Performance of VIDISCA-454 in Feces-Suspensions and Serum

Virus discovery combining sequence unbiased amplification with next generation sequencing is now state-of-the-art. We have previously determined that the performance of the unbiased amplification technique which is operational at our institute, VIDISCA-454, is efficient when respiratory samples are used as input. The performance of the assay is, however, not known for other clinical materials like blood or stool samples. Here, we investigated the sensitivity of VIDISCA-454 with feces-suspensions and serum samples that are positive and that have been quantified for norovirus and human immunodeficiency virus type 1, respectively. The performance of VIDISCA-454 in serum samples was equal to its performance in respiratory material, with an estimated lower threshold of 1,000 viral genome copies. The estimated threshold in feces-suspension is around 200,000 viral genome copies. The decreased sensitivity in feces suspension is mainly due to sequences that share no recognizable identity with known sequences. Most likely these sequences originate from bacteria and phages which are not completely sequenced.     

Increased risk of rhinovirus infection in children during the coronavirus disease‐19 pandemic

BackgroundCoronavirus disease (COVID‐19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), was first detected in Japan in January 2020 and has spread throughout the country. Previous studies have reported that viral interference among influenza virus, rhinovirus, and other respiratory viruses can affect viral infections at the host and population level.MethodsTo investigate the impact of COVID‐19 on influenza and other respiratory virus infections, we analyzed clinical specimens collected from 2244 patients in Japan with respiratory diseases between January 2018 and September 2020.ResultsThe frequency of influenza and other respiratory viruses (coxsackievirus A and B; echovirus; enterovirus; human coronavirus 229E, HKU1, NL63, and OC43; human metapneumovirus; human parainfluenza virus 1, 2, 3, and 4; human parechovirus; human respiratory syncytial virus; human adenovirus; human bocavirus; human parvovirus B19; herpes simplex virus type 1; and varicella‐zoster virus) was appreciably reduced among all patients during the COVID‐19 pandemic except for that of rhinovirus in children younger than 10 years, which was appreciably increased. COVID‐19 has not spread among this age group, suggesting an increased risk of rhinovirus infection in children.ConclusionsRhinovirus infections should be continuously monitored to understand their increased risk during the COVID‐19 pandemic and viral interference with SARS‐CoV‐2.     

Metagenomics Study of Viral Pathogens in Undiagnosed Respiratory Specimens and Identification of Human Enteroviruses at a Thailand Hospital

Numerous pathogens cause respiratory infections with similar symptoms. Routine diagnostics detect only a limited number of pathogens, leaving a gap in respiratory illness etiology surveillance. This study evaluated next-generation sequencing for unbiased pathogen identification. Respiratory samples collected in Thailand, Philippines, Bhutan, and Nepal, that were negative by several molecular and immunofluorescence assays, underwent viral cultivation. Samples which demonstrated cytopathic effect in culture (N = 121) were extracted and tested by Luminex xTAG respiratory viral panel (RVP) assay and deep sequencing by Roche 454 FLX Titanium system. Using RVP assay, 52 (43%) samples were positive for enterovirus or rhinovirus and another three were positive for respiratory syncytial virus B, parainfluenza 4, and adenovirus. Deep sequencing confirmed the Luminex assay results and identified additional viral pathogens. Human enteroviruses, including Enterovirus A type 71 and 12 types of Enterovirus B (EV-B) were identified from a hospital in Bangkok. Phylogenetic and recombination analysis showed high correlation of VP1 gene-based phylogeny with genome-wide phylogeny and the frequent genetic exchange among EV-B viruses. The high number and diversity of enteroviruses in the hospital in Bangkok suggests prevalent existence. The metagenomic approach used in our study enabled comprehensive diagnoses of respiratory viruses.     

Experimental rhinovirus infection in human volunteers exposed to ozone

We studied 24 young adult male volunteers experimentally inoculated with type 39 rhinovirus to determine whether the course of viral infection was modified by exposure to moderate levels of ozone (0.3 ppm for 6 h per day) over the 5 days after virus inoculation. No differences in rhinovirus titers in nasal secretions, recruitment of neutrophils into nasal secretions, levels of interferon in nasal lavage fluid, in vitro lymphocyte proliferative responses to rhinovirus antigen, or levels of convalescent serum neutralizing antibody to type 39 rhinovirus were demonstrated in relation to ozone exposure. The level and pattern of ozone exposure used in this experiment had no demonstrable adverse effects on the immune responses necessary to limit and terminate rhinovirus infection of the upper respiratory tract.     

Rhinovirus and influenza type A infections as precipitants of asthma.

Specimens from 49 persons, 3 to 60 years of age, who were prone to attacks of apparently infectious asthma were cultured for viruses and bacteria during episodes of symptomatic respiratory infection from September 1972 to June 1973. Seventy-one of 128 episodes (55 per cent) of symptomatic respiratory infection occurred with asthma. Shedding of respiratory pathogens was associated with 43 of the 128 episodes, and 19 of the 43 etiologically defined episodes occurred with wheezing. Seven of 15 rhinovirus symptomatic respiratory infections were linked with asthma, as were 4 of 5 influenza type A (H3N2) and 2 of 3 respiratory syncytial virus infections. A total of 21 different rhinovirus serotypes have been identified from asthmatic patients during 2 years of study; 14 of these were associated with wheezing, indicating that the ability to cause asthma is not restricted to only a few types. Other viruses that were shed during wheezing-associated symptomatic respiratory infections included para-influenza type 3, adenovirus type 7, and 2 unidentified viruses. Although infectious asthma was not easily defined in adults and some older children, viruses that appeared to precipitate asthma were distributed among patients of widely differing ages.     

Identification of New Respiratory Viruses in the New Millennium

The rapid advancement of molecular tools in the past 15 years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. The inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. In 2001, this led to the discovery of human metapneumovirus (hMPV) and soon following that the outbreak of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (HCoV) NL63 and HCoV-HKU1. Human bocavirus, with its four separate lineages, discovered in 2005, has been linked to acute respiratory tract infections and gastrointestinal complications. Middle East Respiratory Syndrome coronavirus (MERS-CoV) represents the most recent outbreak of a completely novel respiratory virus, which occurred in Saudi Arabia in 2012 and presents a significant threat to human health. This review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since 2001.     

Genetic diversity and molecular epidemiology of human rhinoviruses in South Africa

Background

Rhinoviruses (RV) are a well-established cause of respiratory illness. RV-C has been associated with more severe illness. We aimed to characterize and compare the clinical presentations and disease severity of different RV type circulating in South Africa.

Method

We performed two analyses of RV-positive specimens identified through surveillance in South Africa across all age groups. First, RV-positive specimens identified through severe acute respiratory illness (SARI) surveillance in four provinces was randomly selected from 2009 to 2010 for molecular characterization. Second, RV-positive specimens identified through SARI, influenza-like illness (ILI) and control surveillance at hospitals and outpatient clinics in during 2012–2013 were used to determine the association of RV type with severe disease. Selected specimens were sequenced, and phylogenetic analysis was performed.

Results

Among the 599 sequenced specimens from 2009 to 2010 and 2012 to 2013, RV-A (285, 48%) and RV-C (247, 41%) were more commonly identified than RV-B (67, 11%), with no seasonality and a high genetic diversity. A higher prevalence of RV infection was identified in cases with SARI [515/962 (26%); aRRR = 1·6; 95% CI 1·21; 2·2] and ILI [356/962 (28%); aRRR = 1·9; 95% CI 1·37; 2·6] compared with asymptomatic controls (91/962, 22%). There was no difference in disease severity between the different type when comparing SARI, ILI and controls.

Conclusion

All three type of RV were identified in South Africa, although RV-A and RV-C were more common than RV-B. RV was associated with symptomatic respiratory illness; however, there was no association between RV type and disease severity.     

儿科重症监护室先天性心脏病合并呼吸道感染患儿的病毒性病原学研究

目的：总结儿科重症监护室中先天性心脏病合并呼吸道感染患儿的病毒性病原谱。方法收集2010年6月至2012年6月因呼吸道感染入住本院儿科重症监护室的患儿咽拭子标本622份,其中先天性心脏病合并呼吸道感染患儿咽拭子34份。应用多重聚合酶链反应(PCR)技术对呼吸道病毒进行检测,并对照分析合并先天性心脏病患儿的病毒性病原学特点。结果①34份先天性心脏病组咽拭子标本中,呼吸道病毒检测阳性20份(58.8%),588份非先天性心脏病组咽拭子标本中,呼吸道病毒检测阳性368份(62.6%)。②先天性心脏病组中,最常见的病毒分别为人鼻病毒(human rhinovirus,HRV)8份,呼吸道合胞病毒(respiratory syncytial virus, RSV)6份,人博卡病毒(human bocavirus,HBoV)4份,腺病毒(adenovirus,ADV)2份;非先天性心脏病组中,最常见的病毒分别为 HRV 160份,RSV 104份,ADV 72份,HBoV50份;其他病毒阳性率较低。③先天性心脏病组中,混合病毒感染有2份(2/20,10.0%),非先天性心脏病组中,混合病毒感染有110份(110/368,29.9%)。结论本地区儿科重症监护室中先天性心脏病合并呼吸道感染患儿的病原体中病毒性病原体检出率高,以鼻病毒、呼吸道合胞病毒、人博卡病毒和腺病毒最常见,病毒谱和非先天性心脏病组相似。     

A recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in Germany

Acute respiratory infection is a significant cause of morbidity and mortality in children worldwide. Accurate identification of causative agents is critical to case management and to prioritization in vaccine development. Sensitive multiplex diagnostics provide us with an opportunity to investigate the relative contributions of individual agents and may also facilitate the discovery of new pathogens. Recently, application of MassTag polymerase chain reaction (PCR) to undiagnosed influenza-like illness in New York State led to the discovery of a novel rhinovirus genotype. Here we report the investigation, by MassTag PCR, of pediatric respiratory-tract infections in Germany, studying 97 cases for which no pathogen was identified through routine laboratory evaluation. Respiratory viruses were identified in 49 cases (51%); of the 55 identified viruses, 41 (75%) were rhinoviruses. The novel genotype represented 73% of rhinoviruses and 55% of all identified viruses. Infections with the novel genotype were associated with upper-respiratory-tract symptoms but, more frequently, with bronchitis, bronchiolitis, and pneumonia.     

Influenza and rhinovirus infections among health-care workers

BACKGROUND AND OBJECTIVE: Health-care workers (HCWs) are at higher risk of acquisition and transmission of respiratory virus infections. Nosocomial transmission of influenza has been documented but whether this is so for other respiratory viruses has not been assessed. METHODS: Epidemiological, clinical and viral laboratory surveillance was carried out on HCWs presenting with acute respiratory infection in a university hospital. RESULTS: Over a 2-year period, 203 subjects were recruited: rhinovirus was the most frequently detected virus (37.7% in flu negative samples) and influenza A/B was positive in only 12.3% of subjects. Only 19.7% of HCWs were immunized against influenza. High detection of rhinovirus occurred even during the peak of the influenza season and half of the infected subjects reported an influenza-like illness. CONCLUSION: Rhinovirus infection occurred frequently in this study population and probably contributes to influenza misdiagnosis. Educational interventions about different viruses causing respiratory symptoms and an increase in standards of infection control besides influenza immunization among HCWs is needed.     

Culturing of respiratory viruses in well‐differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses

Background

Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses.

Method

Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air–liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique.

Results

Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively).

Conclusion

We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch''s postulates.     

Longitudinal study of acute respiratory diseases in Rio de Janeiro: occurrence of respiratory viruses during four consecutive years.

The occurrence of different viruses in nasopharyngeal secretions from children less than 5 years old with acute respiratory infections (ARI) was investigated over a period of 4 years (1982-1985) in Rio de Janeiro. Of the viruses known to be associated with ARI, all but influenza C and parainfluenza types 1, 2 and 4 were found. Viruses were found more frequently in children attending emergency or pediatric wards than in outpatients. This was clearly related to the high incidence of respiratory syncytial virus (RSV) in the more severe cases of ARI. RSV positive specimens appeared mainly during the fall, over four consecutive years, showing a clear seasonal occurrence of this virus. Emergency wards provide the best source of data for RSV surveillance, showing sharp increase in the number of positive cases coinciding with increased incidence of ARI cases. Adenovirus were the second most frequent viruses isolated and among these serotypes 1, 2 and 7 were predominant. Influenza virus and parainfluenza virus type 3 were next in frequency. Influenza A virus were isolated with equal frequency in outpatient departments, emergency and pediatric wards. Influenza B was more frequent among outpatients. Parainfluenza type 3 caused outbreaks in the shanty-town population annually during the late winter or spring and were isolated mainly from outpatients. Herpesvirus, enterovirus and rhinovirus were found less frequently. Other viruses than RSV and parainfluenza type 3 did not show a clear seasonal incidence.     

成人重症肺炎患者EB病毒和人鼻病毒混合感染的病原学分子鉴定

目的对1例有禽类接触史的成人重症肺炎患者进行病原学研究,确定其病原体。方法采集患者的下呼吸道标本,应用荧光PCR方法检测禽流感H5N1和SARS病毒及甲型H1N1和季节性流感病毒;应用多重PCR方法检测12种常见呼吸道病毒;应用普通PCR方法检测EB病毒和巨细胞病毒,扩增阳性的病毒基因片段测序后,与GenBank数据库参考序列进行同源性分析。结果荧光PCR法检测禽流感病毒H5N1病毒和SARS病毒核酸阴性,甲型H1N1和季节性流感病毒核酸阴性;多重PCR检测人鼻病毒、EB病毒均阳性;普通PCR检测EB病毒核酸阳性。基因序列比对显示,EB病毒核苷酸序列与GenBank数据库中参考序列同源性为99.5%～100%;人鼻病毒的核苷酸序列与GenBank数据库中A组鼻病毒(73血清型)同源性最高,为94.7%。结论该例重症肺炎患者为EB病毒和A组(73血清型)人鼻病毒混合感染,排除禽流感H5N1和SARS感染。     

Pathogenesis of lower respiratory tract symptoms in experimental rhinovirus infection

To investigate the pathogenesis of lower respiratory tract symptoms during rhinovirus infection, 19 healthy young adult volunteers were exposed to a currently unnumbered rhinovirus strain (HH). Spirometry and bronchoprovocation with histamine sulfate were performed prior to and on Days 4, 5, and 21 after exposure to the virus. Fiberoptic bronchoscopy for visualization and culture of the tracheobronchial mucosa was done on Day 4 or Day 5. Fourteen of 19 volunteers were infected with rhinovirus; 9 of the 14 had clinical illness. Rhinovirus was isolated from the bronchial brush specimens in 5 of the 13 infected volunteers bronchoscoped, all of whom had clinical illness. Rhinovirus was not isolated at bronchoscopy from any of the 5 infected volunteers without clinical illness (p = 0.025, Fisher's exact test). Spirometry and histamine bronchoprovocation were unchanged during experimental rhinovirus infection. Rhinovirus may invade the lower respiratory tract in symptomatic infections, and thereby cause lower respiratory tract symptoms.     

Rhinoviruses and Their Receptors: Implications for Allergic Disease

Human rhinoviruses (RVs) are picornaviruses that can cause a variety of illnesses including the common cold, lower respiratory tract illnesses such as bronchitis and pneumonia, and exacerbations of asthma. RVs are classified into three species, RV-A, B, and C, which include over 160 types. They utilize three major types of cellular membrane glycoproteins to gain entry into the host cell: intercellular adhesion molecule 1 (ICAM-1) (the majority of RV-A and all RV-B), low-density lipoprotein receptor (LDLR) family members (12 RV-A types), and cadherin-related family member 3 (CDHR3) (RV-C). CDHR3 is a member of cadherin superfamily of transmembrane proteins with yet unknown biological function, and there is relatively little information available about the mechanisms of RV-C interaction with CDHR3. A coding single nucleotide polymorphism (rs6967330) in CDHR3 could promote RV-C infections and illnesses in infancy, which could in turn adversely affect the developing lung to increase the risk of asthma. Further studies are needed to determine how RV infections contribute to pathogenesis of asthma and to develop the optimal treatment approach to control asthma exacerbations.     

114例疑似COVID-19患者呼吸道病毒感染分析

目的 分析114例COVID-19疑似患者呼吸道病毒感染情况, 探讨多种病毒同时检测在疫情防控中的价值。方法 利用Real Time RT-PCR技术检测发热门诊COVID-19疑似患者SARS-CoV-2核酸,利用基因芯片恒温扩增技术检测SARS-CoV-2核酸阴性患者其他常见的18种呼吸道病毒。结果 114例COVID-19疑似患者SARS-CoV-2核酸均为阴性,有21例感染了非其它呼吸道病毒,感染率达18.42%,21例患者中共检测出10种呼吸道病毒,包括冠状病毒NL63/229型、呼吸道合胞病毒、柯萨奇病毒A16型、乙型流感病毒、人副流感病毒1型、人副流感病毒3型、人偏肺病毒、甲型流感病毒、甲型流感病毒季节性H3亚型、肠道病毒/鼻病毒。其中乙型流感病毒感染患者最多,有6例,呼吸道合胞病毒有5例。有3例患者同时感染2种病毒:呼吸道合胞病毒与柯萨奇病毒A16型混合感染、冠状病毒NL63/229型与人副流感病毒1型混合感染、甲型流感病毒与甲型流感病毒季节性H3亚型混合感染。结论 在应对本次SARS-CoV-2疫情中,针对COVID-19疑似患者中,要注意鉴别SARS-CoV-2与其它呼吸道病毒,及时有效地排除疑似病例。     

Epidemiology of respiratory virus infections

Respiratory viral infections are very common in young children. They sometimes occur as primary infections (and sometimes re-infections) by influenza and parainfluenza virus, respiratory syncytial virus (VRS), adenovirus, rhinovirus and coronavirus. The clinical pictures are very varied and without strict clinico-virological correlation. In adults the role of the site (frail lung, aged persons) and the type of virus play an important part. Many viral infections develop in an epidemiological way (influenza, VRS bronchiolitis, rhinovirus infections...) and several epidemics by different viruses overlap from September-October to March-April making it very difficult to decide the precise cause. Epidemics are followed thanks to networks of medical practitioners (GROG, SENTINELLE...) and by data from hospitalised patients, but precise identification of epidemic viruses is only possible and validated by virological analysis of samples taken from patients.