Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization

Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.Human respiratory syncytial virus (RSV) is a negative-strand RNA virus of genus Pneumovirus, family Paramyxoviridae. RSV is the most important viral agent of serious respiratory tract illness in infants and children worldwide (1–3). Worldwide, nearly all children are infected by RSV at least once by the age of 2 y. RSV disease ranges from rhinitis to bronchiolitis or pneumonia. The RSV genome consists of a single-stranded negative-sense 15.2-kb RNA and has 10 genes in the order 3′ NS1-NS2-N-P-M-SH-F-G-M2-L 5′ (for a review, see ref. 4). The M2 gene encodes two separate proteins, M2-1 and M2-2, from overlapping ORFs.RSV vaccines and new antiviral drugs are in preclinical and clinical development; however, no RSV vaccines or antiviral drugs suitable for routine use are commercially available. The goal of the present study was to design and generate new vaccine candidates for RSV using the recently described strategy of codon-pair deoptimization (CPD) (5). By this strategy, one or more ORFs in a virus or other pathogen are recoded by rearranging existing synonymous codons so as to increase the presence of underrepresented codon pairs within the ORF. CPD can be done without changing codon use although, in the present study, codon use was occasionally changed slightly when we manually edited the sequence to remove features such as long homo-oligomers. Amino acid coding was completely unaffected, and nontranslated genome regions were unchanged. A major effect of CPD is thought to be a reduction in the efficiency of translation although this effect is not well-defined (6–11). Because CPD involves hundreds or thousands of nucleotide substitutions, deattenuation is thought to be unlikely. Because amino acid coding is unaffected, CPD strains provide the same repertoire of epitopes for inducing cellular and humoral immunity as the WT pathogen. Recently, the CPD approach has been used successfully to attenuate poliovirus, influenza A virus, Streptococcus pneumonia, and HIV type 1 (5, 10–13).In the present work, four CPD RSV genomes were designed, synthesized, and recovered by reverse genetics. The CPD recombinant (r) RSVs were attenuated and temperature-sensitive in vitro. Furthermore, we demonstrated that the CPD rRSVs were attenuated and immunogenic in mice and African green monkeys (AGMs). In the AGM model, CPD rRSVs replicated at a level comparable with two pediatric RSV vaccine candidates, which are currently being tested in clinical phase I studies in infants and young children. Thus, CPD represents a new strategy for the generation of novel live-attenuated RSV vaccine candidates.     

Treatment of respiratory failure due to respiratory syncytial virus pneumonia with natural surfactant

We describe two infants suffering from severe pneumonia caused by respiratory syncytial virus (RSV) infection and needing mechanical ventilation with both high ventilator settings and a high fraction of inspired oxygen. The severity of the respiratory failure and the possibility of decreased and/or altered surfactant production led us to treat these infants with intratracheal instillation of natural surfactant. This resulted in an improvement of lung compliance and a decrease in the amount of oxygen required to maintain acceptable oxygen saturations. Intratracheal surfactant instillation might, therefore, be useful in the treatment of severe RSV pneumonia. Pediatr Pulmonol 1996; 22:412–415. © 1996 Wiley-Liss, Inc.     

Human metapneumovirus and respiratory syncytial virus infections in older children with cystic fibrosis

BACKGROUND: Human metapneumovirus (hMPV) has been isolated from children with acute respiratory infection worldwide. Its epidemiology remains to be defined in children with cystic fibrosis (CF). We describe the epidemiology and clinical impact of hMPV in CF children and compared it to respiratory syncytial virus (RSV). METHODS: CF children ages 7-18 years were studied prospectively during the 1998 -1999 RSV season. Nasopharyngeal specimens were collected during acute respiratory illnesses and tested for respiratory viruses. Blood specimens were drawn early, mid, and end of the RSV season, and tested for serological evidence of hMPV and RSV infections. Rates of lower respiratory tract illnesses (LRTI) and hospitalizations for pulmonary exacerbations were compared during the time intervals they developed serological evidence of infection to their non-infection intervals. RESULTS: Six of 44 CF children had a virus positive respiratory illness in 56 LTRI events and 18 hospitalizations. Serological evidence of hMPV and RSV infections occurred in 16 and 20 CF children, respectively; 8 had infections with both viruses. A greater proportion of CF children had >or=1 LRTI during their infection intervals compared to their non-infection intervals (13/25 vs. 5/25; P=0.03). A trend for higher rates of LRTI was observed in the infection intervals compared to non-infection intervals (9.5 +/- 11.0 vs. 4.2 +/- 9.9 per 1,000 child-days; P=0.06), and it was significantly greater with a more conservative estimate (one event per child per interval; 7.4 +/- 7.7 vs. 2.6 +/- 5.4 per 1,000 child-days; P 相似文献   

Systematic literature review of the signs and symptoms of respiratory syncytial virus

Respiratory syncytial virus (RSV) is responsible for over 30 million lower respiratory tract infections (LRTIs) and 3 million hospitalizations worldwide each year. Despite the risk RSV poses to young children, older adults, and individuals with comorbidities or suppressed immunity, there is limited understanding of RSV symptom presentation across these at-risk groups, and there is no vaccine for RSV. We conducted two systematic literature reviews (SLRs) of studies that document signs and symptoms (S&S) of RSV in (1) children aged ≤5 years and (2) immunocompromised adolescents and adults, and adults at high risk for severe RSV due to age or comorbidities. Symptom duration and hospital length of stay (LOS) were explored. Electronic database searches were performed following PRISMA guidelines. Studies captured RSV S&S across community and hospital settings. Clinicians and caregivers reported (n = 25 studies) nasal discharge/congestion, cough, shortness of breath, feeding abnormalities, and fever in ≥40% of children across studies and settings. Median hospital stays for children ranged from 2 days in the United States to 7.5 days in China. High-risk adults with RSV (n = 6 studies) commonly (≥40% of adults) reported cough, sputum, dyspnea, and fever/feverishness. Median length of hospital stay in adults ranged from 6 to 15 days across studies. Caregivers and clinicians reported similar RSV S&S in young children, including upper and lower respiratory and systemic symptoms. In high-risk and immunocompromised adults, the most frequent (in multiple publications) and commonly reported RSV S&S were primarily LRTI symptoms. RSV symptoms could last for weeks and are variable based on geography.     

Successful therapy with ribavarin of late onset respiratory syncytial virus pneumonitis complicating allogeneic bone transplantation

Summary A 21-year-old patient developed interstitial pneumonitis nine months post bone marrow transplant for acute myeloblastic leukaemia. Immunofluorescence of broncheoalveolar lavage fluid revealed the presence of respiratory syncytial virus (RSV). Aerosolized ribavarin therapy resulted in rapid resolution of the pneumonitis with full recovery without any side effects. Ribavarin therapy should be considered early in the management of BMT patients who develop RSV pneumonitis.     

2009-2016年广州市呼吸道合胞病毒感染的流行特征及临床特点分析北大核心CSCD

目的分析急性上呼吸道感染患者中呼吸道合胞病毒(respiratory syncytial virus,RSV)的流行特征及临床特点。方法以南方医科大学珠江医院2009年11月至2016年6月收治的具有发热呼吸道症状患者为研究对象,采集鼻拭子标本3446份,提取标本RNA,采用qRT-PCR方法进行RSV检测,并对RSV阳性标本进行临床上常见的7种(14个亚型)呼吸道病毒的检测,以了解混合感染情况。结果共检测患者标本3446份,其中RSV阳性标本672份,总阳性率为19.5%。4个季节中RSV检出率差异具有统计学意义(χ2=133.184,P<0.001),并以春节最高,冬季次之,夏秋两季最少。RSV感染患者随着年龄的增长检出率逐渐降低,并以3岁及以下儿童检出率最高,占RSV阳性标本数的90.8%(610/672),儿童患者检出率明显高于成年患者(χ2=266.433,P<0.001),男性患者检出率明显高于女性患者(χ2=4.940,P=0.026),住院患者检出率明显高于门诊患者(χ2=60.433,P<0.001)。混合感染标本160份,其中双病毒感染标本143份,3种病毒混合感染16份,4种病毒混合感染1份,并以鼻病毒和呼吸道合胞病毒混合感染率最高,住院患者混合感染率高于门诊患者(χ2=20.896,P<0.001)。与非RSV感染者相比,RSV感染者咳嗽、咳痰、气促/呼吸困难等临床症状发生率均较高(χ2_(咳嗽)=108.934,χ2_(咳痰)=60.626,χ2_(气促/呼吸困难)=38.139,均P<0.001),而咽喉痛、头痛、乏力、寒战/畏寒和肌肉酸痛等发生率相对较低(χ2_(咽喉痛)=46.499,χ2_(头痛)=29.516,χ2_(乏力)=16.972,χ2_(寒战/畏寒)=9.616,χ2_(肌肉酸痛)=8.801,均P<0.001)。此外,RSV感染组与非RSV感染组在临床诊断上也存在统计学差异(χ2=212.157,P<0.001)。结论RSV是引起3岁以下儿童呼吸道病毒感染的主要病原体,也是引起3岁以下儿童呼吸道病毒感染住院的主要原因,咳嗽、咳痰及气促/呼吸困难的发生率较高,临床上应加强对该年龄段儿童的防护。     

Modulation of monocyte Mo2 surface antigen expression by exposure to respiratory syncytial virus

The Mo2 antibody recognizes a monocyte-specific cell surface antigen of unknown function. Upward modulation of Mo2 surface epitope density was demonstrated in response to 72-hr culture of monocytes with respiratory syncytial virus (RSV), but this was not seen after culture with phytohemagglutinin or other respiratory viruses. Monoclonal antibody probes for RSV proteins were used to demonstrate probable replication of RSV proteins in peripheral blood monocytes and pulmonary alveolar macrophages. These data provide possible clues to the biologic role of the Mo2 antigen.     

Lower number of plasmacytoid dendritic cells in peripheral blood of children with bronchiolitis following respiratory syncytial virus infection

Objectives

Dendritic cells (DCs) are key mediators of allergic airway inflammation. Thus, it is important to understand the relationship between respiratory syncytial virus (RSV) infection and DCs, especially in children with RSV bronchiolitis.

Methods

We collected peripheral blood from 71 children with RSV bronchiolitis at the time of admission and 28 children who were followed up 3 months following admission. Flow cytometry was performed to detect dendritic cell immunophenotypes.

Results

Patients with RSV bronchiolitis exhibited significantly higher number of myeloid DCs and lower number of plasmacytoid DCs at the time of admission and 3 months following discharge, compared with healthy controls. These children had a significantly higher myeloid/plasmacytoid ratio 3 months after discharge compared with healthy controls.

Conclusions

Among children with RSV bronchiolitis, there is an imbalance in peripheral blood myeloid/plasmacytoid ratio. The low number of plasmacytoid DCs in peripheral blood indicates the development of bronchiolitis due to RSV infection.     

Acute respiratory distress syndrome caused by respiratory syncytial virus

Acute respiratory distress syndrome (ARDS) complicating severe respiratory syncytial virus (RSV) infection has been described in only a few infants. In contrast to the low mortality rates usually associated with RSV infections (<5%), mortality rates in the range of 40–70% have been reported in pediatric patients with ARDS. However, studies on patients with ARDS are usually lumped with respect to causation, and the disease course of RSV-induced ARDS has not been previously studied. We examined the pulmonary function abnormalities of 37 infants with RSV-induced respiratory failure who were admitted to our pediatric intensive care unit for assisted ventilation. Measurements included respiratory mechanics, maximum expiratory flow-volume curves, and lung volumes. These allowed the calculation of a Murray lung injury score (modified for pediatric use) in which radiographic findings, ventilator settings, lung compliance, and blood gas results were considered. We identified ten infants with severe restrictive lung disease who fulfilled the clinical criteria for classification as ARDS. All had lung injury scores above 2.5, compatible with a diagnosis of ARDS. Twenty-seven infants had obstructive patterns of lung function consistent with a clinical diagnosis of RSV bronchiolitis. The patients with RSV-induced ARDS were significantly younger, and had a longer time on assisted ventilation (P < 0.05) and a higher proportion of predisposing illnesses (P < 0.05, odds ratio = 6.67, two-tailed Fisher's exact test) when compared with the patients who had obstructive disease. Only one patient (who had immunodeficiency) died, and all others were successfully managed on conventional mechanical ventilation. We conclude that RSV-induced respiratory failure represents a relatively benign cause of ARDS in pediatric patients. Our observations support the notion of differentiating ARDS with respect to causation, especially when novel and experimental therapy is considered and mortality rates are analyzed. Pediatr. Pulmonol. 1997; 23:176–183 © 1997 Wiley-Liss, Inc.     

呼吸道合胞病毒诱导一氧化氮产生的机制

目的探讨呼吸道合胞病毒(RSV)感染时一氧化氮(NO)的水平变化及其产生的调控机制,为临床治疗RSV疾病提供有益的思路。方法以RSV感染人肺上皮细胞A549细胞,并设立不同的感染时间(4h、8h、16h和24h),同时分别给予PDTC(核转录因子NF-κB的特异性抑制剂)或AG(诱导型一氧化氮合酶i NOS的特异性抑制剂)处理。收集各组细胞和细胞培养上清,用Western blot法检测细胞核内活性NF-κBp65蛋白的表达,半定量RT-PCR和免疫细胞化学法检测i NOS mR-NA和蛋白表达,硝酸还原酶法检测细胞培养上清中NO含量。结果RSV感染4h后,核内活性NF-κBp65蛋白、i NOS mR-NA和蛋白、细胞培养上清中NO含量均升高,各指标的变化与正常对照相比,差异均有显著性,并且与RSV感染存在时间依赖关系。加入PDTC后可明显抑制NF-κB活化,同时下调i NOS mRNA和蛋白表达。加入AG后,则明显降低细胞培养上清中NO含量。结论RSV感染可诱导产生大量的NO,其主要受i NOS的调节。NF-κB活化对i NOS基因表达具有重要的正调控作用。提示RSV诱导NO生成可能通过活化NF-κB所致。     

Prediction of duration of hospitalization in respiratory syncytial virus infection

Identification of variables that predict duration of RSV-associated hospitalization may be useful in the identification of preventive and therapeutic strategies. A recently published prediction model (Michigan model) for the duration of hospitalization in RSV infection demonstrated good discrimination between children with and without an increased likelihood of a hospital stay >or= 7 days, based on variables such as log weight, congenital heart disease, failure to thrive, premature birth, bronchopulmonary dysplasia, other pulmonary diseases, miscellaneous conditions, early mechanical ventilation, and early ribavirin treatment (receiver operating characteristic (ROC) area, 0.89). Validation of this model is of particular interest for Europe, since the mean duration of hospitalization in The Netherlands is approximately twice that in the USA. The objective of the current study was 1) to validate the Michigan model for RSV hospitalized patients in a large university hospital in The Netherlands, and 2) to develop our own prediction model for a prolonged hospital stay. Data from 177 children younger than 12 months of age admitted with confirmed RSV infection to the Sophia Children's Hospital Rotterdam between 1992-1995, were used for valiation of the Michigan model and derivation of the Rotterdam model. Mean duration of hospitalization for the Rotterdam database was 10.3 (+/-6.3) days, with a median of 9 days; 138 (78%) patients had a hospital stay >or= 7 days. The Michigan model performed poorly when applied to the Rotterdam database, with an ROC area of 0.65 (95% CI, 0.57-0.73). The Rotterdam prediction model (hospital stay >or= 9 days, the median in our database) considered weight and need for oxygen supplementation. The ROC area was 0.65 (95% CI, 0.57-0.73). When using data from patients for the 1995-1996 season, the ROC area was 0.52 (95% CI, 0.34-0.72). The Michigan and the Rotterdam models failed to identify a considerable number of patients who had a prolonged hospital stay, with a low false-positive rate. We conclude that neither the Michigan, nor the Rotterdam model reliably predicted the duration of hospitalization based on demographic and clinical variables.     

Human respiratory syncytial virus diversity and epidemiology among patients hospitalized with severe respiratory illness in South Africa, 2012–2015

BackgroundWe aimed to describe the prevalence of human respiratory syncytial virus (HRSV) and evaluate associations between HRSV subgroups and/or genotypes and epidemiologic characteristics and clinical outcomes in patients hospitalized with severe respiratory illness (SRI).MethodsBetween January 2012 and December 2015, we enrolled patients of all ages admitted to two South African hospitals with SRI in prospective hospital‐based syndromic surveillance. We collected respiratory specimens and clinical and epidemiological data. Unconditional random effect multivariable logistic regression was used to assess factors associated with HRSV infection.ResultsHRSV was detected in 11.2% (772/6908) of enrolled patients of which 47.0% (363/772) were under the age of 6 months. There were no differences in clinical outcomes of HRSV subgroup A‐infected patients compared with HRSV subgroup B‐infected patients but among patients aged <5 years, children with HRSV subgroup A were more likely be coinfected with Streptococcus pneumoniae (23/208, 11.0% vs. 2/90, 2.0%; adjusted odds ratio 5.7). No significant associations of HRSV A genotypes NA1 and ON1 with specific clinical outcomes were observed.ConclusionsWhile HRSV subgroup and genotype dominance shifted between seasons, we showed similar genotype diversity as noted worldwide. We found no association between clinical outcomes and HRSV subgroups or genotypes.     

干扰素和更昔洛韦治疗婴幼儿呼吸道合胞病毒肺炎疗效分析简

目的 探讨干扰素和更昔洛韦治疗婴幼儿呼吸道合胞病毒肺炎疗效分析和对免疫功能的影响.方法 将90例呼吸道合胞病毒肺炎患儿随机分为两组,对照组45例给予干扰素治疗,观察组45例给予更昔洛韦治疗,比较两组疗效.婴幼儿患者T淋巴细胞亚群百分率、IL-2R的百分率、IL-2水平.结果 观察组的临床总有效率达95.6%,明显高于对照组（86.7%）,P＜0.05;两组在治疗RSVP前后,免疫指标大小有明显的差异,P＜0.05,但两组在治疗RSVP后,两组免疫指标大小并无明显差异,P3＞0.05.结论更昔洛韦对于治疗婴幼儿呼吸道合胞病肺炎毒疗效更佳.