Acute Disease ; Case-Control Studies ; Child, Preschool ; Coronavirus NL63, Human ; isolation & purification ; Female ; Humans ; Infant ; Male ; Respiratory Tract Infections ; virology

PURPOSE: This study was performed to investigate the epidemiologic characteristics of human bocavirus (HBoV)-associated lower respiratory tract infections (LRTIs) in children. METHODS: Nasopharyngeal aspirate samples were obtained from 658 children who had been hospitalized for LRTIs in Seoul National University (SNU) Children's Hospital and SNU Bundang Hospital from March 2000 to September 2005. Multiplex RT-PCR was performed to detect 11 respiratory viruses including respiratory syncytial virus, adenovirus, rhinovirus, parainfluenza viruses 1 and 3, influenza viruses A and B, human metapneumovirus, HBoV, human coronavirus (HCoV) OC43/229E, and HCoV-NL63. Clinical data were reviewed retrospectively. RESULTS: Overall, respiratory viruses were detected in 325 (49.4%) among 658 patients. HBoV was detected in 62 cases (9.4%) and was responsible for 19.1% of virus-positive cases. HBoV was prevalent among infants and young children aged from 3 months to 5 years with the mean age of 25.3 months. Co-detection of HBoV and other respiratory viruses was observed in 37.1% which is significantly higher than average co-detection rate (12.3%) among overall virus-positive cases (P=0.000). HBoV was identified mainly in late spring and early summer from May to July. CONCLUSION: This study describes epidemiologic features of HBoV in Korean children compared with those associated with other respiratory viruses. HBoV was prevalent among LRTIs in childhood, especially in late spring and early summer season in Korea.
Adenoviridae ; Aged ; Bronchiolitis ; Child ; Coronavirus ; Coronavirus NL63, Human ; Human bocavirus ; Humans ; Infant ; Korea ; Metapneumovirus ; Orthomyxoviridae ; Paramyxoviridae Infections ; Pneumonia ; Respiratory Syncytial Viruses ; Respiratory Tract Infections ; Rhinovirus ; Seasons

A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63.
Colorimetry ; methods ; Coronavirus Infections ; diagnosis ; virology ; Coronavirus NL63, Human ; genetics ; isolation & purification ; DNA Primers ; genetics ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Reverse Transcription ; Sensitivity and Specificity

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.
Adenoviridae ; Coinfection ; Coronavirus NL63, Human ; Human bocavirus ; Humans ; Immunoglobulins ; Metapneumovirus ; Mucocutaneous Lymph Node Syndrome ; Orthomyxoviridae ; Paramyxoviridae Infections ; Respiratory Syncytial Viruses ; Respiratory Tract Infections ; Retrospective Studies ; Rhinovirus

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.
Adenoviridae ; Coinfection ; Coronavirus NL63, Human ; Human bocavirus ; Humans ; Immunoglobulins ; Metapneumovirus ; Mucocutaneous Lymph Node Syndrome ; Orthomyxoviridae ; Paramyxoviridae Infections ; Respiratory Syncytial Viruses ; Respiratory Tract Infections ; Retrospective Studies ; Rhinovirus

Viruses are the most common cause of lower respiratory tract infections (LRTIs) in infants and young children and are a major public health problem in this age group. Viruses were identified in 54.9-70.4% of hospitalized infants and children with LRTIs in Korea. The viral pathogens identified included respiratory syncytial virus (RSV) A and RSV B, influenza (Inf) A, Inf B, parainfluenza (PIV)1, PIV2, human bocavirus (hBoV), human rhinovirus (hRV), adenovirus (ADV), human metapneumovirus (hMPV), human coronavirus (hCoV)-OC 43, hCoV-229E, hCoV-NL63, hCoV-HKU1, and human enterovirus (hEV). Coinfections with > or =2 viruses were observed in 11.5-22.8% of children. The occurrence of LRTIs was the highest in the first year of life. The specific viruses are frequently associated with specific clinical syndromes of LRTIs. LRTIs caused by RSV were predominant among younger infants. hRV accounted for a larger proportion of LRTIs in young infants than ADV and hBoV. hMPV was frequently detected in children >24 months old. The number of hMPV infections peaked between February and May, whereas hRV was detected throughout the year. Thus far, hCoV is a less common respiratory pathogen in cases of ALRI and URI in Korean children.
Adenoviridae ; Child ; Coinfection ; Coronavirus ; Coronavirus NL63, Human ; Enterovirus ; Human bocavirus ; Humans ; Infant ; Influenza, Human ; Korea ; Metapneumovirus ; Paramyxoviridae Infections ; Public Health ; Respiratory Syncytial Viruses ; Respiratory System ; Respiratory Tract Infections ; Rhinovirus

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.
Animals ; Coronavirus Infections ; metabolism ; virology ; Coronavirus NL63, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Mice ; Mice, Inbred BALB C ; Protein Engineering ; Protein Structure, Tertiary ; Receptors, Virus ; metabolism ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

The spike (S) glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Four fragments coding spike proteins (S1, S2, RL and RS) from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus (Tiantan strain), and recombinant vaccinia viruses expressing four segments of spike proteins were generated (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS), respectively. Their expression location in cell and level were characterized using indirect immune fluorescence assay (IFA) and Western-Blot, respectively. The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification (glycosylation), and S1, RL and RS were mainly distributed in the cell membrane, while the S2 was mainly distributed in the cytoplasm. Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.
Base Sequence ; Blotting, Western ; Coronavirus NL63, Human ; chemistry ; Fluorescent Antibody Technique, Indirect ; Humans ; Membrane Glycoproteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Plasmids ; Recombinant Proteins ; biosynthesis ; Spike Glycoprotein, Coronavirus ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; biosynthesis ; genetics

PURPOSE: The aim of this study was to evaluate the severity of disease in children with acute bronchiolitis according to the type of infected virus. METHODS: From November 2007 to May 2015, 768 patients under 2 years of age who underwent real time-polymerase chain reaction of nasopharyngeal aspirates admitted to the Department of Pediatrics of Dongguk University Ilsan Hospital for acute bronchiolitis were enrolled. Severe bronchiolitis was defined as presence of one or more kinds among tachypnea, chest retraction, needs of O2 inhalation or ventilator care. RESULTS: The severity of bronchiolitis was increased with shorter fever duration (P < 0.001) and previous wheezing episodes (P = 0.005). In the case of single infection, respiratory syncytial virus (RSV) A only increased the severity of acute bronchiolitis (P = 0.012). However, the severity of illness decreased when RSV A coinfected with adenovirus (P = 0.034), human rhinovirus (P = 0.038), or human coronavirus NL63 (P = 0.042). On the other hand, when human rhinovirus was coinfected with enterovirus (P = 0.013) or parainfluenza 3 (P = 0.019), the severity was increased. When human metapneumovirus coinfected with human bocavirus, the severity was increased (P = 0.038). CONCLUSION: Acute bronchiolitis was associated with increased severity only when RSV A infected solely, but several viruses increased or decreased the severity when coinfection occurred. Therefore, it may be helpful in predicting the course of the acute bronchiolitis according to the affected virus.
Adenoviridae ; Bronchiolitis ; Child ; Coinfection ; Coronavirus NL63, Human ; Enterovirus ; Fever ; Hand ; Human bocavirus ; Humans ; Infant ; Inhalation ; Metapneumovirus ; Paramyxoviridae Infections ; Pediatrics ; Respiratory Sounds ; Respiratory Syncytial Viruses ; Rhinovirus ; Tachypnea ; Thorax ; Ventilators, Mechanical

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagy-inducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM's inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.
Apoptosis Regulatory Proteins ; antagonists & inhibitors ; genetics ; immunology ; Autophagy ; Beclin-1 ; Coronavirus NL63, Human ; genetics ; immunology ; Gene Expression Regulation ; HEK293 Cells ; HeLa Cells ; Host-Pathogen Interactions ; immunology ; Humans ; Immune Evasion ; Immunity, Innate ; Interferon-gamma ; genetics ; immunology ; Lysosomes ; metabolism ; virology ; MCF-7 Cells ; Membrane Fusion ; Membrane Proteins ; antagonists & inhibitors ; genetics ; immunology ; Microtubule-Associated Proteins ; genetics ; immunology ; Papain ; genetics ; immunology ; Phagosomes ; metabolism ; virology ; RNA, Small Interfering ; genetics ; immunology ; Signal Transduction ; Virus Replication