Evaluation of live trivalent vaccine of measles AIK-C strain, mumps Hoshino strain and rubella Takahashi strain, by virus-specific interferon-gamma production and antibody response

A trivalent measles-mumps-rubella live virus vaccine, containing measles AIK-C strain, mumps Hoshino strain, and rubella Takahashi strain, was evaluated in 229 children, aged 1 to 5 years. The vaccine induced a high seroconversion rate: 221 (98.7%) out of 224 subjects initially seronegative for measles virus, 167 (93.3%) out of 179 initially seronegative for mumps virus, and 212 (99.1%) out of 214 initially seronegative for rubella virus. It also induced a sufficient cellular immunity against each of the three viruses in over 90% of the subjects, as judged by virus-specific interferon-gamma (IFN-gamma) production. Virus-specific IFN-gamma production was observed 10 days after vaccination by stimulation with measles virus and rubella virus and 14 days after vaccination by stimulation with mumps virus. Mumps-virus-specific IFN-gamma production was observed in 7 out of 12 recipients without seroconversion for mumps virus. And measles-virus-specific IFN-gamma production was demonstrated in one out of three recipients without seroconversion for measles virus. A significant correlation was observed between the serum antibody and IFN-gamma production six weeks after vaccination for measles virus (r = 0.201, P less than 0.01) and for mumps virus (r = 0.174, P less than 0.05) but not for rubella virus (r = -0.045, P less than 0.05). The incidence of febrile reactions of greater than or equal to 37.5 C was quite low, 14.4%, and that of greater than or equal to 39 C occurred in only 1.3% of the recipients. These results suggested that the trivalent vaccine induced sufficient humoral and cellular immunity and yet resulted in no more untoward reaction than observed from the measles vaccine alone.     

Immunogenic activity of temperature sensitive mutation of herpes simplex virus type-1 in mono- and polyvalent conditions with different attenuated strains of virus. II. Analysis of humoral immunologic response induced by mutants of temperature sensitive herpes simplex type-1 virus in polyvalent systems

The ability of mutant ts HSV-1 on 45 passage to induction of IgG antibody response in the presence of other attenuated viral strain, like measles, mumps, rubella was evaluated, and the effect of mutant ts HSV-1 on humoral immunity induced by attenuated measles and mumps strains was also tested. The obtained results clearly indicated that even immunization with other RNA virus strains had no effect on IgG antibody response to mutant ts HSV-1. Significant differences were observed in IgG antibody responses to mumps and measles between groups of guinea pigs immunized with these viruses alone or together with other viruses. This was especially observed in the case of mumps virus. It clearly shows that change of virus concentration rates in polyvalent schema of immunization is needed.     

Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins

We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly alpha-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K(d)) was determined to be 13 microM at 20 degrees C and 35 microM at 37 degrees C. Our data are consistent with a model in which an alpha-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.     

Detection of measles,mumps and rubella viruses by immuno‐colorimetric assay and its application in focus reduction neutralization tests

Measles, mumps and rubella are vaccine‐preventable diseases; however limited epidemiological data are available from low‐income or developing countries. Thus, it is important to investigate the transmission of these viruses in different geographical regions. In this context, a cell culture‐based rapid and reliable immuno‐colorimetric assay (ICA) was established and its utility studied. Twenty‐three measles, six mumps and six rubella virus isolates and three vaccine strains were studied. Detection by ICA was compared with plaque and RT‐PCR assays. In addition, ICA was used to detect viruses in throat swabs (n = 24) collected from patients with suspected measles or mumps. Similarly, ICA was used in a focus reduction neutralization test (FRNT) and the results compared with those obtained by a commercial IgG enzyme immuno assay. Measles and mumps virus were detected 2 days post‐infection in Vero or Vero‐human signaling lymphocytic activation molecule cells, whereas rubella virus was detected 3 days post‐infection in Vero cells. The blue stained viral foci were visible by the naked eye or through a magnifying glass. In conclusion, ICA was successfully used on 35 virus isolates, three vaccine strains and clinical specimens collected from suspected cases of measles and mumps. Furthermore, an application of ICA in a neutralization test (i.e., FRNT) was documented; this may be useful for sero‐epidemiological, cross‐neutralization and pre/post‐vaccine studies.     

Paramyxovirus accessory proteins as interferon antagonists

A new role of the Paramyxovirus accessory proteins has been uncovered. The P gene of the subfamily Paramyxovirinae encodes accessory proteins including the V and/or C protein by means of pseudotemplated nucleotide addition (RNA editing) or by overlapping open reading frame. The Respirovirus (Sendai virus and human parainfluenza virus (hPIV)3) and Rubulavirus (simian virus (SV)5, SV41, mumps virus and hPIV2) circumvent the interferon (IFN) response by inhibiting IFN signaling. The responsible genes were mapped to the C gene for SeV and the V gene for rubulaviruses. On the other hand, wild type measles viruses isolated from clinical specimens suppress production of IFN, although responsible viral factors remain to be identified. Both human and bovine respiratory syncytial viruses (RSVs) counteract the antiviral effect of IFN with inhibiting neither IFN signaling nor IFN production. Bovine RSV NS1 and NS2 proteins cooperatively antagonize the antiviral effect of IFN. Studies on the molecular mechanism by which viruses circumvent the host IFN response will not only illustrate co-evolution of virus strategies of immune evasion but also provide basic information useful for engineering novel antiviral drugs as well as recombinant live vaccine.     

Studies on live rubella vaccine. V. Quantitative aspects of interference between rubella, measles and mumps viruses in their trivalent vaccine

Rubella vaccine combined with measles and mumps vaccines was administered in a single injection to children of 1 to 5 years of age. All three vaccines were serologically effective, and the clinical reactions caused by measles vaccine were considerably alleviated, when 6 x 10(3) PFU of rubella and 10(4) TCD50 per dose of mumps vaccines were combined with 5 x 10(4) TCD50 of measles vaccine. When a larger amount of mumps vaccine (3 x 10(5) TCD50/dose) was used, it caused interference with the rubella and measles viruses, i.e., the antibody response to rubella virus became poor and the incidence of clinical reactions to measles virus decreased. On the other hand, when 5 x 10(5) TCD50/dose of measles vaccine was combined with 10(4) TCD50/dose of mumps vaccine, the clinical reactions to measles virus were decreased but were almost the same as those induced by this vaccine alone.     

Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay.

The quantitative PCR infectivity assay is a combination of virus propagation and quantitative PCR. Previously [Schalk JAC, van den Elzen C, Ovelgonne H, Baas C, Jongen PMJM. Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR. J Virol Methods 2004;117:179-87.], we used this assay to estimate the titer of infectious measles virus in trivalent, live, measles, mumps, rubella vaccines (MMR). Here we describe the further improvement and development of the assay for simultaneous potency estimation of measles, mumps and rubella viruses. The potency of measles and mumps virus is estimated within one assay after 1 day of cell culture. The potency of rubella virus is estimated in a separate assay after 2 days of cell culture. Compared to conventional CCID50 and plaque assays, the quantitative PCR infectivity assay has the advantage in being fast because the assay is not dependent on the formation of cytopathic effect. Furthermore assay design is simplified: serological neutralization can be omitted because PCR is virus-specific and, under the conditions used, the individual components of trivalent measles, mumps, rubella vaccines do not interfere with each other. The assay was validated and compared to the performance of a plaque assay.     

Recent mumps outbreaks in vaccinated populations: no evidence of immune escape

Recently, numerous large-scale mumps outbreaks have occurred in vaccinated populations. Clinical isolates sequenced from these outbreaks have invariably been of genotypes distinct from those of vaccine viruses, raising concern that certain mumps virus strains may escape vaccine-induced immunity. To investigate this concern, sera obtained from children 6 weeks after receipt of measles, mumps, and rubella (MMR) vaccine were tested for the ability to neutralize a carefully selected group of genetically diverse mumps virus strains. Although the geometric mean neutralizing antibody titer of the sera was lower against some virus strains than others, all viruses were readily neutralized, arguing against immune escape.     

Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells

Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.     

Interference between strains in live virus vaccines, I: Combined vaccination with measles, mumps and rubella vaccine

One to four different lots of four commercially available trivalent measles-mumps-rubella vaccines were tested for their efficacy as measured by the induction of antibodies to the three vaccine viruses. All of the products were satisfactory although a 100% seroconversion rate was attained regularly only with rubella vaccine. The live virus in the different measles and mumps vaccine components and especially the relative amounts of measles to mumps virus varied widely. Obviously, the efficacy of a vaccine should not be judged only by the virus content. Of main importance is the further adjustment of the dosage of the interfering vaccine virus strains in relation to their attenuation.     

Role of biased hypermutation in evolution of subacute sclerosing panencephalitis virus from progenitor acute measles virus.

We identified an acute measles virus (Nagahata strain) closely related to a defective virus (Biken strain) isolated from a patient with subacute sclerosing panencephalitis (SSPE). The proteins of Nagahata strain measles virus are antigenically and electrophoretically similar to the proteins of Edmonston strain measles virus. However, the nucleotide sequence of the Nagahata matrix (M) gene is significantly different from the M genes of all the acute measles virus strains studied to date. The Nagahata M gene is strikingly similar to the M gene of Biken strain SSPE virus isolated several years later in the same locale. Eighty percent of the nucleotide differences between the Nagahata and Biken M genes are uridine-to-cytosine transitions known as biased hypermutation, which has been postulated to be caused by a cellular RNA-modifying activity. These biased mutations account for all but one of the numerous missense genetic changes predicted to cause amino acid substitutions. As a result, the Biken virus M protein loses conformation-specific epitopes that are conserved in the M proteins of Nagahata and Edmonston strain acute measles viruses. These conformation-specific epitopes are also absent in the cryptic M proteins encoded by the hypermutated M genes of two other defective SSPE viruses (Niigata and Yamagata strains). Nagahata-like sequences are found in the M genes of at least five other SSPE viruses isolated from three continents. These data indicate that Biken strain SSPE virus is derived from a progenitor closely resembling Nagahata strain acute measles virus and that biased hypermutation is largely responsible for the structural defects in the Biken virus M protein.     

Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses

Outbreaks of SARS-CoV, influenza A (H5N1, H1N1) and measles viruses in recent years have raised serious concerns about the measures available to control emerging and re-emerging infectious viral diseases. Effective antiviral agents are lacking that specifically target RNA viruses such as measles, SARS-CoV and influenza H5N1 viruses, and available vaccinations have demonstrated variable efficacy. Therefore, the development of novel antiviral agents is needed to close the vaccination gap and silence outbreaks. We previously indentified mucroporin, a cationic host defense peptide from scorpion venom, which can effectively inhibit standard bacteria. The optimized mucroporin-M1 can inhibit gram-positive bacteria at low concentrations and antibiotic-resistant pathogens. In this investigation, we further tested mucroporin and the optimized mucroporin-M1 for their antiviral activity. Surprisingly, we found that the antiviral activities of mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses were notably increased with an EC₅₀ of 7.15 μg/ml (3.52 μM) and a CC₅₀ of 70.46 μg/ml (34.70 μM) against measles virus, an EC₅₀ of 14.46 μg/ml (7.12 μM) against SARS-CoV and an EC₅₀ of 2.10 μg/ml (1.03 μM) against H5N1, while the original peptide mucroporin showed no antiviral activity against any of these three viruses. The inhibition model could be via a direct interaction with the virus envelope, thereby decreasing the infectivity of virus. This report provides evidence that host defense peptides from scorpion venom can be modified for antiviral activity by rational design and represents a practical approach for developing broad-spectrum antiviral agents, especially against RNA viruses.     

ELISA法检测流行性腮腺炎病毒感染特异性IgM抗体的研究

用流行性腮腺炎(流腮)病毒Enders株接种鸡胚尿囊腔培养,尿囊液经聚乙二醇6000处理制备流腮病毒抗原,用ELISA法检测流腮患者血清中特异性IgM抗体,其敏感性,特异性、重复性和稳定性都很高。 79份流腮患者血清,检出特异性IgM72份,阳性率为91％,32例非流腮患者IgM全部阴性、两者有极显著差异(P<0.01)。 10份血清作血清倍比稀释至1∶3200测IgM仍全部阳性,1∶6400稀释仅1例阴性,1∶12800稀释5例中仍有2例阳性。 10份血清作流腮抗原特异性抗体阻断试验,光密度抑制率均大于50％,平均为87％,10份标本作2-ME和SPA阻断后检测IgM抗体,结果2-ME阻断标本全部阴转,而SPA阻断标本仍阳性,证实所检测为流腮特异性抗体。 24份标本2次重复检测流腮IgM,其阴、阳性结果一致,这期间抗原放4℃ 1个月,提示抗原的稳定性和方法的重复性都很好。本方法敏感性明显高于血凝抑制试验,其阳性率分别为91％和61％,两者有显著差异。而且所用试剂简单经济,操作简便,快速,适用于临床早期诊断,易于广泛推广应用。     

采用基因微阵列技术对常见呼吸道病毒进行检测的初步研究

建立一种高通量的基因微阵列检测技术,对常见呼吸道病毒感染进行监控.根据公开发表的8个病毒科38种常见呼吸道病毒的序列,计算其保守区域,设计病毒的特异性检测探针,制备呼吸道病毒检测基因微阵列.利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与基因微阵列上的探针杂交,清洗、扫描后进行结果分析.采用流感病毒、麻疹病毒、腮腺炎病毒和风疹病毒作为报告病毒,并对80例上呼吸道感染患者的咽拭子标本进行验证测试.初步结果表明,该呼吸道病毒微阵列基因芯片检测是可行的,在利用基因微阵列技术对病毒监控方面进行了有益的尝试,得到了有经验的信息.     

Immunogenic activity of HSV-1 temperature sensitive mutant's proteins in mono- and polyvalent systems of immunization

Immunogenic activity of herpes simplex type 1 temperature sensitive mutant's (ts HSV-1 mutant) proteins was tested in two systems: monovalent and polyvalent with other attenuated virus strains (measles and mumps). The guinea pigs were used as animal model. In monovalent system the humoral response in animals infected with ts HSV-1 mutant (1 or 2 doses) was studied and compared to results received for HSV-1 native strain. In polyvalent system the immunological response induced by ts HSV-1 mutant in the presence of RNA virus strains was tested.     

Mechanism of selective inhibition of respiratory syncytial virus by a benzodithiin compound (RD3-0028)

RD3-0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time-dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3-0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3-0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3-0028 (5-20 microg/ml). These resistant viruses were more than 80-fold less sensitive to RD3-0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.     

Susceptibility of endothelial cells derived from different blood vessels to common viruses

Summary We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alteration in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. This work was supported by Public Health Service grants HL28220, HL 29492, and HL 24914 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

Complete nucleotide sequence of a mumps virus SP strain isolated in China

The complete nucleotide sequence of the mumps virus SP, which was isolated in China, was determined. As with other mumps viruses, its genome was 15 384 nucleotides (nts) in length and encoded seven proteins. The full-length nucleotide sequence of the SP isolate differed from other strains by 4%-6.8% at the nucleotide sequence level. Due to variations of amino acids over the full genome (including the HN and N genes), this isolate exhibited significant variations in the antigenic sites. This report is the first to describe the full-length genome of a genotype F strain and provide an overview of the diversity of genetic characteristics of a circulating mumps virus.     

Comparison of Subacute Sclerosing Panencephalitis and Measles Viruses: an Electron Microscope Study

The ultrastructure of CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) viruses was compared with that of CV-1 cells infected with the wild or Edmonston strain of measles virus. Both SSPE viruses and the measles viruses produced two types of nucleocapsid structures: smooth filaments, 15 to 17 nm in diameter, and granular filaments, 22 to 25 nm. The smooth and granular filaments produced by SSPE and measles virus did not differ in appearance. In CV-1 cells infected with SSPE viruses, smooth filaments formed large intranuclear inclusions and granular filaments occupied a large area of the cytoplasm, but always spared the area under the cell membrane. Particles budding from the surface of these cells contained no nucleocapsids. In CV-1 cells infected with measles virus, only small aggregates of smooth filaments were seen in the nuclei. Granular filaments in the cytoplasm predominantly occupied the area under the cell membrane, and were aligned beneath the cell membrane in a parallel fashion and assembled into budding particles. These differences between SSPE and measles virus may be regarded as quantitative, but they do distinguish SSPE viruses from measles virus. Moreover, the formation of large nuclear inclusions filled with smooth filaments appears to be a characteristic process of SSPE, but not of measles, since this type of inclusion is invariably seen in SSPE brain tissues, brain cultures derived from them, and CV-1 cells infected with SSPE viruses.