Infectivity and Shedding of Mouse Kidney Parvovirus After Oronasal Inoculation of C57BL/6, CD1, and NSG Mice

Abstract

Mouse kidney parvovirus (MKPV), the etiology of murine inclusion body nephropathy, has been identified globally in mice used for research, with an estimated prevalence of 10% in academic colonies. In immunodeficient strains, MKPV causes significant morbidity and mortality, and severe renal pathology. In contrast, in immunocompetent mice, the infection is subclinical and causes minimal pathology. We investigated viral infectivity and shedding in inbred C57BL/6NCrl (B6), outbred Crl:CD1(ICR) (CD1), and highly immunocompromised NOD. Cg - Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice. Four doses, ranging from 1.16 × 10 3 to 1.16 × 10 6 viral copies per microliter, of an MKPV inoculum were administered oronasally to 3 mice per dose per mouse type. All 3 types (B6, CD1, and NSG) had persistent infection with prolonged shedding in urine and feces. Viral copy number in the urine generally increased over time, while shedding in the feces was more variable. Among the 3 populations, CD1 mice developed viral shedding in urine earliest (4 wk after inoculation) and at higher levels (greater than 1 × 10 7 viral copies per microliter). B6 mice become viruric later (7 wk after inoculation), with lesser virus shed (1 × 10 6 viral copies per microliter or less). In CD1 and B6 mice, peak urine shedding occurred at 11 to 14 wk after inoculation, after which levels gradually declined until 35 wk after inoculation (study endpoint). In contrast, NSG mice did not become viruric until 10 wk after inoculation and continued to shed large amounts of virus (greater than 1 × 10⁷ viral copies per microliter) in urine until the study endpoint. Two commercial immunofluorescent serologic assays failed to detect serum antibodies to MKPV nonstructural protein 1 as late as 58 wk after inoculation, whereas immunohistochemistry of infected renal tissue successfully detected anti-MKPV serum antibodies. These results increase our knowledge of the biology of MKPV and have practical application for development of effective screening programs for this pathogen.

Figure 1.

(A) Relative viral copies (no. per microliter; real-time PCR analysis) in the urine until 35 wk after inoculation. Data points reflect the mean (± 1 SD) for all mice of the indicated population that received any of the 4 MKPV doses at that time point (n = 4 samples for each mouse type). Data underwent logarithmic transformation prior to graphing. (B) Relative total viral load, reported as area under the curve (VL-AUC). VL-AUC differed significantly (P < 0.05) between NSG and B6 mice.

Figure 2.

Relative viral copies (no. per microliter; real-time PCR analysis) in the urine until 35 wk after inoculation for each inoculum dose for (A) CD1, (B) B6, and (C) NSG mice. Each data point represents a single pooled sample from 3 mice.

Figure 3.

(A) Relative viral copies (no. per microliter; real-time PCR analysis) in the feces until 35 wk after inoculation. Data points reflect the mean (± 1 SD) for all mice of the indicated population that received any of the 4 MKPV doses at that time point (n = 4 samples per stock or for each mouse type). Data has undergone logarithmic transformation prior to graphing. (B) Relative total viral load, calculated as area-under-the-curve (VL-AUC). VL-AUC differed significantly (P < 0.05) between NSG and CD1 mice and between NSG and B6 mice.

Figure 4.

Relative viral copies (no. per microliter; real-time PCR analysis) in the feces until 35 wk after inoculation for each inoculum dose. (A) CD1 mice. (B) B6 mice. (C) NSG mice. Each data point represents a single sample randomly collected from a group of 3 mice.

Figure 5.

Kidney tissues from (A–C) an NSG mouse naturally infected with MKPV and (D–F) an MKPV-negative NSG mouse. (A and D) Hematoxylin and eosin staining. (B and E) Immunohistochemistry results using serum (dilution, 1:100,000) from a CD1 mouse collected at 58 wk after the administration of a 1:10,000 MKPV inoculum. (C and F) Immunohistochemistry results using serum (dilution, 1:100,000) from an experimentally naive MKPV-negative CD1 mouse. Panels A through C show typical histologic evidence of inclusion body nephropathy, including tubular cells with enlarged nuclei containing inclusions, visible with both staining methods (arrows), whereas samples of MKPV-negative tissue (D through F) are histologically normal. The MKPV-positive tissue is characterized by strong immunoreactivity (brown chromogen) located in enlarged nuclei, with milder immunoreactivity in the cytoplasm of the same tubules. Images are representative of positive immunohistochemistry results listed in Table 1. Scale bar, 20 μm.