Studies indicate that West African and Congo basin isolates of monkeypox virus (MPXV) are genetically distinct. Here, we show Congo basin MPXV-ZAI-V79 is more virulent for cynomolgus monkeys as compared to presumed West African MPXV-COP-58. This finding may explain the lack of case-fatalities in the U.S. 2003 monkeypox outbreak, which was caused by a West African virus. Virulence differences between West African and Congo basin MPXV are further supported by epidemiological analyses that observed a similar prevalence of antibodies in non-vaccinated humans in both regions, while >90% of reported cases occurred in the Congo basin, and no fatal cases were observed outside of this region. To determine the basis for this difference in virulence, we sequenced the genomes of one human West African isolate, and two presumed West African isolates and compared the sequences to Congo basin MPXV-ZAI-96-I-16. The analysis identified D10L, D14L, B10R, B14R, and B19R as possible virulence genes, with D14L (ortholog of vaccinia complement protein) as a leading candidate.

The Poxvirus Bioinformatics Resource Center (PBRC) has been established to provide informational and analytical resources to the scientific community to aid research directed at providing a better understanding of the Poxviridae family of viruses. The PBRC was specifically established as the result of the concern that variola virus, the causative agent of smallpox, as well as related viruses, might be utilized as biological weapons. In addition, the PBRC supports research on poxviruses that might be considered new and emerging infectious agents such as monkeypox virus. The PBRC consists of a relational database and web application that supports the data storage, annotation, analysis and information exchange goals of the project. The current release consists of over 35 complete genomic sequences of various genera, species and strains of viruses from the Poxviridae family. Sequence and annotation information for these viruses has been obtained from sequences publicly available from GenBank as well as sequences not yet deposited in GenBank that have been obtained from ongoing sequencing projects. In addition to sequence data, the PBRC provides comprehensive annotation and curation of virus genes; analytical tools to aid in the understanding of the available sequence data, including tools for the comparative analysis of different virus isolates; and visualization tools to help better display the results of various analyses. The PBRC represents the initial development of what will become a more comprehensive Viral Bioinformatics Resource Center for Biodefense that will be one of the National Institute of Allergy and Infectious Diseases' ‘Bioinformatics Resource Centers for Biodefense and Emerging or Re-Emerging Infectious Diseases’. The PBRC website is available at http://www.poxvirus.org.

Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for cell binding and transduction, differences were observed in lectin competition, resulting in distinct tropisms in human cancer cell lines.Adeno-associated virus (AAV) is a member of the Parvoviridae, a virus family characterized by a single-stranded linear DNA genome enclosed by a capsid with icosahedral symmetry. A hallmark of the AAV life cycle is its dependency on a helper virus for efficient productive replication. Adenovirus was originally identified as the AAV helper virus, but other viruses, such as herpesvirus and cytomegalovirus (7), can provide helper functions for AAV replication. Virus stocks have therefore served as a rich source for the discovery of AAV isolates; AAV1, AAV2, AAV3, AAV4, AAV6, and bovine AAV were all found as contaminants of adenovirus preparations (1-3, 8, 10-12, 15).In this article, we describe the analysis of virus stocks from the American Type Culture Collection (ATCC) for the presence of AAV DNA. Our goals were to identify ATCC virus isolates that contain AAV contaminations and, if novel, to characterize the isolates.Virus stocks supplied by the ATCC were analyzed for the presence of AAV DNA by a PCR-based assay, as described previously (5). AAV DNA was detected in 13 of 137 samples analyzed. AAV contaminations were frequently detected in adenovirus isolates (26%) but not in herpesvirus, retrovirus, coronavirus, orthomyxovirus, poxvirus, or reovirus stocks. The entire coding regions for the AAV Rep and Cap open reading frames were PCR amplified and subcloned, and several clones from each isolate were sequenced and analyzed. Adenovirusfree stocks of the novel recombinant AAVs were produced by standard cotransfection protocols (14).Ten of the AAVs detected in simian adenovirus stocks displayed at least 96% homology on the DNA level and 98% identity in the capsid amino acid sequence either to AAV1, to AAV6, or to each other. To determine whether these minor sequence changes could affect the biological activity of the isolates, 2 of the 10 isolates, AAV , and AAV(VR-355), isolated from ATCC respectively (GenBank accession numbers DQ180604 and DQ180605), were studied in greater detail. The VP1 capsid proteins of AAV and AAV(VR-355) differ by 7 and 6 amino acids, respectively, from that of AAV6. The locations of the divergent amino acids within the capsid were identified by superimposing the AAV(VR-195) and AAV(VR-355) VP1 sequences onto a pseudoatomic structure for AAV6 (Fig. 1). Divergent amino acids were found to cluster on the capsid surface around the threefold axis of symmetry, an area ...

The 36 001 base pair DNA sequence of human adenovirus serotype 1 (HAdV-1) has been determined, using a 'leveraged primer sequencing strategy' to generate high quality sequences economically. This annotated genome (GenBank AF534906) confirms anticipated similarity to closely related species C (formerly subgroup), human adenoviruses HAdV-2 and-5, and near identity with earlier reports of sequences representing parts of the HAdV-1 genome. A first round of HAdV-1 sequence data acquisition used PCR amplification and sequencing primers from sequences common to the genomes of HAdV-2 and-5. The subsequent rounds of sequencing used primers derived from the newly generated data. Corroborative re-sequencing with primers selected from this HAdV-1 dataset generated sparsely tiled arrays of high quality sequencing ladders spanning both complementary strands of the HAdV-1 genome. These strategies allow for rapid and accurate low-pass sequencing of genomes. Such rapid genome determinations facilitate the development of specific probes for differentiation of family, serotype, subtype and strain (e.g. pathogen genome signatures). These will be used to monitor epidemic outbreaks of acute respiratory disease in a defined test bed by the Epidemic Outbreak Surveillance (EOS) project.

A collection of randomly generated temperature-sensitive (ts) vaccinia virus (strain IHD-W) mutants were reported by S. Dales et al., (1978, Virology, 84, 403-428) in 1978 and characterized by electron microscopy. We have performed further genetic analysis on the Dales collection of mutants to make the mutants more useful to the scientific community. We obtained the entire Dales collection, 97 mutants, from the American Type Culture Center (ATCC). All 97 mutants were grown and reassessed for temperature sensitivity. Of these, 16 mutants were either very leaky or showed unacceptably high reversion indices even after plaque purification and therefore were not used for further analysis. The remaining 81 ts mutants were used to perform a complete complementation analysis with each other and the existing Condit collection of ts vaccinia virus (strain WR) mutants. Twenty-two of these 81 Dales mutants were dropped during complementation analysis due to erratic or weak behavior in the complementation test. Of the 59 mutants that were fit for further investigation, 30 fall into 13 of Condit's existing complementation groups, 5 comprise 3 previously identified complementation groups independent of the Condit collection, and 24 comprise 18 new complementation groups. The 59 mutants which were successfully characterized by complementation will be accessioned by and made available to the scientific community through the ATCC.