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During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases.

New genotyping scheme facilitates classification of virus sequences., Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes fatal encephalitis in humans. The initial outbreak of NiV infection occurred in Malaysia and Singapore in 1998–1999; relatively small, sporadic outbreaks among humans have occurred in Bangladesh since 2001. We characterized the complete genomic sequences of identical NiV isolates from 2 patients in 2008 and partial genomic sequences of throat swab samples from 3 patients in 2010, all from Bangladesh. All sequences from patients in Bangladesh comprised a distinct genetic group. However, the detection of 3 genetically distinct sequences from patients in the districts of Faridpur and Gopalganj indicated multiple co-circulating lineages in a localized region over a short time (January–March 2010). Sequence comparisons between the open reading frames of all available NiV genes led us to propose a standardized protocol for genotyping NiV; this protcol provides a simple and accurate way to classify current and future NiV sequences.

Measles, mumps, rubella, and varicella are acute viral diseases that are preventable by vaccination. In the United States (US), routine measles, rubella, and mumps vaccination of children was initiated in 1963, 1969, and 1977, respectively. The Advisory Committee on Immunization Practices (ACIP) recommended routine use of the combination measles, mumps and rubella (MMR) vaccine in 1977 and a 2-dose schedule at ages 12–15 months and 4–6 years in 1989 [1]. As a result of high MMR vaccination coverage (≥90% in children aged 19–35 months), incidence declined; endemic transmission of measles was declared eliminated in 2000 and documented for rubella and congenital rubella syndrome (CRS) in 2004 [2, 3]. However, import-associated cases continue to occur in the US, because measles and rubella remain endemic in other countries. Reported mumps cases also declined to an average of

A 29-year-old pregnant woman developed progressively worsening encephalopathy, left hemiparesis, and hemodynamic instability over a 6-week period. Initial brain MRI and work-up for infectious and autoimmune causes were normal, although elevated IgG and oligoclonal bands were seen on analysis of the cerebrospinal fluid (CSF). After uncomplicated spontaneous delivery of a preterm healthy infant, her condition worsened. Repeat brain MRI demonstrated generalized volume loss and evidence of corticospinal tract degeneration. She underwent a brain biopsy, which showed characteristic viral inclusions of the type seen in subacute sclerosing panencephalitis (SSPE). The diagnosis was confirmed by immunohistochemistry and electron microscopy, and additional CSF analysis also showed markedly elevated IgG titer for measles. Sequence analysis of the nucleoprotein gene N-450 demonstrated a close relationship to the sequences of viruses in genotype D7. This case documents an ~ 6-month progression to death of SSPE in a pregnant woman.

Monoclonal antibodies (Mabs) against the Urbani strain of the SARS-associated coronavirus (SARS-CoV) were developed and characterized for reactivity to SARS-CoV and SARS-CoV S, N, M, and E proteins using enzyme-linked immunoabsorbent (ELISA), radioimmunoprecipitation, immunofluorescence, Western Blot and microneutralization assays. Twenty-six mAbs were reactive to SARS-CoV by ELISA, and nine were chosen for detailed characterization. Five mAbs reacted against the S protein, two against the M protein, and one each against the N and E proteins. Two of five S protein mAbs neutralized SARS-CoV infection of Vero E6 cells and reacted to an epitope within amino acids 490-510 in the S protein. While two of the three non-neutralizing antibodies recognized at second epitope within amino acids 270-350. The mAbs characterized should prove useful for developing SARS-CoV diagnostic assays and for studying the biology of infection and pathogenesis of disease.

Severe acute respiratory syndrome (SARS) was first described during a 2002–2003 global outbreak of severe pneumonia associated with human deaths and person-to-person disease transmission. The etiologic agent was initially identified as a coronavirus by thin-section electron microscopic examination of a virus isolate. Virions were spherical, 78 nm in mean diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Herein, we show that infection with the SARS-associated coronavirus resulted in distinct ultrastructural features: double-membrane vesicles, nucleocapsid inclusions, and large granular areas of cytoplasm. These three structures and the coronavirus particles were shown to be positive for viral proteins and RNA by using ultrastructural immunogold and in situ hybridization assays. In addition, ultrastructural examination of a bronchiolar lavage specimen from a SARS patient showed numerous coronavirus-infected cells with features similar to those in infected culture cells. Electron microscopic studies were critical in identifying the etiologic agent of the SARS outbreak and in guiding subsequent laboratory and epidemiologic investigations.

From 1997 to 2001, sequence data from 55 clinical specimens were obtained from confirmed measles cases in the United States, representing 21 outbreaks and 34 sporadic cases. Sequence analysis indicated the presence of 11 of the recognized genotypes. The most common genotypes detected were genotype D6, usually identified from imported cases from Europe, and genotype D5, associated with importations from Japan. A number of viruses belonging to genotype D4 were imported from India and Pakistan. Overall, viral genotypes were determined for 13 chains of transmission with an unknown source of virus, and seven different genotypes were identified. Therefore, the diversity of Measles virus genotypes observed in the United States from 1997 to 2001 reflected multiple imported sources of virus and indicated that no strain of measles is endemic in the United States.

During a resurgence of measles in Sao Paulo, Brazil, in 1997, >40,000 cases (peak incidence rate of 246/ 100,000 inhabitants) and 42 measles-related deaths were reported. Reverse transcriptase-polymerase chain reaction and nucleotide sequencing were used to analyze specimens from patients who had typical clinical measles infection during this outbreak and from six patients who had had measles in 1995 and 1996. Although wild-type measles viruses (genotypes D5 and D6) were present in Sao Paulo before this resurgence, we detected only D6 viruses. The genotype D6 viruses isolated during this outbreak had identical sequences to genotype D6 viruses isolated in other parts of Brazil and South America in 1997 and 1998, suggesting that a single chain of transmission was responsible. We also identified genotype A viruses in two vaccine-associated cases from 1995 and 1996. Our findings extend the knowledge of the circulation patterns of measles virus in South America, contributing to measles control efforts in the Americas.

Wild-type measles viruses have been divided into distinct genetic groups according to the nucleotide sequences of their hemagglutinin and nucleoprotein genes. Most genetic groups have worldwide distribution; however, at least two of the groups appear to have a more limited circulation. To monitor the transmission pathways of measles virus, we observed the geographic distribution of genetic groups, as well as changes in them in a particular region over time. We found evidence of interruption of indigenous transmission of measles in the United States after 1993 and identified the sources of imported virus associated with cases and outbreaks after 1993. The pattern of measles genetic groups provided a means to describe measles outbreaks and assess the extent of virus circulation in a given area. We expect that molecular epidemiologic studies will become a powerful tool for evaluating strategies to control, eliminate, and eventually eradicate measles.

Surveillance of measles virus detected an epidemiologic link between a refugee from Kenya and a Dutch tourist in New Jersey, USA. Identical genotype B3 sequences from patients with contemporaneous cases in the United States, Canada, and Mexico in November and December 2005 indicate that Kenya was likely to have been the common source of virus.

Rubella virus is the causative agent of rubella, a mild rash illness, and a potent teratogenic agent when contracted by a pregnant woman. Global rubella control programs target the reduction and elimination of congenital rubella syndrome. Phylogenetic analysis of partial sequences of rubella viruses has contributed to virus surveillance efforts and played an important role in demonstrating that indigenous rubella viruses have been eliminated in the United States. Sixteen wild-type rubella viruses were chosen for whole genome sequencing. All 16 viruses were collected in the United States from 1961 to 2009 and are from 8 of the 13 known rubella genotypes. Phylogenetic analysis of 30 whole genome sequences produced a maximum likelihood tree giving high bootstrap values for all genotypes except provisional genotype 1a. Comparison of the 16 new complete sequences and 14 previously sequenced wild-type viruses found regions with clusters of variable amino acids. The 5′ 250 nucleotides of the genome are more conserved than any other part of the genome. Genotype specific deletions in the untranslated region between the non-structural and structural open reading frames were observed for genotypes 2B and genotype 1G. No evidence was seen for recombination events among the 30 viruses. The analysis presented here is consistent with previous reports on the genetic characterization of rubella virus genomes. Conserved and variable regions were identified and additional evidence for genotype specific nucleotide deletions in the intergenic region was found. Phylogenetic analysis confirmed genotype groupings originally based on structural protein coding region sequences, which provides support for the WHO nomenclature for genetic characterization of wild-type rubella viruses.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis in up to 75% of infected humans. Like other paramyxoviruses, NiV employs co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternative reading frame. There is evidence from both in vitro and in vivo studies to show that the P gene products play a role in NiV pathogenesis. We have developed a reverse genetic system to dissect the individual roles of the NiV P gene products in limiting the antiviral response in primary human microvascular lung endothelial cells, which represent important targets in human NiV infection. By characterizing growth curves and early antiviral responses against a number of recombinant NiVs with genetic modifications altering expression of the proteins encoded by the P gene, we observed that multiple elements encoded by the P gene have both distinct and overlapping roles in modulating virus replication as well as in limiting expression of antiviral mediators such as IFN-β, CXCL10, and CCL5. Our findings corroborate observations from in vivo hamster infection studies, and provide molecular insights into the attenuation and the histopathology observed in hamsters infected with C, V, and W-deficient NiVs. The results of this study also provide an opportunity to verify the results of earlier artificial plasmid expression studies in the context of authentic viral infection.

The incidence of measles in China from 1991 to 2008 was reviewed, and the nucleotide sequences from 1507 measles viruses (MeV) isolated during 1993 to 2008 were phylogenetically analyzed. The results showed that measles epidemics peaked approximately every 3 to 5 years with the range of measles cases detected between 56,850 and 140,048 per year. The Chinese MeV strains represented three genotypes; 1501 H1, 1 H2 and 5 A. Genotype H1 was the predominant genotype throughout China continuously circulating for at least 16 years. Genotype H1 sequences could be divided into two distinct clusters, H1a and H1b. A 4.2% average nucleotide divergence was found between the H1a and H1b clusters, and the nucleotide sequence and predicted amino acid homologies of H1a viruses were 92.3%-100% and 84.7%-100%, H1b were 97.1%-100% and 95.3%-100%, respectively. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Cluster H1a and H1b viruses were co-circulating during 1993 to 2005, while no H1b viruses were detected after 2005 and the transmission of that cluster has presumably been interrupted. Analysis of the nucleotide and predicted amino acid changes in the N proteins of H1a and H1b viruses showed no evidence of selective pressure. This study investigated the genotype and cluster distribution of MeV in China over a 16-year period to establish a genetic baseline before MeV elimination in Western Pacific Region (WPR). Continuous and extensive MeV surveillance and the ability to quickly identify imported cases of measles will become more critical as measles elimination goals are achieved in China in the near future. This is the first report that a single endemic genotype of measles virus has been found to be continuously circulating in one country for at least 16 years.

Analysis of 200 paired serum and cerebrospinal fluid (CSF) samples from 180 HIV-positive individuals, 136 of whom had AIDS, revealed intrathecal synthesis of antibodies specific for varicella zoster virus (VZV) in 28 (16%) individuals, measles virus in 15 (8%), herpes simplex virus-1 (HSV-1) in 1 (0.6%), and HSV-2 in none. Of the 28 subjects with a positive VZV antibody specificity index, only 1 had zoster rash at the time of serum and CSF sampling; of the total 180 HIV-positive subjects, 146 (81%) had no history of zoster. Based on an estimated 33.4 million HIV-positive individuals worldwide, subclinical reactivation of VZV in even less than 16% of HIV-positive people suggests the possibility that millions of people have active VZV infection of the central nervous system. In cases of VZV vasculopathy, myelopathy and even zoster sine herpete, the CSF is often positive for anti-VZV antibody, but negative for VZV DNA. To rule out VZV infection of the nervous system, CSF must be tested for VZV DNA and anti-VZV IgG and IgM antibody.

Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution., Author Summary The SARS-CoV caused a worldwide epidemic of SARS in 2002/03 and was responsible for this zoonotic infectious disease. The role of neutralizing antibody (nAb) mediated immune pressure in the evolution of SARS-CoV during the 2002/03 outbreak and a second 2003/04 zoonotic transmission is unknown. Here we demonstrate nAb responses elicited during natural infection clearly have strain-specific components which could have been the driving force for virus evolution in spike protein during intra-species transmission. In vitro immune pressure using 2002/03 strain-specific nAb 80R recapitulate a dominant escape mutation that was present in all 2003/04 animal and human viruses. We investigated how to generate a single broad nAb (BnAb) with activity against various natural viral variants of the 2002/03 and 2003/04 outbreaks as well as nAb escape mutants. Remarkably, amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” of somatic hypermutation and localized to a single VL CDR were successful in generating BnAbs. These results provide an effective strategy for generating BnAbs that should be generally useful for improving immune based anti-viral therapies as well as providing a foundation to directly manipulate virus evolution by blocking escape pathways.

Background: The presence of measles virus (MV) RNA in bowel tissue from children with autism spectrum disorders (ASD) and gastrointestinal (GI) disturbances was reported in 1998. Subsequent investigations found no associations between MV exposure and ASD but did not test for the presence of MV RNA in bowel or focus on children with ASD and GI disturbances. Failure to replicate the original study design may contribute to continued public concern with respect to the safety of the measles, mumps, and rubella (MMR) vaccine. Methodology/Principal Findings: The objective of this case-control study was to determine whether children with GI disturbances and autism are more likely than children with GI disturbances alone to have MV RNA and/or inflammation in bowel tissues and if autism and/or GI episode onset relate temporally to receipt of MMR. The sample was an age-matched group of US children undergoing clinically-indicated ileocolonoscopy. Ileal and cecal tissues from 25 children with autism and GI disturbances and 13 children with GI disturbances alone (controls) were evaluated by real-time reverse transcription (RT)-PCR for presence of MV RNA in three laboratories blinded to diagnosis, including one wherein the original findings suggesting a link between MV and ASD were reported. The temporal order of onset of GI episodes and autism relative to timing of MMR administration was examined. We found no differences between case and control groups in the presence of MV RNA in ileum and cecum. Results were consistent across the three laboratory sites. GI symptom and autism onset were unrelated to MMR timing. Eighty-eight percent of ASD cases had behavioral regression. Conclusions/Significance: This study provides strong evidence against association of autism with persistent MV RNA in the GI tract or MMR exposure. Autism with GI disturbances is associated with elevated rates of regression in language or other skills and may represent an endophenotype distinct from other ASD.

This report describes the genetic characterization of 297 wild-type measles viruses that were isolated in 24 provinces of China between 1995 and 2003. Phylogenetic analysis of the N gene sequences showed that all of the isolates belonged to genotype H1 except 3 isolates, which were genotype A. The nucleotide sequence and predicted amino acid homologies of the 294-genotype H1 strains were 94.7%–100% and 93.3%–100%, respectively. The genotype H1 isolates were divided into 2 clusters, which differed by approximately 2.9% at the nucleotide level. Viruses from both clusters were distributed throughout China with no apparent geographic restriction and multiple co-circulating lineages were present in many provinces. Even though other measles genotypes have been detected in countries that border China, this report shows that genotype H1 is widely distributed throughout the country and that China has a single, endemic genotype. This important baseline data will help to monitor the progress of measles control in China.

Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. By using gene trap insertional mutagenesis, we identified Rab9, which mediates late-endosome-to- trans -Golgi-network trafficking, among several candidate host genes whose disruption allowed the survival of Marburg virus-infected cells, suggesting that Rab9 is utilized in Marburg replication. Although Rab9 has not been implicated in human immunodeficiency virus (HIV) replication, previous reports suggested that the late endosome is an initiation site for HIV assembly and that TIP47-dependent trafficking out of the late endosome to the trans -Golgi network facilitates the sorting of HIV Env into virions budding at the plasma membrane. We examined the role of Rab9 in the life cycles of HIV and several unrelated viruses, using small interfering RNA (siRNA) to silence Rab9 expression before viral infection. Silencing Rab9 expression dramatically inhibited HIV replication, as did silencing the host genes encoding TIP47, p40, and PIKfyve, which also facilitate late-endosome-to- trans -Golgi vesicular transport. In addition, silencing studies revealed that HIV replication was dependent on the expression of Rab11A, which mediates trans -Golgi-to-plasma-membrane transport, and that increased HIV Gag was sequestered in a CD63 + endocytic compartment in a cell line stably expressing Rab9 siRNA. Replication of the enveloped Ebola, Marburg, and measles viruses was inhibited with Rab9 siRNA, although the nonenveloped reovirus was insensitive to Rab9 silencing. These results suggest that Rab9 is an important cellular target for inhibiting diverse viruses and help to define a late-endosome-to-plasma-membrane vesicular transport pathway important in viral assembly.

Measles virus (MV) infection of monocytes induces leukocyte function-associated antigen-1 (LFA-1), an integrin that mediates intercellular adhesion to the endothelium. Thus, an increase in LFA-1 expression could lead to enhanced monocyte adherence and virus dissemination to endothelial cells (ECs) and potentially be an important means of distinction between MV strains. We identified both vaccine and wild-type strains that induced LFA-1 and others that failed to induce. Although adhesion of MV-infected monocytes and viral transmission to ECs was demonstrated, strain-specific differences were not correlated with LFA-1 induction. MV infection of ECs was dramatically reduced in the absence of cell contact, suggesting virus dissemination by cell-cell transmission.

The expression of chimeric proteins was performed for the localization of monoclonal antibody (MAb) epitopes and functional domains in the hemagglutinin (H) protein of measles virus. The fusion helper function of the H protein was ablated by a single amino acid substitution at residue 98. Loss of reactivity to MAb 79-XV-V17 and to MAbs 16-CD-11 and 80-II-B2 was attributed to substitutions between residues 211 and 291 and between 451 and 505, respectively. The 80-II-B2 MAb epitope also seemed to be within a domain required for hemadsorption and hemagglutination activities.

A recombinant baculovirus that expresses the nucleoprotein gene of measles virus (Edmonston vaccine strain) under the transcriptional control of the polyhedrin promoter was generated. The expressed protein (B-MVN) comigrated with the authentic viral nucleoprotein as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was phosphorylated. The B-MVN protein proved to be reactive with monoclonal antibodies in radioimmunoprecipitations, and it was immunogenic, eliciting in mice antisera that recognized the native nucleoprotein. In addition, the B-MVN protein was evaluated as a replacement source of antigen for whole virus in enzyme immunoassays (EIAs) for detection of measles virus-specific immunoglobulin M (IgM) and IgG antibodies. A capture IgM EIA with the B-MVN protein as antigen detected specific IgM antibodies in 18 (72%) acute- and all convalescent-phase specimens from 25 clinical measles cases and exceeded 99% specificity with 120 control specimens. An indirect IgG EIA with the B-MVN protein detected specific IgG antibodies in 129 of 131 (98%) serum specimens with antibodies to measles virus, and results obtained from testing 268 additional serum specimens were better correlated with measles virus-neutralizing antibodies than those obtained with a commercial EIA.

The C and V proteins of the measles virus (MV) have been shown to block the signaling of type I and II interferon (IFN-alpha/beta and IFN-gamma). The relative contribution of the C and V proteins to the inhibition of IFN signaling and the extent to which this activity differs in attenuated or wild-type strains of MV remains undefined. This study presents a comparison of the IFN-antagonist activities of C and V proteins from four attenuated and two wild-type strains of MV. The V proteins were more potent inhibitors of IFN-inducible reporter gene expression than the C proteins, and this effect was unrelated to whether the protein originated from an attenuated or wild-type strain. The results also demonstrated the importance of the tyrosine at position 110 in the inhibition of IFN-alpha/beta and IFN-gamma signaling by the V protein, and identified a non-recombinant MV expressing a V protein that was impaired due to a mutation at this residue.

Measles infection and the host immune response to measles virus were compared using naive and immunized rhesus monkeys. The monkeys were experimentally challenged with a wild-type strain of measles virus inoculated intranasally. After pathogenic virus challenge, measles virus was detected in mononuclear cells of peripheral blood, lymph node, and spleen in naive monkeys and viremia peaked on Day 7. However, only one of five vaccinated monkeys had a low virus titer in peripheral blood mononuclear cells at one time point after challenge. No virus was detected in the lymphoid tissues from an immunized monkey that was euthanized on Day 7 of infection. Measles-specific IgM, IgG, neutralizing antibody, and cytotoxic T lymphocytes were detected in vaccinated monkeys before challenge, but antibody titers were significantly lower in immunized monkeys than in naive monkeys after challenge. Measles-specific IgG antibody and cytotoxic T cell responses were still detected more than 1 year after vaccination or infection. This animal model is useful for the further study of measles pathogenesis, immunosuppression, and immunologic memory.

Recently, a new paramyxovirus, now known as Nipah virus (NV), emerged in Malaysia and Singapore, causing fatal encephalitis in humans and a respiratory syndrome in pigs. Initial studies had indicated that NV is antigenically and genetically related to Hendra virus (HV). We generated the sequences of the N, P/C/V, M, F, and G genes of NV and compared these sequences with those of HV and other members of the family Paramyxoviridae. The intergenic regions of NV were identical to those of HV, and the gene start and stop sequences of NV were nearly identical to those of HV. The open reading frames (ORFs) for the V and C proteins within the P gene were found in NV, but the ORF encoding a potential short basic protein found in the P gene of HV was not conserved in NV. The N, P, C, V, M, F, and G ORFs in NV have nucleotide homologies ranging from 88% to 70% and predicted amino acid homologies ranging from 92% to 67% in comparison with HV. The predicted fusion cleavage sequence of the F protein of NV had a single amino acid substitution (K to R) in comparison with HV. Phylogenetic analysis demonstrated that although HV and NV are closely related, they are clearly distinct from any of the established genera within the Paramyxoviridae and should be considered a new genus.

Type I interferons (IFNalpha/beta) are an important part of innate immunity to viral infections because they induce an antiviral response and limit viral replication until the adaptive response clears the infection. Since the nonstructural proteins of several paramyxoviruses inhibit the IFNalpha/beta response, we chose to explore the role of the C protein of measles virus (MV) in such inhibition. Previous studies have suggested that the MV C protein may serve as a virulence factor, but its role in the pathogenesis of MV remains undefined. In the present study, a recombinant MV strain that does not express the C protein (MV C-) and its parental strain (Ed Tag) were used. Growth of MV C- was restricted in human peripheral blood mononuclear cells and HeLa cells, but in the presence of neutralizing antibodies to IFNalpha/beta, MV C- produced titers that were equivalent to those of Ed Tag. In addition, expression of the MV C protein from plasmid DNA inhibited the production of an IFNalpha/beta responsive reporter gene and, to a lesser extent, inhibited an IFNgamma responsive reporter gene. The ability of the MV C protein to suppress the IFNalpha/beta response was confirmed using a biologic assay. After IFNbeta stimulation, HeLa cells infected with Ed Tag produced five-fold less IFNalpha/beta than cells infected with MV C-. While the mechanism of inhibition remains unclear, these data suggest that the MV C protein plays an important role in the pathogenesis of MV by inhibiting IFNalpha/beta signaling.

The C protein of measles virus (MV C) is a basic protein of 186 amino acids (aa) that plays at least two roles in infected cells, interference with the innate immune response and modulation of viral polymerase activity. In this study, Northern blots were used to demonstrate that C proteins from three vaccine strains and three wild-type isolates of MV downregulated both mRNA transcription and genome replication in a plasmid-based mini-genome assay. The effect on transcription always paralleled the effect on replication; however, the six MV C proteins varied considerably in their ability to inhibit polymerase activity. Though the amino-terminal 45 aa of the C protein are more variable among different MV strains than the remaining 75% of the protein, the ability of the MV C proteins to inhibit polymerase activity was not regulated by substitutions in the amino terminus, but rather by the more conserved region containing aa 46–167. Naturally occurring substitutions at positions 147 and 166, but not 88 and 186, were found to regulate MV C protein activity. Deletion of the carboxyl-terminal 19 aa did not affect the polymerase-modulating activity. Though we did not find a link between the aa changes in MV C and attenuation, these data provide new information regarding the functions of this non-structural protein.

Immunization of newborn infants with standard measles vaccines is not effective because of the presence of maternal antibody. In this study, newborn rhesus macaques were immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin (H) and fusion (F) proteins, using the replication-competent WR strain of vaccinia virus or the replication-defective MVA strain. The infants were boosted at 2 months and then challenged intranasally with measles virus at 5 months of age. Some of the newborn monkeys received measles immune globulin (MIG) prior to the first immunization, and these infants were compared to additional infants that had maternal measles-neutralizing antibody. In the absence of measles antibody, vaccination with either vector induced neutralizing antibody, cytotoxic T cell (CTL) responses to measles virus and protection from systemic measles infection and skin rash. The infants vaccinated with the MVA vector developed lower measles-neutralizing antibody titers than those vaccinated with the WR vector, and they sustained a transient measles viremia upon challenge. Either maternal antibody or passively transferred MIG blocked the humoral response to vaccination with both WR and MVA, and the frequency of positive CTL responses was reduced. Despite this inhibition of vaccine-induced immunity, there was a reduction in peak viral loads and skin rash after measles virus challenge in many of the infants with preexisting measles antibody. Therefore, vaccination using recombinant vectors such as poxviruses may be able to prevent the severe disease that often accompanies measles in infants.

A systematic study was made to measure the generation of defective interfering particles upon up to 13 serial passages of two measles vaccine strains, Edmonston and Edmonston–Zagreb, through either simian (Vero) or human (WI-38) cell lines. Results for the Vero cell passage were nearly identical for both viruses. Infectivity titers dropped by nearly 8 logs to undetectable levels at passage 4 and cycled between maximum and minimum levels every 4 passages. Samples with the lowest infectivity titers produced the greatest reduction in titer of standard virus and contained an approximately 900-nucleotide subgenomic RNA for the Edmonston strain and two subgenomic RNAs of 4300 and 3000 nucleotides for the Edmonston–Zagreb vaccine strain. A defective interfering RNA-specific reverse transcription-polymerase chain reaction (RT-PCR) detected subgenomic RNAs at all passage levels. In contrast, samples obtained after passage of these viruses in WI-38 did not reduce the yield of standard virus and did not contain subgenomic RNAs in both Northern blot and RT-PCR assays. These results clearly show that cell type rather than virus strain affects defective interfering particle generation for measles virus.

Recombinant measles nucleoprotein (N) and synthetic peptides spanning the length of the N-protein-coding region were used with a proliferation assay to identify human T-cell epitopes in vaccinated and naturally infected adults. A number of epitopes were mapped to specific regions of the measles virus N. The proliferative response of at least two donors was mediated by CD4 + T cells in association with HLA DR antigens. Over 70% of all donors tested responded to peptides representing amino acids 271–290, 367–386, 400–420, and 483–502, suggesting that these peptides may be broadly recognized within an HLA diverse population. The most frequently recognized T-cell epitopes in both naturally infected and vaccinated donors were located in the genetically heterogeneous carboxy-terminal half of the N. Analysis of patterns of peptide reactivity among vaccinated and naturally infected subjects identified several regions of potential difference between these two groups. Peptides 221–240 and 237–256 were recognized among 100% of naturally infected donors but among only 37.5% of vaccinated donors and therefore may be of further interest in studies to investigate induction of lifelong versus transient immunity to measles. Use of chimeric molecules containing multiple well-characterized T- and B-cell epitopes or genetic alteration of attenuated vaccine virus to enhance critical T-cell responses may eventually lead to the development of a vaccine candidate that can more closely model the patterns of immune response elicited by wild-type virus.