Karin Bok

Norovirus Gastroenteritis in Immunocompromised Patients

Norovirus causes gastroenteritis in immunocompetent and immunocompromised hosts. It is the most common cause of gastroenteritis in U.S. adults seeking emergency department care. Genetic features and emerging treatments are discussed.

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

ABSTRACT 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 Childrens 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 × 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.

Rotavirus antigenaemia and viraemia: a common event?

BACKGROUND Rotavirus infection is thought to be confined to the intestine. Reports of rotavirus RNA in the cerebral spinal fluid and serum of children infected with rotavirus suggest the possibility that rotavirus escapes the intestine into the circulatory system. We assessed whether rotavirus antigen, RNA, or both, were present in serum samples from immunocompetent rotavirus-infected children and animals. METHODS We obtained sera from immunocompetent mice, rats, rabbits, and calves 1-10 days after inoculation with rotavirus or matched vehicle. We obtained sera retrospectively from immunocompetent children diagnosed with rotavirus diarrhoea (n=33), healthy children (n=6) and adults (n=12), children convalescing from rotavirus (n=6), and children with non-rotavirus diarrhoea (n=11). Samples were analysed for the presence of rotavirus antigen or RNA by EIA or RT-PCR, respectively. FINDINGS Rotavirus antigen was present in sera from rotavirus-infected animals, but not in sera from control animals. Infectious rotavirus or rotavirus RNA was detected in sera of mice and calves, respectively. Antigen was present in 22 of 33 serum samples from children with confirmed rotavirus infection but in none of 35 samples from controls. Detection of serum antigen was inversely related to the number of days between symptom onset and sample collection, and directly related to stool antigen concentration. Rotavirus RNA was detected by RT-PCR in three of six rotavirus-positive sera. INTERPRETATION Rotavirus can escape the gastrointestinal tract in children, resulting in antigenaemia and possible viraemia. This finding is important for the understanding of the pathogenesis, immunology, and clinical manifestations of rotavirus infection.

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.

Comparative Evolution of GII.3 and GII.4 Norovirus over a 31-Year Period

ABSTRACT Noroviruses are the most common cause of epidemic gastroenteritis. Genotype II.3 is one of the most frequently detected noroviruses associated with sporadic infections. We studied the evolution of the major capsid gene from seven archival GII.3 noroviruses collected during a cross-sectional study at the Childrens Hospital in Washington, DC, from 1975 through 1991, together with capsid sequence from 56 strains available in GenBank. Evolutionary analysis concluded that GII.3 viruses evolved at a rate of 4.16 × 10−3 nucleotide substitutions/site/year (strict clock), which is similar to that described for the more prevalent GII.4 noroviruses. The analysis of the amino acid changes over the 31-year period found that GII.3 viruses evolve at a relatively steady state, maintaining 4% distance, and have a tendency to revert back to previously used residues while preserving the same carbohydrate binding profile. In contrast, GII.4 viruses demonstrate increasing rates of distance over time because of the continued integration of new amino acids and changing HBGA binding patterns. In GII.3 strains, seven sites acting under positive selection were predicted to be surface-exposed residues in the P2 domain, in contrast to GII.4 positively selected sites located primarily in the shell domain. Our study suggests that GII.3 noroviruses caused disease as early as 1975 and that they evolve via a specific pattern, responding to selective pressures induced by the host rather than presenting a nucleotide evolution rate lower than that of GII.4 noroviruses, as previously proposed. Understanding the evolutionary dynamics of prevalent noroviruses is relevant to the development of effective prevention and control strategies.

Emergence of G9 P[6] Human Rotaviruses in Argentina: Phylogenetic Relationships among G9 Strains

ABSTRACT Because rotavirus diarrhea can be reduced through vaccination and because current vaccine candidates provide protection against only the most common G antigenic types (G1 to G4), detection of uncommon G types is one of the main goals of rotavirus surveillance. After a 2-year nationwide rotavirus surveillance study in Argentina concluded, surveillance was continued and an increase of G9 prevalence in several Argentine cities was detected. During this period G9 strains predominated in the south, and a gradient of decreasing G9 prevalence was observed from south to north (41 to 0%). Sequence analysis of gene 9, encoding the G antigen, showed that Argentine strains cluster with most G9 isolates from other countries, showing less than 2% nucleotide divergence among them, but are distinctive from them in that they present some unique amino acid changes. Our results agree with reports of increased G9 prevalence in other parts of the world, suggesting the need to incorporate G9 into candidate rotavirus vaccines.

Apoptosis in Murine Norovirus-Infected RAW264.7 Cells Is Associated with Downregulation of Survivin

ABSTRACT Noroviruses (NVs) are recognized as a major cause of nonbacterial gastroenteritis in humans. Studies of the human NVs continue to be hampered by the inability to propagate them in any cell culture system. Until recently, most data concerning NV replication were derived from studies of feline calicivirus and rabbit hemorrhagic disease virus, which are cultivable members of the family Caliciviridae. From such studies, it was proposed that caliciviruses induce apoptosis to facilitate the dissemination of viral progeny in the host. The discovery that MNV type 1 (MNV-1) grows in RAW264.7 cells provided the first cell culture system for use in studying the role of apoptosis in NV infection. We first showed that MNV-1 replication triggered apoptosis in infected RAW264.7 cells and then demonstrated that cell death was associated with activation of caspase-9 and caspase-3 through the mitochondrial pathway. This process was dependent on virus replication, since inactivated virus failed to induce signs of apoptosis. In order to better understand the apoptotic process induced by MNV-1 infection of RAW264.7 cells, we investigated the expression profiles of MNV-1-infected versus mock-infected cells. Survivin, a member of the inhibitor of apoptosis protein family, was found to be significantly downregulated in an inverse relationship with the virus genome replication. This study showed that, unlike other viruses that upregulate survivin, MNV-1 is the first virus found to downregulate the levels of survivin. We observed that MNV-1 replication in RAW264.7 cells activated caspases, resulting in apoptosis through the mitochondrial pathway, possibly as a result of downregulation of survivin.

Genetic Variation of Capsid Protein VP7 in Genotype G4 Human Rotavirus Strains: Simultaneous Emergence and Spread of Different Lineages in Argentina

ABSTRACT Rotavirus is the most-common cause of severe diarrhea in young children. Complete rotavirus characterization includes determination of the antigenic type of the two outer capsid proteins, VP7 and VP4, designated G and P types, respectively. During a nationwide rotavirus surveillance study, genotype G4 frequency increased during the second year. To evaluate further the mechanism of emergence and the relationship among G4 strains, the genetic diversity of VP7 capsid protein in these samples was studied in detail. Overall nucleotide sequence divergence ranged from less than 0.1 to 19.5%, a higher divergence than that observed for other rotavirus G types (0.1 to 9%). Sequences were classified into two major lineages (designated I and II) based on their nucleotide distances. The most heterogeneous lineage was further subdivided into four sublineages (designated Ia to Id). Most Argentine sequences were of sublineages Ib and Ic, which were confirmed to be independent sequence clusters by parsimony analysis. This study describes different lineages and sublineages within G4 strains and shows that Argentine strains are distantly related to reference strain ST3. The appearance of at least two G4 genotype (sub)lineages during 1998 demonstrates that the increased frequency of these strains was due to the synchronized emergence of different groups of strains.

Epidemiology and Evolution of Rotaviruses and Noroviruses from an Archival WHO Global Study in Children (1976–79) with Implications for Vaccine Design

Prompted by the discovery of new gastrointestinal viruses, the NIH, NIAID and WHO investigated the etiology of acute diarrhea that occurred from 1976–1979 in a global cohort of infants and young children. Rotaviruses were found to be major pathogens worldwide, whereas the Norwalk virus could not be detected using a radioimmunoassay. The aim of this study is to re-evaluate the role and diversity of rotaviruses and noroviruses in the original cohort using more sensitive current technologies. Stools collected from Asia, Africa, and South America (n = 485) were evaluated for viral genotypes by RT-PCR and sequencing. Rotaviruses were detected in 28.9% and noroviruses in 9.7% of the specimens, with G1 rotaviruses and GII noroviruses accounting for the majority of each respective virus. Various strains in this study predated the currently assigned dates of discovery for their particular genotype, and in addition, two noroviruses (KL45 and T091) could not be assigned to current genotypes. Phylogenetic analyses demonstrated a relative constancy in circulating rotavirus genotypes over time, with several genotypes from this study becoming established in the current repertoire of viral species. Similarly, GII noroviruses have maintained dominance, with GII.4 noroviruses continuing as a predominant genotype over time. Taken together, the complex molecular epidemiology of rotaviruses and noroviruses circulating in the 1970’s is consistent with current patterns, an important consideration in the design of multivalent vaccines to control these viruses.

The Importance of Intergenic Recombination in Norovirus GII.3 Evolution

ABSTRACT Norovirus genotype II.3 (GII.3) strains are a major cause of sporadic gastroenteritis. Intergenic recombination between the capsid and RNA-dependent RNA polymerase (RdRp) genes is common and results in the acquisition of an alternative RdRp genotype. This study aimed to explore the evolution of the GII.3 capsid gene, focusing on the influence of intergenic recombination. The capsid genes from six GII.3 norovirus strains, collected from Australian children between 2001 and 2010, were sequenced and aligned with 66 GII.3 capsid sequences from GenBank, spanning 1975 to 2010. The GII.3 capsid gene evolved at a rate of 4.16 × 10−3 to 6.97 × 10−3 nucleotide substitutions/site/year from 1975 to 2010 and clustered into five temporally sequential lineages. Clustering of the GII.3 capsid gene sequences was associated with intergenic recombination and switches between RdRp genotypes GII.3, GII.a, GII.b, GII.12, and an undefined ancestral RdRp. Comparison of the substitution rate of the GII.3 and GII.b RdRps suggested that RdRp switching allows a higher evolutionary rate, leading to increased genetic diversity and adaptability. Alignment of GII.3 capsid sequences revealed 36 lineage-specific conserved amino acid substitutions, four of which were under positive selection. Many conserved substitutions were within predicted antibody binding regions and close to host attachment factor binding sites. In conclusion, evolution of GII.3 noroviruses was primarily driven by intergenic recombination. The acquisition of new RdRps may lead to a faster mutation rate and increased genetic diversity, improving overall GII.3 fitness.