Extraintestinal spread and replication of a homologous EC rotavirus strain and a heterologous rhesus rotavirus in BALB/c mice

Abstract

Although rotavirus infection has generally been felt to be restricted to the gastrointestinal tract, over the last two decades there have been sporadic reports of children with acute or fatal cases of rotavirus gastroenteritis testing positive for rotavirus antigen and/or nucleic acid in various extraintestinal locations such as serum, liver, kidney, bladder, testes, nasal secretions, cerebrospinal fluid, and the central nervous system. Recently, studies in animals and people have demonstrated that rotavirus antigenemia is a common event during natural infection. In this study, we extend these observations and compare the intestinal and extraintestinal spread of wild-type homologous murine rotavirus EC and a heterologous strain, rhesus rotavirus (RRV), in newborn mice. A strand-specific quantitative reverse transcription-PCR (ssQRT-PCR) assay was used to quantify the ability of different rotavirus strains to spread and replicate extraintestinally. Both strain EC and RRV were detected extraintestinally in the mesenteric lymph nodes (MLN), livers, lungs, blood, and kidneys. Extraintestinal replication, as measured by ssQRT-PCR, was most prominent in the MLN and occurred to a lesser degree in the livers, kidneys, and lungs. In the MLN, strain EC and RRV had similar (P < 0.05) RNA copy numbers, although EC was present at a 10,000-fold excess over RRV in the small intestine. Rotavirus nonstructural protein 4 (NSP4) and/or assembled triple-layered particles, indicated by immunostaining with the VP7 conformation-dependent monoclonal antibody 159, were detected in the MLN, lungs, and livers of EC- and RRV-inoculated mice, confirming the ssQRT-PCR findings. Infectious RRV was detected in the MLN in quantities exceeding the amount present in the small intestines or blood. The cells in the MLN that supported rotavirus replication included dendritic cells and potentially B cells and macrophages. These data indicate that extraintestinal spread and replication occurs commonly during homologous and some heterologous rotaviral infections; that the substantial host range restrictions for rhesus rotavirus, a heterologous strain present in the intestine, are not necessarily apparent at systemic sites; that the level and location of extraintestinal replication varies between strains; that replication can occur in several leukocytes subsets; and that extraintestinal replication is likely a part of the normal pathogenic sequence of homologous rotavirus infection.

FIG. 1.

A 12-h rotavirus replication curve in MA104 cells inoculated with RRV at an MOI of 1. Bars: □, total (+)RNA copy numbers per ml; ▧, total (−)RNA copy numbers detected per ml. Asterisks mark the time points when a significant excess of total (+)RNA over (−)RNA was detected [(+)RNA/(−)RNA ratio of >1.58], indicating rotavirus replication. The triangles connected with the solid line indicate the RRV titer (in PFU/ml) determined by plaque assay. The x axis shows the hours postinoculation, and the y axis indicates the log₁₀ of both (+)RNA and (−)RNA copies/ml and PFU/ml.

FIG. 2 and 3.

(−)RNA and excess (+)RNA copy numbers detected in the feces, small intestines (SI), MLN, blood, livers, lungs, and kidneys in suckling BALB/c mice inoculated with EC or RRV. The black diamond indicates the (−)RNA copy numbers in the inoculum. The solid black dots (•) indicate the total (−)RNA copies/μg of tissue or feces in an individual animal. The black bar indicates the geometric mean of the data set on each day. The asterisks indicate the time points when rotavirus replication [defined as a significant excess of (+)RNA] was detectable. The “‡” symbol indicates that the mean (−)RNA copy numbers are significantly (P < 0.05) higher compared to the same day and tissue in the other inoculation group (EC versus RRV). Groups were evaluated by a two-sample t test. Significance was established if the P value was <0.05. The x axes show the days postinoculation, and the y axes indicate the log₁₀ of the (−)RNA copy numbers per microgram of tissue.

FIG. 3.

FIG. 4.

Whole blood drawn at 3 dpi from four 5-day-old mice inoculated with a 10⁶ ID₅₀ of EC rotavirus. Plasma was separated from the leukocytes, RNA was extracted from the plasma and PBMC fractions, and an ssQRT-PCR assay was used to determine the (−)RNA and excess (+)RNA rotavirus copy numbers in the plasma and leukocyte fractions. The results are reported in rotavirus (−)RNA copies/μg of whole blood. Significant excess (+)RNA was not detected.

FIG. 5.

Serial sections from MLN and lungs were collected from 10⁴ ID₅₀ EC- and 10⁷ PFU RRV-infected and noninfected controls. Rotavirus replication was detected with biotinylated MAbs to rotavirus NSP4 (B4-2) (27) and to VP7 on assembled rotavirus triple-layered particles (MAb 159) (12) and developed with a secondary streptavidin-Texas Red antibody. The cellular nucleus was stained blue with TOTO-3. NSP4 stained EC-infected MLN (A1), RRV-infected MLN (A2), and uninfected MLN (A3). 159 stained EC-infected MLN (B1), RRV-infected MLN (B2), and uninfected MLN (B3). NSP4 stained EC-infected lung (C1), RRV-infected lung (C2), and uninfected control lung (C3). MAb 159 stained RRV-infected liver at ×40 (D1) and ×100 (D2) magnifications. (D3) MAb 159 stained uninfected liver.

FIG. 6.

Cytospin of mechanically disrupted MLN collected 3 dpi from mice inoculated with a 10⁶ ID₅₀ EC when 5 days old. (A1) Merged photograph of NSP4 (Texas Red) (A2) and monoclonal antibody to B220 (FITC) (A3), cell surface markers for B cells and plasmacytoid DC. (B1) Merged photograph of NSP4 (Texas Red) (B2) and monoclonal antibody to CD11b (FITC) (B3), cell surface markers for macrophages, granulocytes, NK cells, and a subset of DC. (C1) Merged photograph of NSP4 (Texas Red) (C2) and monoclonal antibody to CD11c (FITC) (C3), cell surface markers for DC. (D1) Merged photograph of NSP4 (Texas Red) (D2) and monoclonal antibody to CD3 (FITC) (D3), cell surface markers for T cells.