Transfusion-associated hepatitis before the screening of blood for hepatitis risk factors.

BACKGROUND: The true incidence of transfusion-associated hepatitis (TAH) before blood screening is unknown. Our aims were to reevaluate blood recipients receiving unscreened blood and analyze hepatitis viruses circulating more than 45 years ago. STUDY DESIGN AND METHODS: Cryopreserved serum samples from 66 patients undergoing open heart surgery in the 1960s were reevaluated with modern diagnostic tests to determine the incidence of TAH and its virologic causes. RESULTS: In this heavily transfused population receiving a mean of 20 units per patient of predominantly paid-donor blood, 30 of 66 (45%) developed biochemical evidence of hepatitis; of these, 20 (67%) were infected with hepatitis C virus (HCV) alone, four (13%) with hepatitis B virus (HBV) alone, and six (20%) with both viruses. Among the 36 patients who did not develop hepatitis, four (11%) were newly infected with HCV alone, nine (25%) with HBV alone, and one (3%) with both viruses. Overall, 100% of patients with hepatitis and 39% of those without hepatitis were infected with HBV and/or HCV; one patient was also infected with hepatitis E virus. The donor carrier rate for HBV and/or HCV was estimated to be more than 6%; contemporaneously prepared pooled normal human plasma was also contaminated with multiple hepatitis viruses. CONCLUSION: TAH virus infections were a larger problem than perceived 50 years ago and HCV was the predominant agent transmitted. All hepatitis cases could be attributed to HCV and/or HBV and hence there was no evidence to suggest that an additional hepatitis agent existed undetected in the blood supply.

Chimpanzees as an animal model for human norovirus infection and vaccine development.

Noroviruses are global agents of acute gastroenteritis, but the development of control strategies has been hampered by the absence of a robust animal model. Studies in chimpanzees have played a key role in the characterization of several fastidious hepatitis viruses, and we investigated the feasibility of such studies for the noroviruses. Seronegative chimpanzees inoculated i.v. with the human norovirus strain Norwalk virus (NV) did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar to that observed in humans. NV RNA was detected in intestinal and liver biopsies concurrent with the detection of viral shedding in stool, and NV antigen expression was observed in cells of the small intestinal lamina propria. Two infected chimpanzees rechallenged 4, 10, or 24 mo later with NV were resistant to reinfection, and the presence of NV-specific serum antibodies correlated with protection. We evaluated the immunogenicity and efficacy of virus-like particles (VLPs) derived from NV (genogroup I, GI) and MD145 (genogroup II, GII) noroviruses as vaccines. Chimpanzees vaccinated intramuscularly with GI VLPs were protected from NV infection when challenged 2 and 18 mo after vaccination, whereas chimpanzees that received GII VLPs vaccine or a placebo were not. This study establishes the chimpanzee as a viable animal model for the study of norovirus replication and immunity, and shows that NV VLP vaccines could induce protective homologous immunity even after extended periods of time.

Evolutionary dynamics of GII.4 noroviruses over a 34-year period.

Noroviruses are a major cause of epidemic gastroenteritis in children and adults, and GII.4 has been the predominant genotype since its first documented occurrence in 1987. This study examined the evolutionary dynamics of GII.4 noroviruses over more than three decades to investigate possible mechanisms by which these viruses have emerged to become predominant. Stool samples (n = 5,424) from children hospitalized at the Children's Hospital in Washington, DC, between 1974 and 1991 were screened for the presence of noroviruses by a custom multiplex real-time reverse transcription-PCR. The complete genome sequences of five GII.4 noroviruses (three of which predate 1987 by more than a decade) in this archival collection were determined and compared to the sequences of contemporary strains. Evolutionary analysis determined that the GII.4 VP1 capsid gene evolved at a rate of 4.3 x 10(-3) nucleotide substitutions/site/year. Only six sites in the VP1 capsid protein were found to evolve under positive selection, most of them located in the shell domain. No unique mutations were observed in or around the two histoblood group antigen (HBGA) binding sites in the P region, indicating that this site has been conserved since the 1970s. The VP1 proteins from the 1974 to 1977 noroviruses contained a unique sequence of four consecutive amino acids in the P2 region, which formed an exposed protrusion on the modeled capsid structure. This protrusion and other observed sequence variations did not affect the HBGA binding profiles of recombinant virus-like particles derived from representative 1974 and 1977 noroviruses compared with more recent noroviruses. Our analysis of archival GII.4 norovirus strains suggests that this genotype has been circulating for more than three decades and provides new ancestral strain sequences for the analysis of GII.4 evolution.

Norwalk virus N-terminal nonstructural protein is associated with disassembly of the Golgi complex in transfected cells.

Norwalk virus is the prototype strain for members of the genus Norovirus in the family Caliciviridae, which are associated with epidemic gastroenteritis in humans. The nonstructural protein encoded in the N-terminal region of the first open reading frame (ORF1) of the Norwalk virus genome is analogous in gene order to proteins 2A and 2B of the picornaviruses; the latter is known for its membrane-associated activities. Confocal microscopy imaging of cells transfected with a vector plasmid that provided expression of the entire Norwalk virus N-terminal protein (amino acids 1 to 398 of the ORF1 polyprotein) showed colocalization of this protein with cellular proteins of the Golgi apparatus. Furthermore, this colocalization was characteristically associated with a visible disassembly of the Golgi complex into discrete aggregates. Deletion of a predicted hydrophobic region (amino acids 360 to 379) in a potential 2B-like (2BL) region (amino acids 301 to 398) near the C terminus of the Norwalk virus N-terminal protein reduced Golgi colocalization and disassembly. Confocal imaging was conducted to examine the expression characteristics of fusion proteins in which the 2BL region from the N-terminal protein of Norwalk virus (a genogroup I norovirus) or MD145 (a genogroup II norovirus) was fused to the C terminus of enhanced green fluorescent protein. Expression of each fusion protein in cells showed evidence for its colocalization with the Golgi apparatus. These data indicate that the N-terminal protein of Norwalk virus interacts with the Golgi apparatus and may play a 2BL role in the induction of intracellular membrane rearrangements associated with positive-strand RNA virus replication in cells.

Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus.

The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

In vitro proteolytic processing of the MD145 norovirus ORF1 nonstructural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells.

The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q(330)/G(331), Q(696)/G(697), E(875)/G(876), E(1008)/A(1009), and E(1189)/G(1190)) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-kDa protein (p20); virus protein, genome linked (VPg); proteinase (Pro); polymerase (Pol). A time course analysis of proteolytic processing of the MD145-12 ORF1 polyprotein in an in vitro coupled transcription and translation assay allowed the identification of stable precursors and final mapped cleavage products. Stable precursors included p20VPg (analogous to the 3AB of the picornaviruses) and ProPol (analogous to the 3CD of the picornaviruses). Less stable processing intermediates were identified as p20VPgProPol, p20VPgPro, and VPgPro. The MD145-12 Pro and ProPol proteins were expressed in bacteria as active forms of the proteinase and used to further characterize their substrate specificities in trans cleavage assays. The MD145-12 Pro was able to cleave its five mapped cleavage sites in trans and, in addition, could mediate trans cleavage of the Norwalk virus (GI/I) ORF1 polyprotein into a similar proteolytic processing profile. Taken together, our data establish a model for proteolytic processing in the noroviruses that is consistent with nonstructural precursors and products identified in studies of caliciviruses that replicate in cell culture systems.

Isolation of enzymatically active replication complexes from feline calicivirus-infected cells.

A membranous fraction that could synthesize viral RNA in vitro in the presence of magnesium salt, ribonucleotides, and an ATP-regenerating system was isolated from feline calicivirus (FCV)-infected cells. The enzymatically active component of this fraction was designated FCV replication complexes (RCs), by analogy to other positive-strand RNA viruses. The newly synthesized RNA was characterized by Northern blot analysis, which demonstrated the production of both full-length (8.0-kb) and subgenomic-length (2.5-kb) RNA molecules similar to those synthesized in FCV-infected cells. The identity of the viral proteins associated with the fraction was investigated. The 60-kDa VP1 major capsid protein was the most abundant viral protein detected. VP2, a minor structural protein encoded by open reading frame 3 (ORF3), was also present. Nonstructural proteins associated with the fraction included the precursor polypeptides Pro-Pol (76 kDa) and p30-VPg (43 kDa), as well as the mature nonstructural proteins p32 (derived from the N-terminal region of the ORF1 polyprotein), p30 (the putative "3A-like" protein), and p39 (the putative nucleoside triphosphatase). The isolation of enzymatically active RCs containing both viral and cellular proteins should facilitate efforts to dissect the contributions of the virus and the host to FCV RNA replication.