Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

Abstract

Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3= consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCESerial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. R otaviruses, which form one genus within the family Reoviridae, are divided into at least seven species/groups (A to G/H) (1, 2). Although group A to C rotaviruses have been detected in humans with diarrhea, group A rotavirus is the single most important etiologic agent causing severe diarrhea in infants and young children worldwide, resulting in approximately 453,00 deaths (37% of deaths attributable to diarrhea and 5% of all deaths) among children Ͻ5 years of age in 2008 (3). In the United States alone, rotavirus (RV) infections are estimated to cause approximately 20 to 30 deaths, 50,000 to 67,000 hospitalizations, 390,000 to 410,000 physician visits, and a more than $890 million to $1 billion societal cost annually (4, 5). Thus, the introduction of a RV vaccine capable of alleviating this enormous health burden has been an important global public health goal.The RV genome consisting of 11 segments of double-stranded RNA (dsRNA) encodes six structural protein...

Comparative genomic analysis of genogroup 1 (Wa-like) rotaviruses circulating in the USA, 2006–2009

“…Whereas the glycosylation site is thought to facilitate viral growth in cell culture systems, the function of the integrin binding site is unknown (Graham et al, 2005). The N238D mutation, however, appears in virulent murine RVA strains produced by serial passage of avirulent RVA strains in mice (Tsugawa et al, 2014). It is interesting to note that even the Rotarix™ VP7 protein contains the same glycosylation site at position.…”

“…Out of the 3, H/Y385D substitution at position 385, a neutralization escape site, was non-conservative with a change in charge. Amino acid residues 382 – 400 in the VP5* region also contain a potential membrane interaction loop and may be essential for viral virulence, especially in facilitating viral attachment or penetration (Dormitzer et al, 2004; Trask et al, 2010), In a cell culture-murine model system, a charge change at this position has been shown to be associated with reversion of an avirulent strain to a virulent one (Tsugawa et al, 2014). It is interesting to note that these substitutions are present in all sequences except the Allele A strains.…”

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain

“…In this study, we investigated a change in population structure during serial passages (bottleneck events) using rhesus rotavirus (RRV) as a model virus. Rotavirus, a doublestranded RNA (dsRNA) virus with 11 genome segments, exists as quasispecies (15) and is transmitted via the fecal-oral route and causes severe diarrhea among children under five years old. Despite recommendations to vaccinate and improve hygiene (10), rotavirus outbreaks have occurred sporadically both in developed and developing countries (11)(12)(13)(14).…”

Molecular characterization of the rotavirus enterotoxin NSP4 gene of strains causing diarrhoea in children aged 0-5 years in northern India

“…The findings have further reinforced its role as a toxin and virulence factor (Tian et al, 1994;Dong et al, 1997;Hagbom et al, 2011;Halaihel et al, 2000). Some studies have associated mutations in the gene with altered virulence (Zhang et al, 1998, Ball et al, 2013Tsugawa et al, 2014). NSP4 is also being considred as a vaccine candidate (Yu J and Langridge 2001).…”