No abstract

Norwalk virus infection is a common cause of gastroenteritis in humans. The clinical features and virologic and immunologic responses following oral administration of Norwalk virus to 50 volunteers were monitored. New ELISAs using recombinant virus particles as the antigen source were used to assess the pattern of virus shedding and the specific immune responses. Forty-one subjects (82%) became infected; 68% were symptomatic and 32% were asymptomatic. The proportion of subjects infected was similar for those with and without preexisting antibody (82% vs. 60%; P > .2). The magnitude of seroconversion was highest in subjects who had vomiting. The peak of viral shedding was between 25 and 72 h, and virus first appeared in stool at 15 h. Specimens collected 7 days after inoculation remained positive. These results show a higher infection rate, more subclinical infections, and longer virus excretion following Norwalk virus inoculation than previously recognized.

Clonal diversity is a consequence of cancer cell evolution driven by Darwinian selection. Precise characterization of clonal architecture is essential to understand the evolutionary history of tumor development and its association with treatment resistance. Here, using a single-cell DNA sequencing, we report the clonal architecture and mutational histories of 123 acute myeloid leukemia (AML) patients. The single-cell data reveals cell-level mutation co-occurrence and enables reconstruction of mutational histories characterized by linear and branching patterns of clonal evolution, with the latter including convergent evolution. Through xenotransplantion, we show leukemia initiating capabilities of individual subclones evolving in parallel. Also, by simultaneous single-cell DNA and cell surface protein analysis, we illustrate both genetic and phenotypic evolution in AML. Lastly, single-cell analysis of longitudinal samples reveals underlying evolutionary process of therapeutic resistance. Together, these data unravel clonal diversity and evolution patterns of AML, and highlight their clinical relevance in the era of precision medicine.

Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1-14 and GII/1-17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.