Effect of respiratory syncytial virus infection on mice with protein malnutrition

Respiratory syncytial virus (RSV) pulmonary infection was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. As in previously published experiments with Sendai virus, animals on the deficient diet became clinically malnourished, and certain aspects of their cell-mediated immunity were altered. The course of RSV infection in protein-deprived mice was essentially identical to that in normally nourished animals. The titer of virus recovered from lung homogenates over time, as well as the histologic picture of bronchiolitis, were identical under all experimental conditions. This model, unlike that of Sendai virus infection, fails to demonstrate an effect of protein malnutrition on RSV infection.     

Genetic susceptibility to respiratory syncytial virus infection in inbred mice

Differences in the severity of respiratory syncytial virus (RSV)-induced lower respiratory disease in infants have been attributed to multiple environmental and genetic factors. To identify the genetic factor(s) influencing RSV susceptibility, we examined RSV infection in eight inbred mouse strains. Lung RSV titers differed significantly between mouse strains: the RSV titers were 15-fold higher in AKR/J (permissive) mice compared with C57BL/6J (resistant) mice at 4 days after inoculation. This strain-specific difference in RSV titers suggested that susceptibility to RSV infection was attributable to genetic differences between strains. To examine the mode of inheritance of RSV susceptibility, F1 and backcross (F1 x AKR/J) progeny were infected and RSV titers determined. RSV titers in the F1 progeny were similar to those found in the resistant (C57BL/6J) parent, suggesting resistance was inherited as a dominant trait. The distribution of RSV titers in backcross progeny were discordant with that predicted for a single gene effect, suggesting susceptibility was influenced by more than one gene. These data suggest that RSV susceptibility is a multigenic trait that should be amenable to resolution by genomic analysis.     

Humoral immunity to respiratory syncytial virus infection in the elderly.

The relationship between serum immunoglobulins and the severity and risk of Respiratory Syncytial Virus (RSV) infection in the institutionalized elderly was prospectively assessed during the winter of 1989-1990 at a 591 bed nursing home. Forty RSV infections were identified out of 149 respiratory illnesses by isolation of the virus or by a greater than or equal to 4-fold rise in RSV-specific IgG by EIA. Acute serum RSV IgG levels were similar in those with RSV infection and those with non-RSV illness. Additionally, among the RSV-infected elderly there was no correlation between severity of clinical symptoms and level of acute IgG titers by EIA or virus neutralization. The results of this study suggest that humoral immunity does not play a major role in reducing the risk of infection nor modulating the clinical severity of illness in elderly persons with RSV infections.     

Development of cell-mediated cytotoxic immunity to respiratory syncytial virus in human infants following naturally acquired infection

With virus-infected autologous and allogenic mononuclear cells as specific targets, the development of cell-mediated cytotoxic reactivity to respiratory syncytial virus (RSV) was studied in peripheral blood lymphocytes (PBL) in groups of infants with acute RSV infection and in other control groups of subjects during a community outbreak of RSV infection. No RSV-specific cellular cytotoxicity was observed in cord blood lymphocytes and in other uninfected controls. The PBL of infants with acute RSV infection exhibited significant cellular cytotoxic response. The activity peaked early, usually within 1 week after infection. The response appeared to be age-dependent. Over 65% of infants 6-24 months of age and about 35-38% of infants under 5 months of age exhibited cellular cytotoxicity to RSV. Cellular cytotoxic reactivity was observed against autologous and less frequently against allogenic RSV-infected target cells. These findings suggest the appearance of virus-specific cell-mediated cytotoxic immune response after acute RSV infection. The development of cellular cytotoxic responses may play a role in the mechanisms of protection against or the pathogenesis of RSV infection in man.     

加巴喷丁体内外抗RSV感染的作用

目的探讨加巴喷丁对呼吸道合胞病毒(respiratory syncytial virus,RSV)的抗病毒作用。方法体外实验通过MTS、TCID50、q RT-PCR方法检测不同浓度加巴喷丁对RSV感染的抑制作用,包括细胞活力、病毒复制及细胞因子的变化。体内实验选取4~6周龄C57BL/6小鼠,随机分为空白对照组、RSV感染组、低剂量及高剂量加巴喷丁处理组,连续腹腔注射给药,每日观察体质量变化,HE染色观察小鼠肺部的病理变化,q RT-PCR方法检测肺部病毒载量。结果 1、2、5、10 mmol/L不同浓度加巴喷丁显著增加RSV感染A549细胞的活力;5、10 mmol/L浓度的加巴喷丁可显著降低RSV感染A549细胞的病毒载量,10 mmol/L的加巴喷丁可显著抑制病毒的复制,减少CCL3、CCL5、CXCL2及TNF-α、IL-6、IL-8等趋化因子和炎性因子表达,促进干扰素IFN-α、IFN-β表达;动物实验表明90μg、180μg的加巴喷丁处理组可减轻RSV感染小鼠的体质量变化、改善肺部病理损伤和降低病毒载量。结论加巴喷丁可通过抑制病毒复制,调节趋化因子及炎性因子释放,促进干扰素分泌的方式发挥体内抗病毒作用,对RSV感染的C57BL/6小鼠有一定的治疗作用,可改善肺部病理,为进一步临床应用提供实验依据。     

Otitis and respiratory distress episodes following a respiratory syncytial virus infection

Objective  To document, over two consecutive respiratory syncytial virus (RSV) seasons, the occurrence of acute otitis media (AOM) and recurrence of respiratory distress in children < 2 years of age hospitalized for respiratory distress.
Methods  Patients were examined during hospitalization and at 6 weeks and 6 months after discharge. RSV testing was performed on all patients, and hospitalized patients were evaluated daily for the occurrence of AOM.
Results  In total, 347 children were enrolled; 54.8% were RSV positive, and 45.2% were RSV negative. Children were most frequently diagnosed as having bronchiolitis (71.9%) or asthmatic bronchitis (17.9%); other diagnoses included pneumonia, laryngitis, and rhinitis. During hospitalization, AOM was diagnosed in 16.8% of RSV-positive versus 8.3% of RSV-negative children ( P  < 0.05). Six weeks after discharge, AOM was reported in 10.4% of RSV-positive as compared with 5.8% of RSV-negative patients. Six months later, AOM was reported in 2.9% of the RSV-positive and 7.6% of the RSV-negative patients. A second episode of acute respiratory distress, which either required (9) or did not require (35) hospitalization, occurred in 18.4% of the total population, with similar proportions of RSV-positive and RSV-negative children (17% versus 18.6%).
Conclusion  We conclude that RSV appears to be an important contributing factor for the occurrence of AOM in young children hospitalized with respiratory distress. The occurrence of a second episode of acute respiratory distress did not appear to correlate with the previous RSV infection, but longer-term follow-up is required.     

Serum antibody decay in adults following natural respiratory syncytial virus infection

Serum antibody decay following RSV infection in adults was examined to evaluate the durability of the immune response. Twenty subjects with RSV infection and 10 subjects who remained RSV uninfected had blood samples obtained over 16-25 months analyzed by microneutralization assay and enzyme immunoassay. The mean titers of infected subjects rose approximately eightfold post-infection. The mean rate of antibody decline was -0.20 log 2 titer per month which led to a > or =fourfold drop in titer in 75% of subjects at 1 year. In contrast, titers of uninfected subjects were relatively stable. The partial immunity resulting from a boost in serum antibody following natural RSV infection in adults appears to be short lived.     

Disease severity in respiratory syncytial virus infection: Role of host genetic variation

Respiratory syncytial virus (RSV) infection is the most common cause of bronchiolitis and pneumonia in the pediatric population worldwide. The immunopathology of RSV infection varies considerably and severe disease occurs only in a minority of the population. There are many factors (host, viral, and environmental) that contribute to the complicated disease phenotype. In this regard, host factors are decisive for pulmonary susceptibility to RSV infection. Host genetic diversity certainly affects the balance between control of viral replication and tissue damage during RSV infection, consequently impacting on diseases outcome. In this review, we discuss the role of host genetic variation in disease caused by RSV aiming to highlight genetic risk factors for one of the most common diseases in early childhood. Our findings clearly indicate that the response of each individual to infection is influenced by genetic diversity mainly linked to the regulation of host immune responses. Future genetic association and functional studies using more powerful and consistently reproducible approaches will likely be able to confirm, refine, and expand our developing concept of RSV disease pathogenesis.     

Respiratory syncytial virus infection in children admitted to hospital but ventilated mechanically for other reasons

One thousand five hundred sixty‐eight RSV infections were documented prospectively in 1,541 pediatric patients. Of these, 20 (1.3%) had acquired the RSV infection while treated by mechanical ventilation for reasons other than the actual RSV infection (group ventilated mechanically). The clinical characteristics of children who were infected with respiratory syncytial virus (RSV) infection while ventilated mechanically for other reasons are described and compared with a matched control group. Sixty percent of the group ventilated mechanically had at least one additional risk factor for a severe course of infection (prematurity 50%, chronic lung disease 20%, congenital heart disease 35%, immunodeficiency 20%). The median age at diagnosis in the group ventilated mechanically was 4.2 months. The matched pairs analysis (group ventilated mechanically vs. control group) revealed a higher proportion of patients with hypoxemia and apnoea in the group ventilated mechanically; more patients in the control group showed symptoms of airway obstruction (wheezing). At least one chest radiography was performed in 95% of the patients (n = 19) in the group ventilated mechanically versus 45% (n = 9) in the control group (P = 0.001). The frequency of pneumonia was 40% in the group ventilated mechanically and 20% in the control group. Despite existing consensus recommendations, only two patients (10%) of the group ventilated mechanically had received palivizumab previously. Significantly more patients in the group ventilated mechanically received antibiotic treatment (85% vs. 45%, P = 0.008), and attributable mortality was higher in the group ventilated mechanically (15% [n = 3] vs. 0% in the control group, P = 0.231). Children treated by long term mechanical ventilation may acquire RSV infection by transmission by droplets or caregivers and face an increased risk of a severe course of RSV infection. The low rate of immunoprophylaxis in this particular risk group should be improved. J. Med. Virol. 81:160–166, 2009. © 2008 Wiley‐Liss, Inc.     

Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay, indirect immunofluorescence, and virus isolation: a comparative study

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.     

Antibody responses of children to the C-terminal peptide of the SH protein of respiratory syncytial virus and the immunological characterization of this protein

The SH protein of RSV, a small integrated hydrophobic membrane protein, consists of 64 amino acid residues in the polypeptide of subgroup A and 65 amino acid residues in the polypeptide of subgroup B. We synthesized five peptides, representing the SH protein of each RSV subgroup comprised of the following amino acid residues: 2–16, 12–26, 35–49, 45–60, and for subgroup A, 51–64 and for subgroup B, 51–65. Peptides 2–16 and 51–64/65 represented the N-terminal and C-terminal ends of the protein, respectively. In RIPA, under reducing conditions with mercaptoethanol, hyperimmune guinea pig (GP) serum against C-terminal peptide of the two subgroups precipitated the homologous 7.5 kDa and 21–30 kDa SH proteins. Under nonreducing conditions, the GP antipeptide sera precipitated all three SH proteins, suggesting that the 13–15 kDa protein exists as a dimer. The subgroup A 7.5 and 13–15 kDa proteins had apparent molecular weights about 1–2 kDa higher than the corresponding subgroup B proteins. The C-terminal peptides of subgroups A and B were used to characterize the immune response of 11 children, age 1 month to 1 year, with presumed primary RSV infection. Three of 4 children with subgroup A infection and 4 of 7 children with subgroup B infection developed homologous 4-fold rises in antibody to C-terminal peptide (aa 51–64/65) during convalescence. Except for one child with subgroup A and one child with subgroup B infection, the other 5 children developed heterologous rises also. The antibody levels to C-terminal peptide were low suggesting that the SH protein was a weak stimulus of antibody in children with naturally acquired infection. Thus, it appears that the C-terminal peptides of the SH protein are not useful as ELISA antigens in characterizing the subgroup-specific immune responses to RSV infection. © 1993 Wiley-Liss, Inc. © 1993 Wiley-Liss, Inc.     

Chemokine-receptor upregulation and disease severity in respiratory syncytial virus infection

Respiratory Syncytial Virus (RSV) infection is an important cause of severe infant bronchiolitis, partly due to lower airway inflammation orchestrated by virus-induced chemokine secretion. Chemokine receptors may therefore be therapeutic targets. We investigated RSV-induced chemokine receptor (CCR) 1, 2 and 5 surface expressions in a cellular model and in infants. RSV infection increased human monocytic CCR1, 2 and 5 expression, as assessed by FACS, via replication-dependent mechanisms. CCR1 and CCR5 levels peaked at 36 h and CCR2 levels at 48 h. Monocytes from infants with RSV-bronchiolitis significantly increased CCR1 expression after ex vivo RSV infection compared to controls. Expression of CCR5 also increased, and correlated with CCR1 expression (r=0.78, p<0.0001). CCR1 upregulation correlated with disease severity markers. Monocyte CCR1 receptors were functionally active as stimulation resulted in calcium influx. CCR1/5 blocking strategies may be useful in decreasing cellular inflammation in RSV infection.     

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.     

The association between MICA/MICB polymorphism and respiratory syncytial virus infection in children

MICA/MICB gene polymorphisms are related to several cancers and infectious diseases, but there are no reports on the association between MICA/MICB gene polymorphisms and respiratory syncytial virus (RSV) infection. To clarify the association between MICA/MICB gene polymorphisms and infection of RSV in children, we collected fresh blood samples from paediatric patients with and without pneumonia after RSV infection. The MICA/MICB alleles were characterized by PCR sequence‐specific primers (PCR‐SSP) and PCR sequence‐based genotyping (PCR‐SBT), and then, the frequency of the MICA/MICB alleles and haplotypes was calculated. The results showed that the frequencies of MICA*002:01 and MICA‐A9 in RSV‐infected patients were significantly lower than in controls (9% vs. 20%, pc = 0.04). The allele frequency of MICA*002:01 in pneumonia patients (8%) and nonpneumonia patients (9%) was significantly lower than in controls (20%, pc = 0.02). MICA*002:01‐MICB*008(Δrel = 0.616), MICA*009‐MICB*016 (Δrel = 0.506), and MICA*045‐MICB*014 (Δrel = 0.700) showed linkage disequilibrium in patients infected with RSV. The haplotype frequency of MICA*002:01‐MICB*005:02 in RSV‐infected patients was significantly lower than in controls (10% vs. 16%, pc = 0.033). In conclusion, allele MICA*002:01/A9 and haplotype MICA*002:01‐MICB*005:02 were negatively associated with RSV respiratory tract infections.     

肺表面活性蛋白A基因多态性与呼吸道合胞病毒下呼吸道感染的相关性研究

目的探讨肺表面活性蛋白（SP）-A1-1101C/T和SP-A2-1649G/C位点基因多态性与呼吸道合胞病毒下呼吸道感染（RSV-LRTI）的相关性。方法应用聚合酶链反应-限制性酶切片段长度多态性（PCR-RFLP）分析法检测200例病例组和150例健康对照组2个位点的基因多态性,并进行基因型、等位基因型频率分析;采用DNA测序法进行测序分析。结果 1.病例组SP-A1-1101C/T位点TT、CT基因型频率分别为76.0%、24.0%,T、C等位基因频率分别为88.0%、12.0%;对照组TT、CT基因型频率分别为80.7%、19.3%,T、C等位基因频率分别为90.3%、9.7%,两组基因型及等位基因频率差异无统计学意义（χ2=1.088、0.953,P〉0.05）。2.病例组SP-A2-1649G/C位点CC、CG、GG基因型频率分别为41.5%、50.5%、8.0%,C、G等位基因频率分别为66.8%、33.2%;对照组CC、CG、GG基因型频率分别为43.3%、49.3%、7.4%,C、G等位基因频率分别为68.0%、32.0%,两组基因型及等位基因频率无统计学意义（χ2=0.141、0.122,P〉0.05）;但该位点基因型和等位基因在轻度和重度患儿间的差异有统计学意义（χ2=6.664、5.207,P〈0.05）,携带G等位基因的个体患重度RSV-LRTI的风险是患轻度风险的1.656倍,（OR=1.656,95%CI：1.072～2.559,P=0.023〈0.05）。结论温州地区汉族儿童存在SP-A1-1101C/T、SP-A2-1649G/C基因多态性,未发现其与RSV-LRTI疾病易感性存在关联,但携带SP-A2-1649G等位基因的个体患重度RSV-LRTI的风险是患轻度风险的1.656倍,表明G等位基因可能是影响RSV-LRTI疾病严重程度的一个候选基因。     

Influence of respiratory syncytial virus infection on cytokine and inflammatory responses in allergic mice

BACKGROUND: Th2 lymphocyte responses are associated with inflammation and disease during allergic responses. Exposure to particular environmental factors during the expression of allergy could result in more pronounced Th2-like immune responses and more severe disease. One factor might be a respiratory virus infection. OBJECTIVE: The aim of our study was to investigate the influence of respiratory syncytial virus (RSV) infection on the expression of ovalbumin (OVA)-induced allergy in BALB/c mice. METHODS: We determined OVA-specific IgE in serum, cytokine profiles and histopathological lesions in lungs of OVA-allergic mice after RSV infection. RESULTS: OVA sensitization and challenge induced OVA-specific IgE in serum, Th2 cytokine mRNA expression, and mononuclear and eosinophilic inflammation in the lungs. RSV inoculation during the challenge period enhanced OVA-induced IL-4 and IL-5 mRNA expression in lung tissue. RSV further enhanced the OVA-induced hypertrophy of mucous cells and eosinophilic infiltration in lung tissue. Surprisingly, RSV infection decreased Th2 cytokine secretion and eosinophilic influx in bronchoalveolar lavage of OVA-allergic mice. Because inactivated RSV did not influence these responses, replication of RSV appeared essential for the modification of OVA-induced Th2 cytokine expression. RSV did not change OVA-specific IgE levels in serum. Furthermore, the RSV-induced IL-12 mRNA expression in lung tissue of OVA-allergic mice was diminished, but IFN-gamma mRNA expression was not affected. CONCLUSION: RSV infection enhanced particular OVA-induced Th2 cytokine mRNA responses and pulmonary lesions in allergic mice and thus aggravated allergic respiratory disease.     

Type 1-like immune response is found in children with respiratory syncytial virus infection regardless of clinical severity

The immunological response of infants younger than six months to infection with respiratory syncytial virus (RSV) was studied in relation to clinical severity. IL-6 and IL-8 were found more frequently and at higher levels in the plasma samples of more severely ill patients and no significant differences were found in the levels of cytokines differentiating between Type 1 and Type 2 responses. Cellular infiltrates in nasopharyngeal washings consisted mainly of polymorphonuclear granulocytes and monocytes. Eosinophils, IgE positive cells and tryptase positive cells were found sporadically. Analyses of RSV stimulated T cell cultures established from peripheral blood mononuclear cells, for intracellular and secreted cytokines showed that, irrespective of clinical severity, the responses were dominated by the production of IFN-gamma, and that only low levels of IL-4 and IL-10 were detectable. Collectively these data do not indicate an association between clinical severity and a Type 2-like T cell response.