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.

Importance: Serial 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.

FIG 1

Schematic diagram of serial passage history in the present study. Murine RV EB strain was passaged serially in 4- to 5-day-pld mouse pups (CD-1 or BALB/c; in vivo) or in cell cultures (1⁰ AGMK cell or MA104 cell line; in vitro) alternately and repeatedly. With regard to in vivo passage, eight pups were inoculated orally and inspected daily for diarrhea by gentle palpation of their abdomens. When diarrhea was observed, infected pups were euthanized. In cases of no diarrhea, the animals were euthanized at 96 h postinfection. Thereafter, the intestines of a selected mouse were homogenized, and 50 μl of 10% homogenates diluted with PBS was used as a next-passage inoculum. With regard to in vitro passage, when ca. 75% of the infected cells displayed CPE within 3 to 5 days postinfection, the cultures were frozen and thawed once. Afterward, the lysates were passaged undiluted or diluted 10⁻¹ to 10⁻⁵ (100-μl aliquots). Po indicates original murine RV EB strain. It was adapted to grow in 1⁰ AGMK cell roller tube cultures and subsequently plaque purified on a secondary AGMK cell monolayer. After plaque purification, murine RV EB strain was passaged serially in MA104 cell roller tube cultures 10 to 20 times. Capital (A, B, C, E, G, and R) and lowercase (aa, bb, cc, gg, ii, jj, kk, and ll) letters indicate mouse- and cell culture-passaged viruses. Numbers (7, 8, 17, 18, 27, and 40) indicate the passage level. kk18-pl-4-3-1-1 was plaque purified kk18 consecutively four times in MA104 cells.

FIG 2

Summary of mutations detected in more than three virus samples during serial passages in mice or in cell cultures. “nt” and “aa” indicate nucleotides and amino acids, respectively. A blue background indicates nonsynonymous substitution. Yellow and gray backgrounds indicate mutations only detected in mouse-passaged virus and in cell culture-passaged virus, respectively. More than 10 mutations detected both in mice and cell cultures are not presented in this figure. All of the mutations detected in eleven genes are summarized in Fig. 3A to K, respectively.

FIG 3

Summary of mutations and substitutions detected in each of 11 genes (VP1 to VP4, VP6, VP7, and NSP1 to NSP5/6 [A to K, respectively]) during serial passages in mice and/or in cell cultures. “nt” and “aa” indicate nucleotides and amino acids, respectively. Blue and yellow-green backgrounds indicate nonsynonymous substitutions and untranslated regions, respectively. Yellow, gray, and pink backgrounds indicate mutations only detected in mouse-passaged virus, in cell culture-passaged virus, or both, respectively. ND, no sequencing data.

FIG 3

Summary of mutations and substitutions detected in each of 11 genes (VP1 to VP4, VP6, VP7, and NSP1 to NSP5/6 [A to K, respectively]) during serial passages in mice and/or in cell cultures. “nt” and “aa” indicate nucleotides and amino acids, respectively. Blue and yellow-green backgrounds indicate nonsynonymous substitutions and untranslated regions, respectively. Yellow, gray, and pink backgrounds indicate mutations only detected in mouse-passaged virus, in cell culture-passaged virus, or both, respectively. ND, no sequencing data.

FIG 3

Summary of mutations and substitutions detected in each of 11 genes (VP1 to VP4, VP6, VP7, and NSP1 to NSP5/6 [A to K, respectively]) during serial passages in mice and/or in cell cultures. “nt” and “aa” indicate nucleotides and amino acids, respectively. Blue and yellow-green backgrounds indicate nonsynonymous substitutions and untranslated regions, respectively. Yellow, gray, and pink backgrounds indicate mutations only detected in mouse-passaged virus, in cell culture-passaged virus, or both, respectively. ND, no sequencing data.

FIG 4

Schematic diagram of substitutions detected on VP4 (A), VP7 (B), and NSP4 (C). Numbers indicate the amino acid position on each of three genes (1, 22, 34–36). Red, blue, and green arrowheads indicate substitutions detected in murine RVs passaged in mouse, cell culture, or both, respectively. Big, middle, and small arrowheads indicate substitutions occurring, respectively, in >5, in 3 to 4, or in 1 to 2 strains detected during serial passages in mice and/or in cell cultures. (A) Substitution at aa 538 detected in both passages in mice and passages in cell cultures (see Fig. 3J).