北京市2007-2010年风疹病原学监测

目的 对北京地区风疹病原学进行监测.方法 收集风疹疑似病例血清标本和尿液和(或)咽拭子,用于血清学检测和病毒分离.病毒分离物和临床标本中的核酸利用real-time PCR方法进行检测.结果 2007-2010年,共收集99个风疹疑似病例血清标本和临床标本,血清学确诊55个病例(56%),病毒分离确诊51个病例(52%).回顾性检测疑似风疹病例及尿液和(或)咽拭子核酸,共确诊72个风疹病例(73%).分离的风疹病毒基因型主要为1E基因型.结论 北京地区主要流行的风疹病毒为1E基因型别.

Abstract：

Objective To clarify the pathogen for rubella in Beijing from 2007 to 2010. Methods Beijing Center for Disease Preventipn and Control ( CDC ) collected the specimens (including blood, urine and throat swab specimens) frqm clinically diagnosed rubella cases for serological test and virus isolation. The nucleic acid of rubella virus in clinical specimens and isolations was detected by real-time PCR. Results Fifty-five out of 99 blood specimens were positive for anti- rubella IgM. Fifty-one out of 99 clinically diagnosed rubella cases were confirmed as rubella cases by virus isolation. Seventy-two were confirmed as rubella virus infections with real-time PCR method for detecting the nucleic acid of rubella virus in clinical specimens. Compared with the sequences of reference strains of rubella virus, all of detected rubella virus belonged the IE gene type. Conclusion This study indicates that IE gene type virus was the predominant endemic rubella virus in Beijing.     

An indirect immunocolorimetric assay to detect rubella virus infected cells

An indirect immunocolorimetric assay (ICA) to detect rubella virus infected cells by the naked eye was developed. This assay was as sensitive and specific as an indirect immunofluorescent assay (IFA), could detect as little as 10 plaque forming units (pfu) of rubella virus in the initial inoculum and could detect viruses from throat swabs. This assay detected infection with viruses of all nine rubella virus genotypes available for testing. It could be utilized for virus quantitation and a neutralization assay. In addition to detecting rubella virus infection, this assay also allowed us to confirm measles virus infection. This assay should be useful for virus isolation from clinical samples.     

Use of enzyme immunoassay and nucleic acid hybridization for detecting Sindbis virus in infected mosquitoes

Aedes aegypti mosquitoes were inoculated intrathoracically with prototype Sindbis virus, held at 26.7 degrees C for from 0-95 h and placed at -70 degrees C. Individual mosquitoes were tested for virus by plaque assay in Vero cells, for viral RNA by nucleic acid hybridization using a cloned cDNA probe, and for viral protein by enzyme-linked immunosorbent assay. Virus was detected by plaque assay as early as 8 h after infection. Sindbis virus RNA was detected by nucleic acid hybridization 18 h after infection and by enzyme-linked immunosorbent assay 10 h after infection. The results of these comparisons suggest that both nucleic acid hybridization and enzyme-linked immunosorbent assay are applicable to direct detection of Sindbis virus in mosquitoes containing virus at levels usually found during arbovirus epidemics.     

The present situation and limitation of antibody assays for diagnosis of rubella virus infection in pregnant women]

Rubella virus infection during early stages of pregnancy often results in a number of developmental disorders referred to as congenital rubella syndrome(CRS). Both clinical and laboratory diagnosis of suspect cases of CRS can be made with relative ease, particularly when expectant mothers show the typical rubella-specific rash. Serological diagnosis of CRS is accomplished using hemagglutination inhibition (HI) and enzyme-linked immunosorbent(IgM-EIA) assays. Antibody titers as determined by these assays are generally very high following acute apparent rubella infections, thus making serological diagnosis relatively easy in most cases. However, the detection of possible CRS cases can be hampered by clinically inapparent rubella infections during early pregnancy. As much as 30 percent of all acute rubella cases are inapparent infections, and there is the very real potential for such inapparent infections to occur during pregnancy, to result in fetal infections, and consequently to cause CRS. Detection of CRS becomes extremely difficult in such settings. Complicating CRS detection even more are rare rubella re-infections that might occur in early pregnancy, and unknown risk of fetal infection and CRS. In re-infection cases, HI antibody titer becomes elevated due to a secondary immune response, and IgM antibody is produced in a significant number of cases. To determine directly the fetal infection, virus genome detection was developed and applied clinically for the past decade. Using a combination of serological and genomic detection methods, the results of the investigation suggest that when rubella infection during early pregnancy occurs 1) there is a significant risk of fetal infection that results from acute apparent rubella infection, 2) there is a measurable risk of fetal infection resulting from inapparent infections as defined by HI antibody titers > or = 256 and with and IgM-EIA index > or = 7, and 3) high HI antibody titers with low IgM-EIA indices or no detectable IgM antibody in cases of inapparent rubella infections may represent rubella re-infections and result in a low risk of fetal infections.     

Coxsackievirus B3-induced acute pancreatitis: analysis of histopathological and viral parameters in a mouse model.

Coxsackievirus B3 infection in mice was studied histopathologically, by virus isolation and by nucleic acid hybridization after intraperitoneal inoculation of the virus. Extensive viraemia was detected for 1-3 days post-infection. All mice developed necrotizing acute pancreatitis and focal myocarditis. Pancreatitis eventually lead to complete atrophy of the exocrine pancreas. However, the islets of Langerhans and pancreatic ducts remained morphologically intact. Virus could be demonstrated in pancreatic tissue for 1-5 days post-infection by in-situ and spot hybridization as well as by virus isolation. Virus was not detectable on days 7-22 post-infection suggesting an autodigestive aetiology in further destruction of the exocrine pancreas. The mouse model described here permits detailed analysis of viral and host factors in the pathogenesis of enterovirus infections. Since coxsackie B viruses have been proposed to be aetiological agents in human acute pancreatitis, the application of in-situ hybridization allows analysis of enteroviruses directly from pancreatic tissue of clinical routine specimens.     

In situ detection of rubella RNA and antigens in cultured cells

We developed in situ hybridization and immunofluorescent procedures to detect rubella RNA and antigens in tissue-cultured cells infected with rubella virus. cDNA fragments of the rubella virus E1 structural gene were used as probes for in situ hybridization to detect rubella RNA sequences in Vero cells infected with rubella virus. Using antibodies against rubella proteins, indirect immunofluorescence detected rubella virus structural proteins in Vero cells infected with rubella virus. The immunofluorescence method has also been applied to study the expression of rubella polypeptide E1 in transfected COS cells and may be applied to the detection and study of persistent rubella virus infection in human tissues.     

Prenatal diagnosis of viral infections. A two year study in Strasbourg]

We report here the results of a 2-year study on the prenatal diagnosis of viral infections in Strasbourg. This screening was carried out by virus isolation, by PCR assay, or by detection of IgM fetal antibody for 98 pregnant women at risk of transmitting one of the viruses that causes fetal disease such as parvovirus B19 (B19), Herpesviruses [cytomegalovirus (CMV), varicella-zoster virus, herpes simplex virus] and rubella virus. A viral etiology was proven in 7 out 98 cases: PCR applied to B19 DNA detection was positive in 5 amniotic fluids (AF), 2 fetal serums and one ascitic liquid. The diagnosis of 2 cases of CMV infection was obtained by both PCR and virus isolation in AF from twins fetuses. The detection of specific IgM in maternal serum or fetal serum is useful to achieve the diagnosis but serological tests on other samples have no efficiency. No virus was found in any other specimen, but the genome of Toxoplasma gondii was detected by PCR in 1 of 17 AF samples analyzed at the Institut de Parasitologie. These findings show that PCR assay is a sensitive method for the positive diagnosis of intrauterine infection and promises to careful follow-up of the pregnancy.     

应用简便逆转录环介导等温扩增方法检测麻疹病毒核酸

目的 应用简便快速的核酸检测新方法--逆转录环介导等温扩增方法(RT-LAMP)检测麻疹病毒核酸,并与巢式逆转录多聚酶链式反应(nest-RT-PCR)方法进行比较.方法 比较RT-LAMP方法与nest-RT-PCR方法对吉林省历年麻疹病毒分离株以及临床疑似麻疹咽试子中麻疹病毒核酸检测的检出率.结果 两种方法对23株麻疹分离株麻疹病毒核酸阳性检出率均达到100%.针对18份麻疹病毒分离阴性的临床疑似麻疹咽试子分别利用RT-LAMP和巢式RT-PCR两种方法进行麻疹核酸检测,RT-LAMP方法阳性率为56.52%,巢式RT-PCR方法阳性率为47.83%.结论 RT-LAMP方法比巢式nest-RT-PCR方法更敏感.     

DNA homologies between strains of herpes simplex virus.

Twelve strains of herpes simplex virus from various geographical areas and different sites of isolation were compared by nucleic and hybridization. Within each serological type there was no detectable difference between strains gauged by the extent of the nucleic acid homology and the thermal stability of the hybrid molecules. Based on these results there is less than 5% nucleotide sequence variation within each serological type and less than 20% true homology between the two types.     

Genomic characterization of a persistent rubella virus from a case of Fuch’ uveitis syndrome in a 73 year old man

BackgroundMany cases of Fuchs’ uveitis have been associated with persistent rubella virus infection. A 73-year-old male patient with typical Fuchs’ Uveitis Syndrome (FUS) first experienced heterochromia of the left eye at the age fourteen, when rubella was endemic in the US.ObjectivesThe purposes of this report are to describe the patient’s FUS clinical presentations and to characterize the virus detected in the vitreous fluid.Study designThe patient underwent a therapeutic pars plana vitrectomy in May 2013. A real-time RT-PCR assay for rubella virus was performed on the vitreous fluid by Focus Diagnostics. Additional real-time RT-PCR assays for rubella virus detection and RT-PCR assays for generation of templates for sequencing were performed at the Centers for Disease Control and Prevention (CDC).ResultsThe results from Focus Diagnostics were positive for rubella virus RNA. Real-time RT-PCR assays at CDC were also positive for rubella virus. A rubella virus sequence of 739 nucleotides was determined and phylogenetic analysis showed that the virus was the sole member of a new phylogenetic group when compared to reference virus sequences.ConclusionsWhile FUS remains a clinical diagnosis, findings in this case support the association between rubella virus and the disease. Phylogenetic analysis provided evidence that this rubella virus was likely a previously undetected genotype which is no longer circulating. Since the patient had rubella prior to 1955, this sequence is from the earliest rubella virus yet characterized.     

Persistent fetal rubella vaccine virus infection following inadvertent vaccination during early pregnancy

Inadvertent immunisation of seronegative women with RA27/3 rubella virus live-attenuated vaccine several weeks before and after conception is described. Whereas in 5 cases the vaccine virus was not transmitted vertically, in 1 case vaccination led to the development of persistent fetal infection with prolonged virus shedding for more than 8 months. Sequence analysis carried out on isolates from amniotic fluid, from cord blood leukocytes as well as from infantile urine confirmed an infection by the vaccine strain. At birth, the newborn infant exhibited none of the symptoms compatible with the congenital rubella syndrome and signs indicative for development of late onset disease are not apparent. This observation constitutes the first unequivocal documented case of rubella vaccine virus related to persistent fetal infection.     

先天性HCMV感染胎鼠大脑皮层ET-1 mRNA的研究

目的 对先天性人巨细胞病毒（HCMV）感染的胎鼠大脑皮层内皮素－1（ET－1）mRNA进行测定,以探讨先天性HCVMV感染致脑损害的机制。方法 在建立先天性HCMV中枢神经系统（CNS）感染胎鼠模型的基础上,用逆转录－聚合酶链式反应（RT－PCR）测定受不同病毒剂量感染的胎鼠大脑皮层ET－1 mRNA,并用地高辛标记的ET－1寡核苷酸探针对大脑皮层细胞印片进行原位杂交以检测相应mRNA转录量及胞内定位。结果 在大脑皮层组织的上清液中HCMV分离阳性;病理学研究证实受染胎鼠大脑皮层表现为侵袭性脑膜脑炎性改变,并在神经细胞内发现特异性核内嗜碱性包涵体。RT－PCR和原位杂交研究发现,受染胎鼠大脑皮层内ET－1 mRNA转录量增加,以1．0ml和0．5ml组为显著,而0.25ml组与正常对照组比较无明显差别。结论 HCM可经胎盘垂直传播至胎鼠脑组织。先天性HCMV感染可刺激受染胎鼠CNS ET－1 mRNA的转录,且与母鼠所接种的病毒量存在一定的量效关系。这些结果提示,ET－1在先天性HCMV感染脑损害过程中,早期可导致组织缺血性改变,而晚期则与受损大脑皮层的功能恢复有关。这对了解先天性HCMV感染致NS损伤的机理将提供有价值的参考依据,同时也为临床防治和优生优育提供一种有价值的手段。     

Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections

Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.     

PCR Detection of North American and Central African Isolates of Epizootic Hemorrhagic Disease Virus (EHDV) Based on Genome Segment 10 of EHDV Serotype 1

PCR amplification technology for the detection of epizootic hemorrhagic disease virus (EHDV) ribonucleic acid in cell culture and clinical specimens was developed. With oligoribonucleotide primers selected from genome segment 10 of EHDV serotype 1 (EHDV-1), which codes for two nonstructural proteins (NS3 and NS3a), the PCR-based assay resulted in a 535-bp PCR product. RNAs from North American EHDV-1 prototype, EHDV-2 prototype, and a number of EHDV field isolates, including the Central African isolates of EHDV-5 and EHDV-318 propagated in cell cultures, were detected by this PCR-based assay. The specific 535-bp PCR products were visualized onto agarose gels, and the identity of the PCR products was confirmed by chemiluminescent hybridization with a 352-bp internal probe. The sensitivity of the EHDV PCR assay was increased by chemiluminescent hybridization; by this EHDV-NS3 PCR, 10 fg of EHDV RNA was detected (equivalent to 600 viral particles). Amplification product was not detected when the PCR-based assay was applied to RNAs from North American bluetongue virus prototype serotypes 2, 10, 11, 13, and 17; total nucleic acid extracts from uninfected BHK-21 cells; or unfractionated blood from calves and deer that were EHDV seronegative and virus isolation negative. The described EHDV PCR-based assay with primers derived from segment 10 of EHDV-1 resulted in detection of EHDV RNA from blood and tissues collected from calves and deer with natural and experimental EHDV infections and provides a valuable tool to study the epidemiology of EHDV infection in susceptible ruminants.     

Significance of avidity and immunoblot analysis for rubella IgM-positive serum samples in pregnant women

A total of 512 IgM-positive serum samples from 449 pregnant women referred by microbiologists and medical laboratories for additional testing and final interpretation were collected over a 3-year period. Employment of an IgM capture enzyme immunoassay (EIA) confirmed only 31% of the initial EIA-IgM-positive samples. In order to discriminate acute rubella virus infections, which are associated with increased risk of fetal infection and embryopathy, from persistent or non-specific IgM, IgG avidity index and the presence of IgG with specificity to rubella virus E2 glycoprotein was determined by Western immunoblot. In only six patients (1.3%), a primary infection with rubella virus was diagnosed on the basis of IgM positivity, low avidity IgG, absence of E2-specific IgG in immunoblot concordant with clinical findings as well as consistent changes in follow-up samples. The serological results were not compatible with rubella re-infection. The infection status in 14 patients (3.1%) remained inconclusive even when both avidity assay and immunoblot were used, while restriction to either test did not allow a conclusive interpretation in 11.6% of patients. The use of both assays is clearly better, and therefore, recommended in IgM-positive samples.     

Detection of bovine herpesvirus 1 DNA immobilized on nitrocellulose by hybridization with biotinylated DNA probes.

A molecular hybridization technique using biotinylated DNA probes was used to detect bovine herpesvirus 1 (BHV-1) nucleic acid species immobilized on nitrocellulose. Seventeen recombinant plasmids containing HindIII restriction fragments of the BHV-1 genome were compared for their ability to detect immobilized BHV-1 DNA from purified virus and infected cells. One probe, pCB2, labeled by nick translation with either 3H or biotin, detected as little as 10 pg of viral DNA. In time course experiments, BHV-1 DNA could be detected by 2 h postinfection in 10(6) infected cells. BHV-1 DNA was detected in nasal swabs and exudate from experimentally infected cattle, even when specimens had been stored for over a year. In a retrospective study of a respiratory disease outbreak in a feedlot, hybridization was compared with virus isolation for diagnosis of BHV-1 infections. The sensitivity rate was 0.68 with virus isolation as the referent standard. Blot hybridization provides a novel approach with unique applications for the diagnosis of bovine herpesvirus infections.     

Localization of simian immunodeficiency virus nucleic acid and antigen in brains of fetal macaques inoculated in utero.

Neurological dysfunction has been shown to be associated with human immunodeficiency virus (HIV) infection. The incidence of these abnormalities is greater in HIV-infected children when compared with adults, and the patterns of neurological disease are also known to differ from those observed in the adult population. The reasons for these differences are unclear but are most likely related to the immaturity of the host's immune and central nervous systems at the time of infection. This is thought to be particularly true for infants infected with HIV prenatally. To examine these questions, the brains of fetal rhesus macaques that were infected with SIVmac251 at various time points in utero were examined. Direct fetal inoculations were performed on gestational day (GD) 65 (n = 8; early second trimester), GD 110 (n = 4; early third trimester) and GD 130 (n = 2; mid third trimester), with harvest of fetal tissues on GD 80, 100, 130, or 145. Eleven sham controls were included with harvest at correlative time points. Specimens were examined by routine histology, immunohistochemistry, and in situ hybridization to localize viral antigens and SIV nucleic acid. Histologically, scattered glial nodules, spongiosis, and mineralization were found in the basal ganglia and deep white matter in 4 of the 14 fetuses (3 inoculated on GD 65 and one on GD 110). These fetuses and those without histological lesions had viral nucleic acid and SIV antigen in the stroma of the choroid plexus, meninges, and external granular layer of the cerebellum and in columns of cells in the cortical plate. In contrast to juvenile and adult macaques, very few SIV-positive perivascular mononuclear cells were present. These findings suggest that SIV has a different distribution in the brain of fetal macaques after direct infection when compared with adult or juvenile animals. Furthermore, the results of these studies suggest that differences in neurological disease between pediatric and adult patients with acquired immune deficiency syndrome are most likely related to the time of infection.