Bettina Bankamp

Genetic Characterization of Nipah Virus, Bangladesh, 2004

Until 2004, identification of Nipah virus (NV)-like outbreaks in Bangladesh was based on serology. We describe the genetic characterization of a new strain of NV isolated during outbreaks in Bangladesh (NV-B) in 2004, which confirms that NV was the etiologic agent responsible for these outbreaks.

Ultrastructural Characterization of SARS Coronavirus

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.

Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus.

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection.

Inhibition of interferon induction and signaling by paramyxoviruses.

Summary: The family Paramyxoviridae comprises a diverse group of viruses that includes several important human and veterinary pathogens. Members of this family have a non‐segmented, single‐stranded, negative sense RNA genome, a conserved gene order, and a similar replication strategy. Paramyxoviruses are divided into two subfamilies, Paramyxovirinae and Pneumovirinae, which comprise five genera and two genera, respectively. Viruses in each genus have developed strategies to circumvent the interferon (IFN) response by using a diverse array of proteins that are encoded within the phosphoprotein genes of the Paramyxovirinae or non‐structural genes of the Pneumovirinae. This review focuses on the specific roles that these viral proteins play in the inhibition of IFN signaling and, to a lesser extent, on the mechanisms by which these proteins inhibit the induction pathways of IFN. An improved understanding of the interactions between viral proteins and the host innate immune response is critical to achieving a thorough comprehension of the pathogenesis of this important group of viruses. Hopefully this knowledge will support the development of more targeted vaccines and therapeutics to better prevent and control viral infection.

Regulation of interferon signaling by the C and V proteins from attenuated and wild-type strains of measles virus.

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.

Genetic Characterization of Measles Vaccine Strains

The complete genomic sequences of 9 measles vaccine strains were compared with the sequence of the Edmonston wild-type virus. AIK-C, Moraten, Rubeovax, Schwarz, and Zagreb are vaccine strains of the Edmonston lineage, whereas CAM-70, Changchun-47, Leningrad-4 and Shanghai-191 were derived from 4 different wild-type isolates. Nucleotide substitutions were found in the noncoding regions of the genomes as well as in all coding regions, leading to deduced amino acid substitutions in all 8 viral proteins. Although the precise mechanisms involved in the attenuation of individual measles vaccines remain to be elucidated, in vitro assays of viral protein functions and recombinant viruses with defined genetic modifications have been used to characterize the differences between vaccine and wild-type strains. Although almost every protein contributes to an attenuated phenotype, substitutions affecting host cell tropism, virus assembly, and the ability to inhibit cellular antiviral defense mechanisms play an especially important role in attenuation.

Improving Global Virologic Surveillance for Measles and Rubella

An important aspect of laboratory surveillance for measles and rubella is the genetic characterization of circulating wild-type viruses to support molecular epidemiologic studies and to track transmission pathways. Virologic surveillance that is sufficient to document the interruption of transmission of measles and rubella viruses will be an essential criterion for verification of elimination. Laboratories in the World Health Organization (WHO) Measles and Rubella Laboratory Network have worked to improve and expand virologic surveillance as many regions move toward elimination of measles and rubella/congenital rubella syndrome. As countries approach elimination, it will be necessary to obtain genetic information from as many chains of transmission as possible. In addition, baseline virologic surveillance, especially for rubella, needs to be improved in many countries. This report contains a summary of recent improvements to the methods used for virologic surveillance.

The C, V and W proteins of Nipah virus inhibit minigenome replication.

Nipah virus (NiV) is a recently emergent, highly pathogenic, zoonotic paramyxovirus of the genus Henipavirus. Like the phosphoprotein (P) gene of other paramyxoviruses, the P gene of NiV is predicted to encode three additional proteins, C, V and W. When the C, V and W proteins of NiV were tested for their ability to inhibit expression of the chloramphenicol acetyltransferase (CAT) reporter gene in plasmid-based, minigenome replication assays, each protein inhibited CAT expression in a dose-dependent manner. The C, V and W proteins of NiV also inhibited expression of CAT from a measles virus (MV) minigenome, but not from a human parainfluenzavirus 3 (hPIV3) minigenome. Interestingly, the C and V proteins of MV, which have previously been shown to inhibit MV minigenome replication, also inhibited NiV minigenome replication; however, they were not able to inhibit hPIV3 minigenome replication. In contrast, the C protein of hPIV3 inhibited minigenome replication of hPIV3, NiV and MV. Although there is very limited amino acid sequence similarity between the C, V and W proteins within the paramyxoviruses, the heterotypic inhibition of replication suggests that these proteins may share functional properties.

Activity of Polymerase Proteins of Vaccine and Wild-Type Measles Virus Strains in a Minigenome Replication Assay

ABSTRACT The relative activities of five measles virus (MV) polymerase (L) proteins were compared in an intracellular, plasmid-based replication assay. When coexpressed with N and P proteins from an attenuated strain, L proteins from two attenuated viruses directed the production of up to eight times more reporter protein from an MV minigenome than the three wild-type L proteins. Northern blot analysis demonstrated that the differences in reporter protein production correlated with mRNA transcription levels. Increased activity of polymerases from attenuated viruses equally affected mRNA transcription and minigenome replication. The higher level of transcription may be a consequence of increased template availability or may be an independent effect of the elevated activity of the attenuated polymerases. Coexpression of wild-type L proteins with homologous N and P proteins did not affect the activity of the wild-type polymerases, indicating that the differential activity was a function of the L proteins alone. Use of a minigenome that incorporated two nucleotide changes found in the genomic leader of the three wild-type viruses did not raise the activity of the wild-type L proteins. These data demonstrate that increased polymerase activity differentiates attenuated from wild-type viruses and suggest that functions involved in RNA synthesis contribute to the attenuated phenotype of MV vaccine strains.

Improving molecular tools for global surveillance of measles virus

BACKGROUND The genetic characterization of wild-type measles viruses plays an important role in the description of viral transmission pathways and the verification of measles elimination. The 450 nucleotides that encode the carboxyl-terminus of the nucleoprotein (N-450) are routinely sequenced for genotype analysis. OBJECTIVES The objectives of this study were to develop improved primers and controls for RT-PCR reactions used for genotyping of measles samples and to develop a method to provide a convenient, safe, and inexpensive means to distribute measles RNA for RT-PCR assays and practice panels. STUDY DESIGN A newly designed, genetically defined synthetic RNA and RNA isolated from cells infected with currently circulating genotypes were used to compare the sensitivity of primer pairs in RT-PCR and nested PCR. FTA® cards loaded with lysates of measles infected cells were tested for their ability to preserve viral RNA and destroy virus infectivity. RESULTS A new primer pair, MeV216/MeV214, was able to amplify N-450 from viruses representing 10 currently circulating genotypes and a genotype A vaccine strain and demonstrated 100-fold increased sensitivity compared to the previously used primer set. A nested PCR assay further increased the sensitivity of detection from patient samples. A synthetic positive control RNA was developed that produced PCR products that are distinguishable by size from PCR products amplified from clinical samples. FTA® cards completely inactivated measles virus and stabilized RNA for at least six months. CONCLUSIONS These improved molecular tools will advance molecular characterization of circulating measles viruses globally and provide enhanced quality control measures.