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Objective To explore novel methods for efficient respiratory viral infection of organoids by microinjection and polarity inversion techniques.Methods Lung tissue samples were obtained from 8-week-old male C57BL/6 mouse,and respiratory epithelial cells were extracted to establish a transwell organoid culture model.The green fluorescent protein(GFP)labeled influenza virus PR8(GFP-PR8)was quantitatively injected into organoids by improving the traditional microinjection platform,and morphologic changes in organoids and the immunofluorescence staining characteristics of tight junction proteins and microtubule proteins were observed.Polarity inversion apical-out(AO)was induced by suspension culture,and the morphological characteristics of polarity inversion was determined by HE staining.Normal and inverted organoids were infected with PR8,and the infection efficiency and expression differences of key pathway genes under different virus concentrations were observed.Results Ordinary organoids showed a significant increase in volume after microinjection.Following PR8 injection,the efficiency of infection was significantly higher in the apical region of organoids,accompanied by noticeable damage,as evidenced by significant down-regulation of tight junction proteins and microtubule protein expression.After suspension culture of the organoids,the polarity of ciliated cells gradually inverted outward over time,and the proportion of AO organoids stabilized on the 6th day.The efficiency of viral infection significantly increased in the inverted organoids,accompanied by significant cellular damage.After PR8 infection at 0.01 MOI,AO organoids showed significant changes in the inflammatory pathway and differentiation-related genes,with the opposite trend observed after higher concentration of PR8 infection.Conclusion Both polarity inversion and microinjection techniques significantly enhance the efficiency of influenza virus infection in organoids,thereby facilitating organoid widespread application in the field of respiratory tract infections.
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Objective:To analyze the status and epidemiological characteristics of respiratory virus infection in children with influenza-like illness in outpatient department, and to provide evidence for the prevention and treatment of children in this area.Methods:Nasopharyngeal swab samples were collected from children who attended the fever clinic of The Children′s Hospital, Zhejiang University School of Medicine due to influenza-like illness from July 2021 to March 2022, and six common respiratory virus nucleic acids were detected by reverse transcription-polymerase chain reaction (RT-PCR). The general information of the children was collected and grouped by gender and age (0-<6 months, 6-<12 months, 1-3< year-old, 3-<6 year-old , and ≥6 year-old), and the chi-square test was used for statistical analysis between the groups to explore the epidemic pattern of respiratory viruses.Results:A total of 739 cases (45.9%, 739/1 609) of respiratory viruses were detected from children with influenza-like illness, including 651 cases (40.5%, 651/1 609) of simple infection and 88 cases (5.5%, 88/1 609) of multiple infections. Respiratory syncytial virus (RSV) was detected in 18.6% (300/1 609), followed by influenza B virus (FluB) in 11.9% (192/1 609), adenovirus (ADV) in 8.3% (134/1 609), parainfluenza virus type 3 (PIV-3) in 7.6% (123/1 609), parainfluenza virus type 1 (PIV-1) in 4.9% (79/1 609), and influenza A virus (FluA) in 0.4% (6/1 609). Multiple infections including double or triple infections, with 81(92.0%, 81/88) cases of double infection and the most common being ADV+RSV (22.7%, 20/88) and 7 (8.0%, 7/88) cases of triple infection. There was a significant difference in the virus detection rate between the age groups (χ2=17.078, P=0.002), with the highest virus detection rate in the 3-<6 years of age group (49.7%, 286/575). Among the detection of simple infection, FluB had the highest detection rate in the ≥ 6 years of age group (26.6%, 98/369), and RSV and PIV-1 had the highest detection rate in the 3-<6 years of age group (20.0%, 115/575 and 5.9%, 34/575). The total monthly virus detection rate increased from 26.8% (37/138) in July to 63.0% (58/92) in January, and decreased to 46.1% (106/230) and 26.8% (37/138) in February and March. The detection rate of RSV was the highest from August to November, the detection rate of FluB was the highest from December to March, the detection rate of ADV increased in December and January, and the detection rate of PIV-3 increased from October to December; the detection rate of PIV-1 did not fluctuate significantly, and FluA was sporadically detected. Conclusions:RSV is the main respiratory virus in children with influenza-like illness. Most respiratory viruses are present as single infections. Multiple infections are more common in double infections. FluB, RSV and PIV-1 infections showed certain age distribution characteristics, especially in children over 3 years of age. The epidemic characteristics of respiratory virus infection show that the epidemic gradually peaks from summer to autumn and winter, and turns into an epidemic decline in spring. RSV was relatively prevalent in autumn, FluB was prevalent in winter and spring, ADV and PIV-3 were prevalent to varying degrees in winter, PIV-1 continued to circulate at a low level, and FluA did not present epidemic characteristics.
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Objective To investigate the clinical and epidemiological characteristics of Saffold virus (SAFV)infection in outpatient and hospitalized children with acute respiratory tract infections(ARI). Methods A total of 1060 clinical specimens were collected from children with ARI in the Affiliated Children's Hospital,Zhejiang University School of Medicine from March 2011 to February 2014, including 256 samples of throat swabs from outpatients,and 804 samples of trachea suctions from hospitalized patients. Real time PCR(RT-PCR)was performed to detect 5'UTR segment of SAFV.SPSS 17.0 software was used to analyze the test results and clinical data.Results The positive detection rates of SAFV in outpatients and hospitalized children with ARI were 2.3%(6/256)and 13.2%(106/804), respectively(χ2=24.147, P<0.01).Among the hospitalized children,the positive detection rates of SAFV in children <1 year,1-<3 years,3-<6 years and 6-12 years were 14.0%,11.2%,11.1% and 8.3%, respectively(χ2=1.845,P>0.05).The positive rates of SAFV in males and females were 12.7% and 17.7%(χ2=0.279,P>0.05).The detection rate of SAFV in autumn was highest(21.2%), followed by that in spring (14.6%),winter(9.5%)and summer(8.8%)(χ2=15.625, P<0.01).The co-infection rates with other respiratory pathogens of hospitalized and outpatients children were 76.4%(81/106)and 66.7% (4/6).Among the hospitalized patients, the rate of co-infection with respiratory syncytial virus was the highest(36.8%),followed by rhinovirus(27.4%), metapneumovirus(10.4%)and parainfluenza virus (10.4%).Among children with ARI,the fever rate of SAFV-positive cases was lower than that of SAFV-negative cases(χ2=4.069,P<0.05).Conclusions The detection rate of SAFV in hospitalized children with ARI is significantly higher than that in outpatients,and SAFV infection was dominated by co-infection. The prevalence of SAFV in the Hangzhou area presents a certain local epidemic pattern.
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Objective To investigate the epidemiology of respiratory viruses in children from Wuxi area.Methods A total of 2 747 cases of children diagnosed with acute respiratory infection in Wuxi during 2011 —2014 were collected.Reverse transcription-polymerase chain reaction was used to detect nine kinds of respiratory viruses,including influenza virus A (Flu A),influenza virus B (Flu B),parainfluenza virus (PIV)Ⅰ-Ⅳ,adenovirus (ADV),respiratory sycytial virus (RSV),human metaneumovirus (hMPV), human bocavirus (HBov),human coronaviruses (hCov)and human rhinovirus (HRV).The categorical data were compared using chi square test.Results A total of 856 among the 2 747 samples were tested positive for respiratory virus nucleic acid,with the positive rate of 31 .16%.The viral distribution was uneven in different seasons,and the infection peaked in winter and spring.The virus detection rate was highest in age 1 to 2 year group (up to 40.18%),and followed by age 6 to 12 year group (32.63%).Flu A virus was the most frequently detected virus,accounting for 7.54% (207/2 747);followed by PIV, accounting for 6.95 % (191/2 747);and Flu B accounted for 4.22%(116/2 747).There were 84 cases of mixed infection of two or more kinds of respiratory viruses,with positive rate of 3.06% (84/2 747 ). Conclusions Our study suggests that Flu A is the most common pathogen in children with acute respiratory infections in Wuxi area during 2011 —2014;virus detection rate is highest in age 1 to 2 year group;and parainfluenza virus is almost detected throughout the year,while the rest of respiratory viruses are commonly seen in winter and spring.
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Objective To observe the clinical effect of interferon aerpsp; in the treatment of pediatric viral upper respiratory tract infection disease.Methods 60 children with upper respiratory tract infection were divided randomly into two groups.The observation group were treated with interferon-α1b high atomization treatment of patients in the control group,with ribavirin injection treatment,two groups of patients were treated for 7 days.After a comparison of clinical outcomes.Results The total efficiency of the observation group was 90％,which was significantly higher than that of the control group(70％) (x2 =5.684,P ＜0.05) ; The fever retrcating time in observation group was (16.39 ± 5.34) h,whichwas significantly shorter than that in control group[(16.39 ± 5.34) h] (t =3.584,P ＜ 0.05).Conclusion For children virus upper respiratory tract infection treated by interferon treatment,atomization,having remarkable curative effect,and can effectively reduce the adverse reaction,enhance safety,clinical is a recommendable method.