Evolution of Human Calicivirus RNA In Vivo:Accumulation of Mutations in the Protruding P2 Domain of the CapsidLeads to Structural Changes and Possibly a NewPhenotype

Abstract

In the present study we report on evolution of calicivirus RNA from a patient with chronic diarrhea (i.e., lasting >2 years) and viral shedding. Partial sequencing of open reading frame 1 (ORF1) from 12 consecutive isolates revealed shedding of a genogroup II virus with relatively few nucleotide changes during a 1-year period. The entire capsid gene (ORF2) was also sequenced from the same isolates and found to contain 1,647 nucleotides encoding a protein of 548 amino acids with similarities to the Arg320 and Mx strains. Comparative sequence analysis of ORF2 revealed 32 amino acid changes during the year. It was notable that the vast majority of the cumulative amino acid changes (8 of 11) appeared within residues 279 to 405 located within the hypervariable domain (P2) of the capsid protein and hence were subject to immune pressure. An interesting and novel observation was that the accumulated amino acid changes in the P2 domain resulted in predicted structural changes, including disappearance of a helix structure, and thus a possible emergence of a new phenotype. FUT2 gene polymorphism characterization revealed that the patient is heterozygous at nucleotide 428 and thus Secretor ؉ , a finding in accordance with the hypothesis of FUT2 gene polymorphism and calicivirus susceptibility. To our knowledge, this is the first report of RNA evolution of calicivirus in a single individual, and our data suggest an immunity-driven mechanism for viral evolution. We also report on chronic virus excretion, immunoglobulin treatment, and modification of clinical symptoms; our observations from these studies, together with the FUT2 gene characterization, may lead to a better understanding of calicivirus pathogenesis.Acute diarrheal diseases are common in humans and are associated with significant morbidity worldwide and substantial mortality in developing countries. In recent years, caliciviruses have emerged as an important cause of gastroenteritis in all age groups and are now recognized as the main cause of gastroenteritis in nursing homes and hospitals (3,8,12,14,15,27).Caliciviruses are classified into four genera: norovirus, sapovirus, vesivirus, and logovirus. Human caliciviruses are found within the norovirus and sapovirus genera, which contain a wide range of genetically distinct strains. The sequence heterogeneity of these strains may be a result of the high mutation rate caused by the lack of proofreading of the polymerase. However, the sequence variation has also raised the possibility that calicivirus RNA undergoes constant genetic drift through immune-response-driven mechanisms, leading to the emergence of new strains. However, at present there is no information on human calicivirus mutation rates over time, nor has the importance of immune-response-driven mutations been reported.The clinical features of a calicivirus infection includes an incubation period of 12 to 48 h characterized by acute onset of nausea, vomiting, abdominal cramps and diarrhea that generally lasts for about 48 h. Recent studies have shown, howe...

Quasispecies dynamics and molecular evolution of human norovirus capsid P region during chronic infection

“…A break point between early isolates obtained between the autumn/winter 2000 (isolates P1, P3 and P5) and late isolates obtained during spring to autumn 2001 (isolates P11, P13 and P15) (Fig. 1) was observed that coincides with recorded changes of clinical symptoms of the host (Nilsson et al, 2003). Isolates obtained between January and March year 2001 (P7 and P9) are located within this break point, separating the early and late isolates.…”

“…In this study faecal samples were collected from August 2000 to March 2003 (termed P1 to P17). These samples have previously been investigated for NoV and found to be positive by electron microscopy and PCR (Nilsson et al, 2003).…”

“…The sample donor is an immunosuppressed patient who became infected with NoV in June 2000, and developed a chronic infection with four to eight episodes of diarrhoea per day lasting for several years. Early in the illness the patient also suffered from nausea and vomiting, which disappeared in later course of the infection (Nilsson et al, 2003). In this study faecal samples were collected from August 2000 to March 2003 (termed P1 to P17).…”

Genetic Analysis of Noroviruses in Chiba Prefecture, Japan, between 1999 and 2004

“…Recently, the in vivo evolution of NV in an immunosuppressed patient was reported by Nilsson et al (25). They reported an accumulation of amino acid substitutions in the P2 region within 1 year and also discussed the predicted structural changes that occurred in the P region.…”

“…PCR was performed with the G1F1 and G1R1 primer pair for the GI strains and the G2F1 and G2R1 primer pair for the GII strains (17). For detection of genetically divergent GII strains, primer G4R2 (CCNGCTGTGAASGCRTTNCCMGC) was used in place of primer G2R1, and for amplification of the 3Ј end of the NV genome, primer dT25VN [(T) 25 V(A/G/C)N(A/G/C/T)] (19) was used as the reverse primer. Primer LVPF (AGTCTCYTGTCGAGTYCTCAC) and primer LVCAPEND (CCAAGGACATCAGAYGCCA) were used to analyze the P region of the GII-4 genotype.…”