长春地区冬季儿童上呼吸道感染病毒病原学检测结果分析

目的了解长春地区冬春季急性上呼吸道感染患儿6种呼吸道病毒感染情况。方法采集门诊119例患儿咽拭子,应用(RT)-PCR方法检测甲型流感病毒(IVA)、A亚型呼吸道合胞病毒(RSVA)、副流感病毒(PIV)3型、腺病毒(ADV)、博卡病毒(HBoV)以及人偏肺病毒(hMPV)等6种常见呼吸道病毒。结果检测出阳性标本53例,阳性率44.54%,以流感病毒最高(19.33%)。经χ2检验,病毒总检出率在性别间差异有统计学意义(χ2=7.96,P<0.05)。结论流感病毒是长春地区2008年冬季患儿急性上呼吸道感染的主要病毒病原,男性患儿较女性患儿对呼吸道病毒易感。     

Development of a PIV-vectored RSV vaccine: preclinical evaluation of safety, toxicity, and enhanced disease and initial clinical testing in healthy adults

MEDI-534 is a bivalent live attenuated vaccine candidate against human respiratory syncytial virus (hRSV) and human parainfluenza virus type 3 (hPIV3) that was previously shown to be immunogenic and to protect rodents and African green monkeys from wild-type (wt) hRSV challenge. We performed further preclinical evaluations to address the safety of MEDI-534 prior to human testing. MEDI-534 did not predispose rodents to enhanced RSV disease following wt-RSV challenge, and the tissue tropism of the chimeric virus was confined to the respiratory tract. Representative clinical trial material did not produce toxicity in rats. In adults, MEDI-534 was highly restricted in replication, did not boost RSV and PIV3 antibody titers, and produced no medically significant vaccine-related adverse events thereby warranting further evaluation in pediatric populations.     

Genetic stability of RSV-F expression and the restricted growth phenotype of a live attenuated PIV3 vectored RSV vaccine candidate (MEDI-534) following restrictive growth in human lung cells

MEDI-534 is the first live, attenuated and vectored respiratory syncytial virus (RSV) vaccine to be evaluated in seronegative children. It consists of a bovine/human parainfluenza virus type 3 (PIV3) backbone with the RSV fusion glycoprotein (RSV-F) expressed from the second position. The PIV3 fusion and hemaglutinin-neuraminidase proteins are human-derived. No small animal appropriately replicates the restrictive growth of bovine PIV3 (bPIV3) based viruses relative to human PIV3 (hPIV3) observed in the respiratory tract of rhesus monkeys and humans, making analysis of the genetic stability of the attenuation phenotype and maintenance of RSV-F expression difficult. Screening of multiple cell-lines identified MRC-5 cells as supporting permissive growth of hPIV3 while restricting bPIV3 and MEDI-534 growth. In MRC-5 cells, the peak titers of MEDI-534 were more than 20-fold lower compared to hPIV3 peak titers. After more than 10 multicycle passages in MRC-5 cells, genetic alterations were detected in MEDI-534 that contributed to a partial loss in restricted growth in MRC-5 cells and a decrease in RSV-F expression. These adaptive mutations did not occur in the RSV-F gene but were found in the polyA sequence upstream of the transgene. MRC-5 adapted MEDI-534 viruses (1) lost some attenuation but did not replicate to the level of hPIV3 in this cell line, (2) did not completely lose RSV-F expression and (3) were able to elicit a protective anti-RSV immune response in hamsters despite lower levels of RSV-F expression. Interestingly analysis of shed MEDI-534 from a recent clinical trial indicates that in some recipients similar mutations arise by day 7 or day 12 post immunization (in press) suggesting that these mutations can arise rapidly in the human host. The utility and limits of MRC-5 cells for characterizing the attenuation and RSV-F expression of MEDI-534 is discussed.     

Evaluation of pre- and post-fusion Human metapneumovirus F proteins as subunit vaccine candidates in mice

Human metapneumovirus (hMPV) is an important respiratory pathogen especially in young children and elderly subjects. Our objective was to assess the immunogenicity and protection conferred by predominant pre- and post-fusion (F) hMPV-F constructs in Balb/C mice. Immunizations without adjuvant were not immunogenic whereas alum-adjuvanted hMPV-F proteins, regardless of their conformations, generated comparable neutralizing antibody titers with undetectable pulmonary viral titers following viral challenge. In conclusion, we found no apparent advantage for mixtures of predominant pre-fusion F proteins over post-fusion conformations for hMPV vaccination in opposite to recent data obtained with the human respiratory syncytial virus.     

Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development.     

急性下呼吸道感染患者支气管肺泡灌洗液非典型病原体与病毒检测

目的探讨急性下呼吸道感染患者非典型病原体与病毒感染情况。方法采集71例社区获得性肺炎和21例急性气管一支气管炎患者支气管肺泡灌洗液（BALF），采用多重PCR技术与Luminex xMAP多重分析技术平台相结合，以荧光微球为载体检测非典型病原体与多种病毒。结果10种（14个亚型）病原体阳性检出率为50．00％，其中病毒阳性检出率为33．69％，以流感病毒A为主，其占病毒阳性检出的67．74％f非典型病原体阳性检出率为16．30％，以肺炎支原体（MP）为多，其占非典型病原体阳性检出的73．33％IMP、病毒和细菌混合感染率为13．04％。结论多重荧光微球检测技术具有高通量、敏感性高、特异性强、检测速度快、价格便宜的优点，可实用于临床病原微生物的快速检测；流感病毒A和肺炎支原体是本地区社区获得性肺炎和急性气管-支气管炎重要的病原体，并有相互混合感染或与细菌混合感染，临床应予重视。     

西宁地区儿童急性呼吸道感染病毒病原学检测分析

目的:探讨西宁市儿童常见呼吸道病毒感染的病原学特点和分布特征。方法:采集141例急性呼吸道感染住院患儿的咽拭子标本,采用逆转录-聚合酶链反应(RT-PCR)进行合胞病毒(RSV),流感病毒A、B型(IFV A、B),副流感病毒Ⅰ、Ⅱ、Ⅲ型(PIVⅠ、Ⅱ、Ⅲ),腺病毒(ADV),冠状病毒(HCoV)OC43/HKU1、229E/NL63,博卡病毒(HBoV),偏肺病毒(hMPV)检测。结果:从141份鼻咽拭子样本中检测出呼吸道病毒阳性22例,阳性率为15.6%。检出4种呼吸道病毒,其构成比分别为ADV 45.5%、PIVⅡ26.1%、HCoV-229E/NL63 17.4%、IFV A 13.0%。不同性别间患儿呼吸道病毒阳性率无统计学差异(P>0.05)。<6个月组阳性率最低(6.3%),3～6岁年龄组阳性率最高(29.2%)。结论:西宁地区急性呼吸道感染病毒病原检出率低于全国水平,ADV为西宁地区急性呼吸道感染的首位病毒病原体,HCoV-229E/NL63是该地区急性呼吸道感染的病原之一。     

河北省2013-2015年急性上呼吸道感染病毒病原学构成研究

目的 了解河北省2013-2015年引起急性上呼吸道感染（acute upper respiratory infection,AURI）的病原学构成和流行病学特征,为呼吸道感染的诊断和防治工作提供科学依据。方法 采集河北省4家医院门诊就诊的AURI患者的咽拭子标本1 551份,提取核酸后采用多重实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）方法检测15种呼吸道病毒。结果 采集1 551份临床咽拭子标本,检测出阳性标本714例,阳性率为46.03%,其中鼻病毒阳性率最高,为186（11.99%）例,其次为副流感病毒3型167（10.77%）例、呼吸道合胞病毒122（7.87%）例、腺病毒108（6.96%）例、乙型流感病毒56（3.61%）例、人偏肺病毒40（2.58%）例、甲型流感病毒39（2.51%）例、人博卡病毒38（2.45%）例、副流感病毒1型35（2.26%）例、冠状病毒229E/NL63型33（2.13%）例、肠道病毒32（2.06%）例、副流感病毒4型31（2.00%）例、冠状病毒OC43型30（1.93%）例、副流感病毒2型11（0.71%）例。病毒混合感染病例176例,检出率为11.35%。5岁以下年龄组病毒阳性率最高为56.07%（337/601）。结论 河北省AURI主要以鼻病毒、副流感病毒3型、呼吸道合胞病毒、腺病毒、流感病毒为主,各病毒有各自的流行特征。     

Evaluation of recombinant respiratory syncytial virus gene deletion mutants in African green monkeys for their potential as live attenuated vaccine candidates

Towards the goal of developing live attenuated respiratory syncytial virus (RSV) vaccines to prevent severe respiratory tract infections caused by respiratory syncytial virus, recombinant RSV containing a deletion of single or multiple NS1, NS2, SH and M2-2 genes have been generated. In this study, recombinants, rA2DeltaM2-2, rA2DeltaNS2, rA2DeltaNS1NS2, rA2DeltaSHNS2, rA2DeltaM2-2NS2 were evaluated in African green monkeys (AGMs) for their infectivity, immunogenicity and protection against wild type (wt) RSV challenge. Replication of rA2DeltaNS2 and rA2DeltaSHNS2 was not attenuated in either the upper or the lower respiratory tracts of AGMs. On the other hands, rA2DeltaNS1NS2 was over-attenuated; it did not replicate in the respiratory tracts of the infected monkeys and did not provide sufficient protection against wild type RSV challenge. rA2DeltaM2-2NS2 was slightly more attenuated than rA2DeltaM2-2 and provided partial protection against wt RSV challenge. rA2DeltaM2-2, and possibly rA2DeltaM2-2NS2, exhibited the attenuated but protective phenotypes in the monkeys that could be further evaluated as potential live attenuated RSV vaccine candidates in the clinical studies.     

Immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hMPV infection

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is an important cause of acute respiratory tract disease. In the 1960s, vaccination with formalin-inactivated paramyxovirus preparations--respiratory syncytial virus (RSV) and measles virus (MV)--resulted in predisposition for enhanced disease upon natural infection. We have produced a formalin-inactivated hMPV preparation (FI-hMPV), which was used to immunize young cynomolgus macaques. Six days after challenge FI-hMPV-primed monkeys had developed eosinophilic bronchitis and bronchiolitis, indicative of a hypersensitivity response. This study indicates that formalin-inactivated hMPV vaccines have the same propensity to predispose for immune-mediated disease as inactivated RSV and MV vaccines.     

Human parainfluenza virus type I (HPIV1) vaccine candidates designed by reverse genetics are attenuated and efficacious in African green monkeys

A set of recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) vaccine candidates was evaluated for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs). Temperature sensitive (ts) and non-ts attenuating (att) mutations in the P/C and L genes were introduced individually or in various combinations into rHPIV1, including the C(R84G) and HN(T553A) mutations identified in the present work and the C(F170S), L(Y942A), and L(L992C) mutations identified previously. The rHPIV1 vaccine candidates exhibited a spectrum of attenuation in AGMs. One genetically and phenotypically stable vaccine candidate, rC(R84G/F170S)L(Y942A/L992C), was attenuated and efficacious in AGMs and is a promising live attenuated intranasal HPIV1 vaccine candidate suitable for clinical evaluation.     

Current status of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) vaccine development: Memorandum from a Joint WHO/NIAID meeting

A Joint WHO/National Institute of Allergy and Infectious Diseases (NIAID) meeting on the current status of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) vaccine development was held in Bethesda, MD, from 30 September to 1 October 1996. The meeting summarized the worldwide impact of RSV and PIV3; presented the current status of development of RSV and PIV3 vaccines; and examined the applications of recombinant DNA technology to the development and characterization of vaccines and to the understanding of viral pathogenesis.     

Evaluation of replication, immunogenicity and protective efficacy of a live attenuated cold-adapted pandemic H1N1 influenza virus vaccine in non-human primates

We studied the replication of influenza A/California/07/09 (H1N1) wild type (CA09wt) virus in two non-human primate species and used one of these models to evaluate the immunogenicity and protective efficacy of a live attenuated cold-adapted vaccine, which contains the hemagglutinin and neuraminidase from the H1N1 wild type (wt) virus and six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. We infected African green monkeys (AGMs) and rhesus macaques with 2×10(6) TCID(50) of CA09wt and CA09ca influenza viruses. The virus CA09wt replicated in the upper respiratory tract of all animals but the titers in upper respiratory tract tissues of rhesus macaques were significant higher than in AGMs (mean peak titers 10(4.5) TCID(50)/g and 10(2.0) TCID(50)/g on days 4 and 2 post-infection, respectively; p<0.01). Virus replication was observed in the lungs of all rhesus macaques (10(2.0)-10(5.4) TCID(50)/g) whereas only 2 out of 4 AGMs had virus recovered from the lungs (10(2.5)-10(3.5) TCID(50)/g). The CA09ca vaccine virus was attenuated and highly restricted in replication in both AGMs and rhesus macaques. We evaluated the immunogenicity and protective efficacy of the CA09ca vaccine in rhesus macaques because CA09wt virus replicated more efficiently in this species. One or two doses of vaccine were administered intranasally and intratracheally to rhesus macaques. For the two-dose group, the vaccine was administered 4-weeks apart. Immunogenicity was assessed by measuring hemagglutination-inhibiting (HAI) antibodies in the serum and specific IgA antibodies to CA09wt virus in the nasal wash. One or two doses of the vaccine elicited a significant rise in HAI titers (range 40-320). Two doses of CA09ca elicited higher pH1N1-specific IgA titers than in the mock-immunized group (p<0.01). Vaccine efficacy was assessed by comparing titers of CA09wt challenge virus in the respiratory tract of mock-immunized and CA09ca vaccinated monkeys. Significantly lower virus titers were observed in the lungs of vaccinated animals than mock-immunized animals (p≤0.01). Our results demonstrate that AGMs and rhesus macaques support the replication of pandemic H1N1 influenza virus to different degrees and a cold-adapted pH1N1 vaccine elicits protective immunity against pH1N1 virus infection in rhesus macaques.     

2013年度贵州省发热呼吸道症候群病毒感染状况调查

摘要:目的　了解贵州省发热呼吸道症候群病毒性疾病的感染状况。方法　调查贵州省2013年度发热呼吸道症候群病例432例,采集呼吸道标本421份,通过相应试剂盒提取核酸,进行实时荧光定量（Real-Time）PCR检测流感病毒、副流感病毒、冠状病毒、呼吸道合胞病毒、腺病毒、博卡病毒,鼻病毒及偏肺病毒。结果　从421份标本中检测出阳性标本128份,阳性率为30.41%。其中流感病毒60份,阳性率为14.25%;冠状病毒8份,阳性率为1.90%;博卡病毒3份,阳性率为0.71%;腺病毒10份,阳性率为2.38%;偏肺病毒9份,阳性率为2.14%;鼻病毒38份,阳性率为9.03%;未检出呼吸道合胞病毒和副流感病毒。检出3例流感病毒和鼻病毒混合感染。结论　呼吸道病毒是导致发热呼吸道症候群的重要病原体,其中以流感病毒最为常见,鼻病毒其次,腺病毒、偏肺病毒等检出率较低。     

成都市发热呼吸道症候群患者病毒感染调查

目的了解成都市发热呼吸道感染病例常见病毒的感染状况,探讨发热呼吸道症候群的病原谱构成情况。方法 2010-07/2011-03,采集成都市3家哨点医院具备发热、咳嗽以及胸部X线片提示肺部炎性改变等临床症状的呼吸道感染病例标本302份;采用多重RT-PCR方法,对302份呼吸道标本同时进行流感病毒、副流感病毒、呼吸道合胞病毒、冠状病毒、人偏肺病毒、人博卡病毒、人腺病毒、人鼻病毒和肠道病毒等常见呼吸道病毒核酸检测。结果 302份标本中检出阳性标本72份,核酸阳性率为23.84%（72/302）,其中两种病毒混合感染6份,占阳性标本的8.33%（6/72）;5岁以下儿童患者标本病毒核酸阳性检出率为39.33%（59/150）,其中偏肺病毒的检出率最高;6～18岁患者标本病毒核酸阳性检出率为15.15%（5/33）,其中呼吸道合胞病毒检出率最高;18岁以上患者标本阳性检出率为11.76%（14/119）,其中流感病毒检出率最高。3月份病毒核酸检出率最高为74.36%（29/39）,不同性别患者之间病毒核酸检出率差异无统计学意义。结论成都市发热呼吸道病毒感染病例的病原体以偏肺病毒、鼻病毒和呼吸道合胞病毒为主。     

A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5)

Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses – one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection.     

Human metapneumovirus: enhanced pulmonary disease in cotton rats immunized with formalin-inactivated virus vaccine and challenged

Cotton rats (Sigmodon hispidus) are susceptible to the recently discovered human metapneumovirus (hMPV), an agent closely related to human respiratory syncytial virus. Since certain respiratory syncytial virus vaccines can induce enhanced disease upon viral challenge, we have done similar experiments with hMPV in cotton rats. Young adult cotton rats were vaccinated with a formalin-inactivated preparation of hMPV strain C-85473, or with a mock preparation of the vaccine on day 0 and again on day 28. All animals were challenged intranasally on day 49 with 10(7) TCID50 of the same hMPV strain. Animals were sacrificed on days 4, 7, and 10 post-challenge and lungs were removed for viral quantitation, histopathology, and cytokine mRNA expression analysis (interferon-gamma (IFN-gamma) and interleukin-4 (IL-4)). Although the vaccinated animals showed almost complete protection from viral replication in the lungs (<10(2.0) TCID50 per gram), there was a dramatic increase in the lung pathology, particularly the interstitial pneumonitis and alveolitis with elevated serum neutralizing antibody titer prior to challenge. Cytokine profiles were distinctive from those observed during primary infection and re-infection. The data raise safety concerns for hMPV vaccine preparations.     

Hospitalization rates for human metapneumovirus infection among 0- to 3-year-olds in Gipuzkoa (Basque Country), Spain

Numerous studies have been published on human metapneumovirus (HMPV) infection, but few have been population based. The main aim of this study was to estimate the incidence rate of hospitalization for community-acquired HMPV infection in infants and children aged <3 years. Between July 2004 and June 2007, 796 episodes (742 patients) of community-acquired acute respiratory infection were hospitalized. HMPV was detected in 90 episodes (11.3%). Fifty-nine episodes occurred in infants aged <1 year. The mean length of hospital stay was 6.2 days (range 2-31 days). Thirteen children required admission to the intensive care unit. Viral co-infections were detected in 46 episodes (51.1%). The incidence rate of hospitalization per 1000 inhabitants was 2.6 (95% CI 2.1-3.2), lower than that for respiratory syncytial virus, but higher than that observed for the influenza and parainfluenza viruses. HMPV is a major respiratory pathogen that leads to a high hospitalization rate.     

Sendai virus recombinant vaccine expressing hPIV-3 HN or F elicits protective immunity and combines with a second recombinant to prevent hPIV-1, hPIV-3 and RSV infections

The human parainfluenza viruses (hPIVs) and respiratory syncytial virus (RSV) are the leading causes of serious respiratory illness in the human pediatric population. Despite decades of research, there are currently no licensed vaccines for either the hPIV or RSV pathogens. Here we describe the testing of hPIV-3 and RSV candidate vaccines using Sendai virus (SeV, murine PIV-1) as a vector. SeV was selected as the vaccine backbone, because it has been shown to elicit robust and durable immune activities in animal studies, and has already advanced to human safety trials as a xenogenic vaccine for hPIV-1. Two new SeV-based hPIV-3 vaccine candidates were first generated by inserting either the fusion (F) gene or hemagglutinin-neuraminidase (HN) gene from hPIV-3 into SeV. The resultant rSeV-hPIV3-F and rSeV-hPIV3-HN vaccines expressed their inserted hPIV-3 genes upon infection. The inoculation of either vaccine into cotton rats elicited binding and neutralizing antibody activities, as well as interferon-gamma-producing T cells. Vaccination of cotton rats resulted in protection against subsequent challenges with either homologous or heterologous hPIV-3. Furthermore, vaccination of cotton rats with a mixture of rSeV-hPIV3-HN and a previously described recombinant SeV expressing the F protein of RSV resulted in protection against three different challenge viruses: hPIV-3, hPIV-1 and RSV. Results encourage the continued development of the candidate recombinant SeV vaccines to combat serious respiratory infections of children.