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OBJECTIVE Human metapneumovirus (hMPV) is semblable to respiratory syncytial virus (RSV) which causes respiratory infections typically characterized by cough, runny nose, fever, and nasal congestion but sometimes progressing to bronchiolitis and pneumonia. Whereas, there is no corresponding drug to inhabit the virus. Studies of new compounds with potential anti-HMPV activity could produce clinical value. Chinese herbal medicine played a great role during COVID-19, therefore we choose some small molecular (JH001) extracted from botany to investigate therapeutic effect on hMPV and the underlying mechanisms. METHODS In this study, 16HBE cells were used as a model to explore in vitro antiviral effect. Cytotoxicity assays were performed before the antiviral tests, cell viability of 16HBE cells handled by different concentration of JH001 was estimated by Cell Counting Kit-8 (CCK-8). Then RT-qPCR, immunofluores?cence, and flow cytometer were used to test the viral titer after cells infected with hMPV. Eventually, 6-8 weeks mice were infected intranasally with 60 μL of hMPV, the control group was treated with 0.9％ saline water, other groups were administered with JH001 and ribavirin, then the lung virus titer and protective effect in lung were judged. RESULTS The obtained JH001 exhibited no cytotoxicity to 16HBE cells during 6.25 - 200 μmol · L-1. RT-QPCR demonstrated that JH001 showed obvious inhabitation to the viral replication and showed great significance compared with saline. And fluo?rescence exhibited distinct decrease of hMPV-N protein, flow cytometer results showed that MFI decrease evidently. Sig?nificant reduction of N-gene expression was observed in those mice treated with JH001 compared with saline group, which indicated that JH001 probably had protective and therapeutic effect on viral replication. CONCLUSION This study illustrated that JH001 might be a promising option for small molecular against hMPV and JH001 might be worthy of fur?ther development and used as a potential therapeutic strategy for other respiratory viruses in the future.
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Objective: The clinical course of human metapneumovirus (hMPV) infection is similar to that of coronavirus 2019 disease (COVID-19). However, community-acquired hMPV infections in adults have not yet been sufficiently investigated. We examined the detection status of hMPV antigens and the clinical features of positive patients during the first wave of COVID-19, which coincided with the epidemic season of hMPV infection in Japan.Methods: In this cross-sectional, observational, and single-center study, we recruited consecutive individuals who visited the Japan Agricultural Cooperatives Kochi Hospital due to fever, respiratory symptoms, or close contact with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected persons during the period from January to May 2020.Results: The positive rate of immunochromatography for hMPV antigens from nasopharyngeal swabs was 9.5% (4/42), and four positive cases were community-acquired pneumonia (CAP) (5.3% of all CAP). The positive rate of hMPV antigens in the CAP group (30.8%, 4/13) was higher than that in the non-pneumonia group (0.0%, 0/19) (p < 0.05). The average age of the four adult patients with CAP was 69.8 years (range 35–93). Mean white blood cell counts and C-reactive protein blood levels were 6,250 cells/μL (3,500–12,180) and 4.30 mg/dL (4.05–7.04), respectively. Chest computed tomography images were diverse and two patients showed dense consolidation. No multi-organ disorder was noted during the clinical course in any of the four cases, and their prognoses were good.Conclusion: hMPV infection may be considered in the differential diagnosis of COVID-19 and CAP in Japan under the preventive measures for SARS-CoV-2 infection, at least during the epidemic season of hMPV infection.
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Objective:To clarify the infection and epidemic characteristics of the human metapneumovirus (HMPV) in Chinese patients with febrile respiratory syndrome (FRS), and to provide important baseline data for clinical diagnosis, treatment, prevention and control of HMPV-induced respiratory tract diseases in China.Methods:FRS cases from January 2009 to June 2021 in 9 provinces in China, including Beijing, Hebei, Jilin, Shandong, Shaanxi, Xinjiang, Anhui, Guangdong, Hunan were retrospectively analyzed for their respiratory samples, clinical and epidemic data.The respiratory samples were detected for HMPV by quantitative real-time PCR.Results:A total of 11 660 cases were tested for HMPV, involving 296 (2.54%) HMPV-positive cases.Among 296 HMPV-positive cases, 218 were single HMPV infection, and 78/296 (26.35%) were co-infected with one or more respiratory viruses.HMPV mainly affected children under 5 years of age (3.10%), and in this population, the proportion of pneumonia in HMPV co-infection cases was significantly higher than that of single HMPV infection.HMPV could be detected all year round, which was more popular in winter and spring, with the peak of HMPV epidemic in March.Conclusions:HMPV is one of the important pathogens causing acute respiratory infection in children, showing a clear seasonal epidemic.HMPV can be infected alone or in combination with other respiratory viruses, which may increase the risk of pneumonia in children.
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Objective: To investigate the effect of simvastatin pretreatment on human metapneumovirus (hMPV) infection and the related mechanism. Methods: Human bronchial epithelial cells (16HBE) were used in vitro experiments and BALB/c mice were used in vivo experiments. They were randomly divided into control group, simvastatin group, hMPV group, hMPV+simvastatin group. After corresponding treatment, the fluorescence of hMPV was observed with microscopy; virus titer and the expressions of autophagy-related gene recombinant human autophagy-related protein 5 (ATG5), Beclin1, and microtubule-associated protein 1 light chain 3 (LC3) were detected by Fluorescence Quantitative Real-time PCR; Western blotting was performed to detect autophagy-related protein ATG5, Beclin1, LC3 and serine/threonine kinase/mammalian rapamycin target protein (AKT/mTOR) pathway proteins. Results: In vitro experiment, the direct and indirect fluorescence expressions of hMPV and the virus titer were lower in hMPV+simvastatin group (0.260 ± 0.018) than those in hMPV group (1.241 ± 0.030), while the mRNA expression levels of autophagy-related gene ATG5, Beclin1 and LC3 were higher in hMPV+simvastatin group [(7.31±0.15), (8.67±0.88) and (6.55±0.30), respectively] than those in control group [(1.00±0.06), (1.00±0.05) and (1.10±0.06), respectively]; the mRNA and protein levels of autophagy-genes were the highest in hMPV+simvastatin group and the differences were statistically significant (P<0.05). The AKT/mTOR pathway was inhibited in hMPV group and hMPV+simvastatin group. In vivo experiment, the virus titer of the lung tissue and of the lung tissue supernatant inoculated in Vero E6 were lower in hMPV+simvastatin group (0.75±0.26 and 0.42±0.17, respectively) than those in hMPV group (2.46±0.53 and 1.80±0.40, respectively). The mRNA and protein expression levels of autophagy-related genes ATG5, Beclin1 and LC3 were the highest in hMPV+simvastatin group (3.89±0.42, 3.13±0.26 and 3.56±0.22, respectively), higher than those in control group and hMPV group, the differences were statistically significant (P<0.05), and the AKT/mTOR pathway was inhibited in this group; HE staining of lung tissue showed an inflammatory response in the hMPV group, and pre-treatment with simvastatin could reduce the inflammation. Conclusion: Pretreatment with simvastatin may reduce human metapneumovirus infection, which is associated with autophagy and the AKT/mTOR pathway.
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Objective To evaluate the immune responses and protection against human metapneu-movirus ( hMPV) conveyed by influenza virus vectors carrying multiple epitope antigens of hMPV. Methods Two recombinant influenza viruses ( rFLU/hMPV/B and rFLU/hMPV/CTL+Th ) carrying hMPV multi-epitope gene segments in NS gene were generated by reverse genetic techniques of eight-plasmid system. BALB/c mice were immunized intranasally with rFLU/hMPV/B and rFLU/hMPV/CTL+Th twice at a two-week interval. Virus-specific antibody titers and splenocyte cytokines were detected two weeks after the boost immunization. Viral loads in lung tissues and turbinates were detected with digital PCR after the immunized mice were challenged with hMPV and influenza virus. Moreover, HE staining was used to observe lung inju-ries. Results Specific antibodies against both the influenza virus and hMPV were induced in mice immu-nized intranasally with rFLU/hMPV/B, while the influenza virus-specific antibody response and hMPV-spe-cific cytotoxic lymphocyte response ( significant IFN-γ secretion ) were detected in mice immunized with rFLU/hMPV/CTL+Th. Additionally, balanced Th1/Th2 responses were elicited by rFLU/hMPV/B and rFLU/hMPV/CTL+Th. Both rFLU/hMPV/B and rFLU/hMPV/CTL+Th conveyed effective protection against subsequent influenza virus and hMPV challenges with significantly alleviated histopathological dama-ges and reduced viral loads. Conclusions Both rFLU/hMPV/B and rFLU/hMPV/CTL+Th can induce spe-cific humoral immune response against hMPV and/or the influenza virus. Moreover, rFLU/hMPV/CTL+Th can also elicit hMPV-specific CTL immune response. These two recombinant strains can also protect BALB/c mice from the challenges with hMPV and influenza virus, suggesting that they are promising vaccine candi-dates.
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Objective@#To evaluate the immune responses and protection against human metapneumovirus (hMPV) conveyed by influenza virus vectors carrying multiple epitope antigens of hMPV.@*Methods@#Two recombinant influenza viruses (rFLU/hMPV/B and rFLU/hMPV/CTL+ Th) carrying hMPV multi-epitope gene segments in NS gene were generated by reverse genetic techniques of eight-plasmid system. BALB/c mice were immunized intranasally with rFLU/hMPV/B and rFLU/hMPV/CTL+ Th twice at a two-week interval. Virus-specific antibody titers and splenocyte cytokines were detected two weeks after the boost immunization. Viral loads in lung tissues and turbinates were detected with digital PCR after the immunized mice were challenged with hMPV and influenza virus. Moreover, HE staining was used to observe lung injuries.@*Results@#Specific antibodies against both the influenza virus and hMPV were induced in mice immunized intranasally with rFLU/hMPV/B, while the influenza virus-specific antibody response and hMPV-specific cytotoxic lymphocyte response (significant IFN-γ secretion) were detected in mice immunized with rFLU/hMPV/CTL+ Th. Additionally, balanced Th1/Th2 responses were elicited by rFLU/hMPV/B and rFLU/hMPV/CTL+ Th. Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th conveyed effective protection against subsequent influenza virus and hMPV challenges with significantly alleviated histopathological damages and reduced viral loads.@*Conclusions@#Both rFLU/hMPV/B and rFLU/hMPV/CTL+ Th can induce specific humoral immune response against hMPV and/or the influenza virus. Moreover, rFLU/hMPV/CTL+ Th can also elicit hMPV-specific CTL immune response. These two recombinant strains can also protect BALB/c mice from the challenges with hMPV and influenza virus, suggesting that they are promising vaccine candidates.
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Abstract INTRODUCTION: Prevalence of influenza A virus (Flu-A), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) was assessed in children with acute respiratory infections (ARIs). METHODS: Nasopharyngeal aspirates and throat swabs were subjected to real-time polymerase chain reaction (PCR) to detect RSV and Flu-A and to conventional PCR to detect hMPV. RESULTS: Of the 156 children assessed, 93 (59.6%) carried at least one virus, with 35.9% positive for RSV, 14.1% for hMPV, and 9.6% for Flu-A. The prevalence of co-infections was 2.6%. CONCLUSIONS: The high detection rate may reflect increased sensitivity of real-time PCR compared to traditional PCR and viral culture.
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Humans , Male , Female , Child, Preschool , Child , Adolescent , Respiratory Tract Infections/virology , Respiratory Syncytial Virus Infections/epidemiology , Paramyxoviridae Infections/epidemiology , Influenza, Human/epidemiology , Orthomyxoviridae/genetics , Respiratory Tract Infections/epidemiology , Nasopharynx/virology , Cross-Sectional Studies , Respiratory Syncytial Virus, Human/genetics , Metapneumovirus/genetics , Real-Time Polymerase Chain Reaction , Iran/epidemiologyABSTRACT
Objective@#To investigate the differences of clinical characteristics and leukotrienes (LTs) level of bronchiolitis children infected with respiratory syncytial virus (RSV), human rhinovirus (HRV) and human metapneumovirus (hMPV), and provide clinical evidence for the treatment of bronchiolitis with LTs receptor antagonist (LTRA) montelukast.@*Methods@#Totally 90 children with bronchiolitis hospitalized from January 2017 to December 2018 were enrolled into this study and viral nucleic acid from respiratory tract specimens were detected; and the patients were divided into three experimental groups: RSV group, HRV group and hMPV group. The clinical data and LTs level in blood and urine of experimental groups were compared; 30 healthy children were enrolled as the control group.@*Results@#There were no significant differences in age, sex, weight, family history and past history of allergy among the three experimental groups. The LTs levels in the experimental groups were higher than that of control group (P<0.05). There was a positive correlation between the LTs levels in blood and urine (r=0.723, P<0.05). In RSV group, LTs level of was the highest, lung function was the worst, and clinical score was the highest, and significantly different from those of HRV group and hMPV group (all P<0.01). However, there was no significant difference between HRV group and hMPV group. The number of days with fever of RSV group and hMPV group were not significantly different, but both were higher than that of HRV group (all P<0.01). The number of days with cough and wheezing among experimental groups had no significant difference(all P>0.05).@*Conclusions@#The bronchiolitis children infected with RSV had the highest LTs level, the worst lung function and the highest clinical score, there was significant difference when compared with HRV group and hMPV group.
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Objective@#To study the molecular prevalence and clinical characteristics of human metapneumovirus and human bocavirus in hospitalized children with community-acquired pneumonia.@*Methods@#Total 333 throat swabs and clinical information of patients were collected between 2017 and 2018 at Department of Pediatrics of No.2 Clinical Teaching Hospital, Chengde Medical University. The IgM of adenovirus (AdV), respiratory syncytial virus (RSV), influenza virus-A/B (Influ-A/B), parainuenza viruses (PIVs)were tested by detection kit, and the positive samples of human metapneumovirus (hMPV), human bocavirus (HBoV), AdV, RSV and human coronavirus (HCoV) were detected by RT-PCR or PCR.@*Results@#43 cases, 19 cases, 3 cases and 2 cases were positive for Influ-B, PIV, RSV and AdV IgM, respectively. Total 80 cases were infected with hMPV (71 cases were single infection, 8 cases were double infection, and 1 case was triple infection), 22 cases were infected with HBoV (14 cases were single infection, 7 cases were double infection, and 1 case was triple infection), 6 cases were infected with AdV (4 cases were single infection, 1 case was double infection, and 1 case was triple infection), only 1 case was single infected with RSV or HCoV, respectively. 39 cases (11.7%) and 41 cases (12.3%) were distributed at <5 years group and ≥5 years group, respectively. 45.0%(18/40)in severe cases and 27.99%(82/293)in mild cases were positive for hMPV, HBoV, AdV, RSV and HCoV, the ratio of viral positive case was significant higher in severe cases than mild cases (P=0.042). The age (P=0.000), peak of fever (P=0.035), duration of hospitalization (P=0.000), neutrophils (P=0.000) and lymphocytes (P=0.000) were significant difference in severe cases compared with mild cases.@*Conclusions@#Multiple respiratory viruses can cause community-acquired pneumonia and more attention should be pay to the surveillance of hMPV and HBoV.
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Objective To construct and rescue recombinant influenza virus strains expressing hu-man metapneumovirus ( hMPV) epitopes. -ethods B cell, CTL and Th epitopes predicted by bioinformat-ics software were coupled together in different combinations. These different array genes were inserted into the NS1 gene of influenza virus strain A/PR/8/34 ( PR8 ) , respectively. Recombinant PR8 influenza virus vectors expressing different hMPV antigenic epitopes were rescued by reverse genetics using eight-plasmid system. Sequencing analysis was conducted to verify whether the rescued viruses carried the chimeric hMPV epitopes. Hemagglutination ( HA) titers, half tissue culture infection dose ( TCID50 ) and growth curves were detected. Results Interval sequences GPGPG and KK were introduced into hMPV epitope combinations to construct multi-epitope antigens (MEA). These MEA were inserted into the PR8 NS gene, respectively. Using 8 plasmid system, three recombinant influenza virus strains were rescued successfully. After cultured for three passages in Madin-Darby canine kidney ( MDCK) cells and one in eggs, these three recombinant strains could proliferate steadily. Whole genome sequencing verified that the three recombinant strains car-ried the chimeric MEA sequences, named as rFLU/hMPV/B, rFLU/hMPV/CTL-Th and rFLU/hMPV/B-Th. HA titers of the recombinant strains were 128, 128 and 256 using turkey erythrocyte, respectively. Their TCID50 were 107. 0/ml, 106. 8/ml and 107. 0/ml, respectively. Growth curve tests also verified that the recombinant strains could proliferate steadily in MDCK cells. Conclusions Three recombinant influenza vi-rus vector strains carrying the B cell, CTL and Th epitopes of hMPV were rescued successfully. This study lays the foundation for further evaluation of the immune effects of these recombinant viruses and their poten-tial application value in vaccine development.
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INTRODUCTION Infections caused by respiratory viruses are important problems worldwide, especially in children. Human metapneumovirus (hMPV) is a respiratory pathogen and causes severe infections with nonspecific symptoms. This study reports the hMPV occurrence and dissemination in southern Brazil and compares the frequency of occurrence of this virus and the human respiratory syncytial virus (hRSV) in the epidemiological weeks in a three-year period (2009-2011). METHODS: In total, 545 nasopharyngeal (NP) specimens from individuals with Severe Acute Respiratory Syndrome (SARS) who were negative for other seven respiratory viruses were analyzed for the presence of hMPV. Human metapneumovirus was detected by direct immunofluorescence and real-time reverse transcription polymerase chain reaction. RESULTS: hMPV was detected in 109 patients from the main geographic regions of the southernmost state of Brazil, presenting similar overall prevalence in males (46.8%) and females (53.2%). Among children who were less than six years old, hMPV was detected in 99 samples of all age groups, with a higher frequency in infants who were less than one year old (45.7%) compared to all other age groups until six years. hMPV and hRSV infection occurred in almost the same epidemiological weeks (EWs) of each year, with peaks of incidence between EW 31/37 and EW 26/38 for the years 2009 and 2011, respectively. hMPV was further detected in several cases of SARS and it was the only virus detected in three deaths. CONCLUSIONS These findings indicate that hMPV is in circulation in southern Brazil and highlight the importance of diagnosing hMPV for influenza-like illness in the population. (AU)
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Humans , Male , Female , Pregnancy , Infant, Newborn , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Respiratory Tract Infections/transmission , Respiratory Tract Infections/virology , Respiratory Syncytial Virus Infections/virology , Metapneumovirus/pathogenicity , Epidemiological Monitoring , Adenoviruses, Human , Pneumovirinae/classification , Paramyxoviridae Infections/virology , Coronavirus , Enterovirus , Severe Acute Respiratory Syndrome , Influenza, Human , Human bocavirusABSTRACT
Human metapneumovirus (hMPV) is a newly described paramyxovirus that was found in the Netherlands in 2001. It is an important pathogen of acute respiratory diseases in infants, young children, elderly and immunocompromised patients. hMPV infection could cause mild upper respiratory tract infection or severe lower respiratory tract disease, including bronchiolitis and pneumonia. hMPV was usually detected using direct immunofluorescence and RT-PCR, but the detection method were different according to the respective experiment requirements. In this paper we review the detection method of hMPV to provide a basis for further study of hMPV.
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Objective@#To reveal the prevalence of human metapneumovirus (HMPV) among pediatric patients with acute respiratory infection (ARI).@*Methods@#Nasopharyngeal aspirates were collected from hospitalized young children (1-5 years) diagnosed as ARI in Department of Respiratory Disease, Shanxi Provincial Children′s Hospital, Taiyuan city. The nucleoprotein (N) gene of HMPV was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The positive products of PCR were sequenced, made homology and phylogenetic analysis.@*Results@#One sample collected in Feb. 2017 and two samples collected in Nov. 2017 were HMPV positive in RT-PCR detection. The homologies of nucleic acid and deduced amino acid sequences of the three positive PCR products with the corresponding HMPV sequences in GenBank were over 83% and 90%, respectively. Phylogenetic analysis revealed that HMPV B1 subgenotype was detected.@*Conclusions@#HMPV B1 subgenotype was the major epidemic HMPV in young children with ARI in Taiyuan city.
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Objective To explore the influence of coinfection with other pathogens on human metapneumovirus (hMPV) infection in children.Methods A total of 11 299 children admitted to the Department of Respiratory Disease,Children's Hospital of Soochow University between June 2010 and May 2015 were enrolled in this study.Sputum specimens were collected and multiple pathogenic joint detection was done,including peripheral blood,and blood routine,C reactive protein (CRP),hepatic function and cellular immunity.Patients' clinical data were collected.Results Among 11 299 hospitalized children,hMPV was positive found in 222 children (1.96％).One hundred and fourteen children (51.4％) had hMPV simple infection and 108 cases of them (48.6％) were coinfected with other pathogens.The hMPV coinfected with bacteria (63 cases,28.4％)was most common.The average age of multiple coinfected children was older than that of simple hMPV infection in children [(2.43 ± 2.47) years old vs.(1.27 ± 1.30) years old],and the difference was significant (Z =-2.360,P ＜ 0.05).Fever seemed to be more common in children coinfected with bacteria or multiple coinfection (63.5％ and 70.0％) compared with those with simple hMPV infection (43.0％),and the differences were significant (x2 =6.827,4.986,all P ＜ 0.05).There was no significant difference in other clinical features among 5 groups (all P ＞ 0.05).Multiple coinfection children had a higher percentage of neutrophils (0.50 ± 0.18) than that in simple hMPV infection children (0.37 ± 0.19),children coinfected with bacteria (0.39 ±0.19) or other virus (0.35 ±0.19),and the differences were significant (all P ＜0.05).CRP was elevated in 30.2％ (19/63 cases) of children coinfected with bacteria,which was significantly higher than that of simple hPMV infection children (16.7 ％,19/114 cases),and the difference was significant (x2 =4.381,P ＜ 0.05).CD3 + CD4 + was significantly lower in multiple coinfection children (0.31 ± 0.07),but there were no significant difference compared with other groups (all P ＞ 0.05).CD19 + CD23 + was significantly higher in children coinfected with other virus com pared with that of simple hMPV infection group,hMPV coinfected with bacteria,hMPV coinfected with Mycoplasma pneumonia and multiple coinfect group (0.37 ± 0.10 vs.0.30 ± 0.09,0.30 ± 0.08,0.29 ± 0.07,0.29 ± 0.09),and the differences were significant (all P ＜ 0.05).Conclusions It is suggested that hMPV seems easily coinfected with other pathogens,especially with bacteria.It should be on high alert that bacteria coinfection is accompanied with high percentage of neutrophils and high level of CRP.Coinfection does not significantly exacerbate the clinical symptoms of hMPV infection,but may exacerbate the cellular immune disorders to a certain extent.
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Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe cases of human respiratory disease. The current outbreak of infection with this virus in South Korea, which began on May 20, 2015, has infected 186 patients and caused 36 deaths within 2 months. In this study, to investigate the viral pathogen causing acute respiratory infections, multiplex/RT-PCR was performed on were obtained from nucleic acid of the Middle East Respiratory Syndrome-negative subjects. Viruses and atypical bacteria were detected in 39 of 337 (11.6%). Frequent viruses were human rhinovirus (n=11, 3.3%), human metapneumovirus (n=9, 2.7%), parainfluenza (n=9, 2.7%) and adenovirus (n=4, 1.2%). Mycoplasma pneumonia (M. pneumonia) was detected in 1.8 % (n=6). Out of 9 human metapneumovirus (hMPV) positive samples, 6 samples were successfully sequenced using F gene. And M. pneumoniae was sequencing of a repetitive region of the P1 gene. Phylogenetic analysis revealed that hMPV clustered into A2b lineage (n=4), B2 lineage (n=2) and M. pneumoniae clustered into two genotypes: Type 1 (n=4), Type 2a (n=2).
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Humans , Adenoviridae , Bacteria , Genotype , Korea , Metapneumovirus , Middle East Respiratory Syndrome Coronavirus , Middle East , Paramyxoviridae Infections , Pneumonia , Pneumonia, Mycoplasma , Repetitive Sequences, Nucleic Acid , Respiratory Tract Infections , RhinovirusABSTRACT
Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe cases of human respiratory disease. The current outbreak of infection with this virus in South Korea, which began on May 20, 2015, has infected 186 patients and caused 36 deaths within 2 months. In this study, to investigate the viral pathogen causing acute respiratory infections, multiplex/RT-PCR was performed on were obtained from nucleic acid of the Middle East Respiratory Syndrome-negative subjects. Viruses and atypical bacteria were detected in 39 of 337 (11.6%). Frequent viruses were human rhinovirus (n=11, 3.3%), human metapneumovirus (n=9, 2.7%), parainfluenza (n=9, 2.7%) and adenovirus (n=4, 1.2%). Mycoplasma pneumonia (M. pneumonia) was detected in 1.8 % (n=6). Out of 9 human metapneumovirus (hMPV) positive samples, 6 samples were successfully sequenced using F gene. And M. pneumoniae was sequencing of a repetitive region of the P1 gene. Phylogenetic analysis revealed that hMPV clustered into A2b lineage (n=4), B2 lineage (n=2) and M. pneumoniae clustered into two genotypes: Type 1 (n=4), Type 2a (n=2).
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Humans , Adenoviridae , Bacteria , Genotype , Korea , Metapneumovirus , Middle East Respiratory Syndrome Coronavirus , Middle East , Paramyxoviridae Infections , Pneumonia , Pneumonia, Mycoplasma , Repetitive Sequences, Nucleic Acid , Respiratory Tract Infections , RhinovirusABSTRACT
Background: Human metapneumovirus (HMPV), discovered in the 21st century, has emerged as an important cause of influenza‑like illness in children and adults causing mild upper respiratory tract infection to severe bronchiolitis and community‑associated pneumonia. The aim of this study was to determine the prevalence of HMPV in the Union Territory of Puducherry, India, as part of National Influenza Surveillance Programme. Materials and Methods: From November 2011 to December 2013, a total of 447 nasopharyngeal samples were collected from patients with acute respiratory infections and tested for HMPV RNA by real‑time polymerase chain reaction. Results: HMPV was identified in 23/447 (5%) samples with 11/23 in the age group of 14–30 years. Most of the HMPV infections were mild with no fatalities. Two patients were co‑infected with the respiratory syncytial virus and one with influenza B virus. The seasonal distribution showed increasing HMPV infection cases in rainy months except for a peak in summer of 2012. Phylogenetic analysis based on the sequences of the nucleoprotein gene of one HMPV strain showed a high degree of sequence identity with Indian strains obtained during 2006 and 2011. Conclusion: This study shows that HMPV infection is more common in adults than in children. Sequence homology suggests the circulation of closely related HMPV strains within the country.
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Objective To investigate the status and clinical and epidemiological characteristics of human metapneumovirus (hMPV) and human bocavirus (HBoV) infections in children with acute respiratory tract infections (ARTIs) in Taiyuan. Methods A total of 549 children with ARTIs from November 2012 to May 2013 and November 2013 to May 2014 were recruited. The pharyngeal swab specimens were collected. The hMPV and HBoV were detected by using real-time PCR. Results In 549 children, 56 children (10.2%) were hMPV positive on swab specimens, 15 children (2.7%) were HBoV positive on swab specimens. The detection rates of hMPV and HBoV in November 2012 to May 2013 were 12.3%and 2.0%, respectively, and in November 2013 to May 2014 were 6.5%and 4.0%, respectively. The detection rate of hMPV was signiifcantly different between two periods (P<0.05), while the detection rate of HBoV has no signiifcant difference between two periods. In different months, the detection rate of hMPV and HBoV showed no signiifcant difference. The highest detection rates of hMPV and HBoV were all in children younger than two years old. The highest detection rate of hMPV was in children with asthmatic bronchitis or bronchiolitis. Conclusion In Taiyuan, during the monitoring periods, the ARITS are associated with childhood hMPV and HBoV infection especially in infants and toddlers. hMPV is one of the most important pathogens in infants and toddlers with wheezing.
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Objetivo: El objetivo de este trabajo fue establecer una metodología para el aislamiento de una cepa de hMPV a partir de hisopados nasales provenientes de pacientes con sintomatología de enfermedad respiratoria aguda. Materiales y métodos: A partir de 12 muestras positivas por fluorescencia para hMPV, se hizo la inoculación en células LLCMK2 (epiteliales de riñón de mono Rhesus) con un medio de mantenimiento que contenía tripsina como proteasa. Se evaluó el efecto citopático diario por 10 días y se tomaron células infectadas que se utilizaron para la prueba de fluorescencia y para confirmar la infección. Se utilizó RT-PCR para corroborar la presencia del virus. Resultados: En 8 de las 12 muestras procesadas, se logró aislar el hMPV, confirmándose la infección por fluorescencia y RT-PCR. Conclusiones: A pesar de ser un virus de crecimiento lento y difícil de aislar según los reportes, en este trabajo se logró aislar el hMPV a partir de muestras clínicas frescas, lo cual resulta de gran utilidad en futuros estudios sobre la patogénesis de la infección respiratoria.
Objective: The aim of this work was to establish a laboratory protocol to isolate human metapneumovirus (hMPV) using as a source confirmed respiratory secretion samples from symptomatic patients. Methods: Twelve respiratory secretion samples confirmed for hMPV by immunofluorescence were inoculated on LLCMK2 primate cells using trypsin to improve the virus binding. The cytopathic effect was evaluated during 10 days, then they were stained with an hMPV specific antibody and culture supernatants were used to detect viral RNA by RT-PCR. Results: We isolated hMPV in 8 out of 12 samples. Monkey cells were susceptible to infection and showed viral replication in both immunocytochemistry and molecular tests and even syncytia formation. Conclusion: Despite the hMPV has low replication rates in culture, causing difficulties to isolate it, in this work we accomplish the viral isolation in eight samples that had been previously confirmed containing hMPV. These clinical isolates are a useful tool to study the respiratory infection pathogenesis.
Subject(s)
Respiratory Tract Infections , Metapneumovirus , Polymerase Chain Reaction , Trypsin , Fluorescent Antibody TechniqueABSTRACT
Objective To evaluate the immune response triggered by an in-house constructed hu-man metapneumovirus multi-epitope antigen ( MEA) in a mouse model .Methods Female SPF BALB/c mice at age 4-6 weeks were used in the study and divided into 7 groups.Mice in the five groups including MEA+oligodeoxynucleotides containing CpG motifs ( CpG ODN) intraperitoneal injection ( i.p.) treatment group, MEA+Alum i.p.treatment group, MEA+Alum+CpG ODN i.p.treatment group, MEA+CpG ODN intranasal (i.n.) treatment group and MEA+Alum+CpG ODN i.n.treatment group were immunized three times on days 0, 14 and 21, and those in the other experimental group were immunized intramuscularly with MEA+Quickantibody5W on days 0 and 21.A control group without treatment was set up accordingly .All mice were sacrificed two weeks after the last immunization .Antibodies including IgG , IgG1, IgG2a and IgA in serum samples were detected by ELISA .MTS assay was performed to analyze the proliferation of lympho-cytes.The cytotoxicity of cytotoxic T lymphocytes (CTL) was measured by LDH assay.Flow cytometry was used to detect T lymphocyte subsets .The cytokines secreted by T helper cells ( Th1 and Th2) were analyzed with Bio-Rad Liquid Chips.Results High titers of IgG, IgG1 and IgG2a antibodies were produced in MEA treated mice except for those in intranasal treatment groups .Serum samples from three groups including the MEA+Alum i.p., MEA+Alum+CpG ODN i.p.and MEA+Quickantibody5W i.m.treatment groups were positive for IgA antibody .The highest titer of IgA antibody was detected in mice from the MEA+Alum+CpG ODN i.p.treatment group, which was 2.15×103.Compared with the control group, significantly enhanced proliferation of lymphocytes was observed in the MEA+Alum i.p., MEA+Alum+CpG ODN i.p.and MEA+Quickantibody5W i.m.treatment groups (P<0.05).Enhanced cytotoxic activities of CTL were observed in mice with ip.and i.m.treatments as compared with those in control group (P<0.05).The levels of CD4+/CD8+T cells were slightly increased in mice from the MEA+CpG ODN i.p., MEA+Alum+CpG ODN i.p. and MEA+Quickantibody5W i.m.treatment groups as compared with those in control group (P<0.05).In-creased secretion of IL-2, IFN-γand Th2-type cytokines including IL-4, IL-5 and IL-10 were detected in mice from the MEA+CpG ODN i.p.treatment group.The MEA+Alum i.p.treated mice showed a slightly increased secretion of IFN-γand significantly increased secretions of IL-4, IL-5 and IL-10.Significantly in-creased secretions of IFN-γ, IL-4, IL-5 and IL-10 were detected in mice from the MEA+Alum+CpG ODN i.p.treatment group.Significantly increased secretions of IFN-γ, IL-5, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected in mice from the MEA+Quickantibody5W i.m.treatment group.Conclusion MEA together with different adjuvants could stimulate high titers of specific antibodies , increase the proliferation of lymphocytes and enhance the cytotoxic activities of CTL .CpG ODN could bal-ance the Th1/Th2-mediated immune responses , and the balance could be enhanced when using CpG ODN in combination with Alum .A similar effect could be achieved by using the commercial adjuvant Quickanti -body5w.This study has paved the way for further investigation on the development of hMPV epitope vaccines and diagnostic reagents for hMPV as well as the epidemiological study of hMPV .