B19 Parvovirus: B19 parvovirus is a small, nonenveloped, single-stranded DNA virus, the only member of the Parvoviridae family that is known to be pathogenic in humans. B19 parvovirus infection is common in childhood, and most adults have been exposed to the virus as determined by serologic assays for anti-viral IgG. B19 parvovirus is the etiologic agent in fifth disease, a childhood exanthem;fifth disease manifests in adulthood as chronic arthropathy. Hematologically, B19 parvovirus causes several diseases: transient aplastic crisis of hemolytic syndromes, leading to severe and sometimes fatal acute anemia, as in patients with sickle cell disease;hydrops fetalis, in which infection of the mother in the second trimester is transmitted in-utero to the developing fetus, leading to severe anemia, congestive heart failure and stillbirth;chronic pure red cell aplasia due to a persistent infection, the result of inability of the host to mount an adequate neutralizing antibody response. The Hematology Branchs notable achievements in B19 parvovirus research include its first propagation in cell culture;elucidation of a detailed transcription map that led to the virus reclassification into a new genus;identification of the cellular receptor, globoside or P antigen, and determination that genetic absence of the receptor leads to insusceptibility in vitro and in vivo;description of the neutralizing epitopes present on the unique region of VP1, which are external to the capsid surface;and production of a recombinant vaccine candidate, based on expression of B19 capsid proteins in a baculovirus system and subsequent self assembly of the proteins into empty capsids, with adjustment of VP1 content to maximize neutralizing antibody responses in animals and humans. In recent years, investigators in the Branch have also developed powerful tools for the study of B19 parvovirus in tissue culture: both an infectious clone, which allows modification of viral proteins at the nucleotide level and therefore detailed molecular mapping of structure-function relationships, and utilization of CD34 cells driven to erythroid differentiation obtained from normal human volunteers as a basis for a productive cell culture system, permitting propagation of the virus under physiologic conditions. We have continued efforts toward production and clinical testing of a parvovirus vaccine for humans. We are collaborating with Phillip Dormitzer of Novartis and investigators at St. Jude Childrens Hospital in utilizing the Novartis yeast methodology for empty capsid production. The eventual aim is a Phase I study of yeast derived empty capsids, enriched for VP1 structural protein, in normal adults to be conducted at the Clinical Center by the Hematology Branch, utilizing the method recently published by Novartis and the GMP facility in Memphis. If no serious toxicity were observed and neutralizing antibodies were elicited, the phase I trial would be followed a phase II/III trial at St. Jude in children with sickle cell disease. Financial constraints have retarded moving forward on this project to date. In our laboratory, we have successfully established four suspension CHO cell lines for stable expression of B19 parvovirus-like particles. These include particles containing VP1, VP1 with the phospholipase gene mutated, and two B19-influenza chimeras: VP1-M2 and VP1-HA2. The latter are developed with the goal of a universal influenza vaccine on a platform of parvovirus capsids. Optimization of expression of the empty capsids was achieved through multiple rounds cell sorting. B19 capsids have been purified using Opti-Prep gradient centrification. Antigenicity and structure were confirmed with immunoblot and by electromicroscopy. To enhance and adjust for expression levels of VP1, which are critical for neutralizing antibody production, we are currently modifying the cell lines using zinc-inducible recombinant plasmids for the appropriate genes. Virus infection and aplastic anemia: There have been repeated failures to identify a viral etiology for seronegative hepatitis (non-A, non-B, and non-C). While seronegative hepatitis is rare in the United States, as many as 20% of hepatitis cases in Asian clinics are seronegative. Seronegative acute hepatitis differs from known viral hepatitis in its demographic features and clinical consequences;in particular, there is a higher rate of severe late complications of fulminant hepatitis and of post-hepatitis aplastic anemia following seronegative acute hepatitis. For bone marrow failure, the pattern is stereotypical: patients are more often male, usually young, and without known risk factors for hepatitis virus exposure;the hepatitis is transient but severe, with marked elevations in bilirubin and serum transaminases;pancytopenia is profound and historically almost always fatal. Due to inability to isolate a putative infectious agent using a wide variety of molecular, immunological and biochemical methods from either bone marrow or blood of patients with post-hepatitis aplastic anemia or in liver samples obtained from patients undergoing liver transplantation for fulminant hepatitis, we have collaborated with other institutions to obtain blood from patients entering the acute phase of seronegative hepatitis. These samples also may be more likely to contain infectious material than are those obtained months following the onset of the hepatitis and its likely clearance by the immune system. Using Solexa deep sequencing, we have isolated viral sequences from samples obtained from a large infectious disease hospital in Chongqing in the west of China. Ninety-two serum specimens were collected from patients with seronegative hepatitis between 1999 and 2007. Ten serapools were screened by Selexa deep sequencing. We discovered a 3780 base pair contig present in all ten pools that yielded high blast xe scores against paroviruses. The complete sequence of the assembled contigs was confirmed by gene amplification of overlapping regions over almost the entire genome. This virus has been designated NIH-CQV provisionally. The contig is composed of two major open reading frames. ORF1 was most homologous to the replication associated protein of bat circovirus, and ORF2 to the capsid protein of porcine parvovirus. Phylogenetic analysis shows that NIH-CQV is at the interface of the Parvoviridae and Circoviridae. The prevelance of NIH-CQV in patients was determined by quantitative PCR: 63 of 90 patient samples (70%) were positive while all 45 healthy controls were negative for virus by this sensitive method. The average virus titer in patient specimens was approximately 1x104copy/ml. Specific antibodies directed to NIH-CQV were detected by immunoblotting. In patients, 84% were positive for IgG and 31% were positive for IgM;in contrast, 78% of healthy controls were positive IgG but none for IgM. These data are highly suggestive that NIH-CQV is a pathogenic virus in humans, and epidemiologic studies in China will be conducted in collaboration with the Chongqing group and others in various cities in order to determine the presence and prevelance of virus and antibodies. Using the same methods, we are currently analyzing specimens from Kenya in collaboration with Joel Montgomery of the CDC in Nairobi. Results to date are suggestive that plasmodium species may be implicated in epidemics of undiagnosed acute encephalitis, based on deep sequencing of cerebral spinal fluid specimens.
