, human metapneumovirus (HMPV), human bocavirus (HBoV), HCoV OC43/229E, and HCoV-NL63. Clinical data were reviewed retrospectively. RESULTS: The median age was 32 months (6 months-10.4 years). Respiratory symptoms were observed in 37 patients (69%). The following respiratory viruses were identified in 12 patients (22%): RV (n=4), PIV-3 (n=2), HBoV (n=2), and adenovirus, RSV, PIV-1, IFV-A, and HCoV-NL63 (n=1). Co-infection with PIV-3 and RV was observed in one patient. Respiratory symptoms were observed in 7 (58.3%) and 30 (71.4%) patients of the virus-positive and virus-negative groups (P>0.05). Response rate to intravenous immunoglobulin administration was 67% (n=8) and 86% (n=36) in the virus-positive and virus-negative groups (P>0.05). CONCLUSION: Respiratory symptoms were commonly observed in KD patients but the association between respiratory viruses and KD were not found. Large multicenter-based investigations are required to confirm the association between acute respiratory viral infections and KD.

Association between Kawasaki disease and acute respiratory viral infections / 소아과

PURPOSE: The etiology of Kawasaki disease (KD) is still unknown. Recently, an association between human coronavirus NL63 (HCoV-NL63) and KD was implicated. Hence, we attempted to determine the association between KD and acute respiratory viral infections. METHODS: Nasopharyngeal aspirate samples were obtained from 54 patients diagnosed with KD at the Seoul National University (SNU) Children's Hospital and SNU-Bundang Hospital between October 2003 and September 2006. Viral diagnoses of 11 respiratory viruses were made using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR): respiratory syncytial virus (RSV), adenovirus, rhinovirus (RV), parainfluenza viruses (PIVs) 1 and 3, influenza viruses (IFVs) A and B, human metapneumovirus (HMPV), human bocavirus (HBoV), HCoV OC43/229E, and HCoV-NL63. Clinical data were reviewed retrospectively. RESULTS: The median age was 32 months (6 months-10.4 years). Respiratory symptoms were observed in 37 patients (69%). The following respiratory viruses were identified in 12 patients (22%): RV (n=4), PIV-3 (n=2), HBoV (n=2), and adenovirus, RSV, PIV-1, IFV-A, and HCoV-NL63 (n=1). Co-infection with PIV-3 and RV was observed in one patient. Respiratory symptoms were observed in 7 (58.3%) and 30 (71.4%) patients of the virus-positive and virus-negative groups (P>0.05). Response rate to intravenous immunoglobulin administration was 67% (n=8) and 86% (n=36) in the virus-positive and virus-negative groups (P>0.05). CONCLUSION: Respiratory symptoms were commonly observed in KD patients but the association between respiratory viruses and KD were not found. Large multicenter-based investigations are required to confirm the association between acute respiratory viral infections and KD.