Identification of a new strain of mouse kidney parvovirus associated with inclusion body nephropathy in immunocompromised laboratory mice

Figures & data

Table 1. Nucleotide sequences of PCR primers and a Taqman probe for detecting MKPV.

Primer ID	Nucleotide sequence (5′→3′)	Strand
	Genome walking
F12	AGCAGTCATGTGTTGTTGTCCTTTGT	Sense
R13	ACGTTCCATTTGCGCTTGCATT	Antisense
F2b	ACCGGACACTTCCTTTGACAGCG	Sense
R2	AGCTGGTGAAATTCCTTGGTGTA	Antisense
F3	CATCCACCCGAGCCAGAAGCCAT	Sense
R3	TAGTTGAGGAGCCGGTGCTGCA	Antisense
F4	GCACCCACAGCCCAAGACCTAG	Sense
R4	CATGAGTATATGGCCATATG	Antisense
F5	ACAAAAATCTCAGTGGTTCATG	Sense
R5	TTGGTGCATAGATGCGGCTGCGT	Antisense
F10	TATGGACCTACACAATGCT	Sense
R15	GTTAACAATAAGATTTTGTATTGTG	Antisense
	5′- and 3′-ITR regions
F1	CTACGAAACTGGGCAGGGCGC	Sense
R16	CCTTTGCATGTGCGCGCACT	Antisense
F14	TTGTTAACATGTGTTAACGTTAAC	Sense
R12	GCATGGCGTACCACTGCCTGTA	Antisense
F15	GCATGTACCTCAACCATAAG	Sense
R14	CCACATGTTCTGTACGACT	Antisense
F16	CCCAGTCGTACAGAACA	Sense
R6	TTAACCCGCCTCCCAATCTTGGCT	Antisense
	PCR and qPCR
MKPV PCR-rF	TGTCAGATATAGAAGAACCAGATC	Sense
MKPV PCR-rR*	GGTGCTGCTTTCATGCAGTCTTCTA	Antisense
MKPV-qF	CCACCTCACAGATGCATACTATCA	Sense
MKPV-qProbe	FAM-AGCTGARATCACAGAAGTSATTATGCAACA-MGB	Sense
MKPV qR	The same as *	Antisense

Figure 1. IBN in the kidneys of B6.Il2rRag2W mice. (A) Representative H&E section of a kidney (400×; inset 20×) from an MKPV-negative mouse. (B) Longitudinal section of kidney from case 1 with chronic IBN and mild hydronephrosis (inset 20×) and mild multifocal neutrophilic pyelonephritis. The kidney has an undulating irregular renal cortex, containing multifocal to coalescing areas of interstitial fibrosis, tubular loss, and tubular degeneration with single cell necrosis, karyomegaly, and intranuclear amphophilic to eosinophilic inclusions (arrowheads: 400×). (C) Longitudinal section of kidney from case 2 with IBN and mild neutrophilic pyelonephritis. Affected convoluted tubules in the renal cortex had vacuolated and degenerated epithelial cells with karyomegalic nuclei and large (∼5–6 µm) amphophilic intranuclear inclusions or small (∼1 µm) densely eosinophilic intranuclear inclusions (arrowheads: 400×). (D) Longitudinal section of kidney from case 3 with moderate to severe IBN (inset 20×). High magnification of the kidney from case 3 exhibited areas of interstitial fibrosis and tubular degeneration with karyomegalic nuclei, chromatin margination and intranuclear inclusions (arrowheads: 400×). (E) MKPV in situ hybridization signals (RNAscope) in renal tubules of case 3 (inset 40×). MKPV nucleic acids were detected (fast red staining) in the nucleus and cytoplasm of affected tubular epithelial cells (400×). (F) Masson trichrome staining of an affected kidney (case 1) showed marked collagen deposition (blue staining) in the renal interstitium (400×; inset 20×).

Figure 2. Gene organization of MKPV MIT-WI1. The 4440 nt genome of MIT-WI1 with 5′ and 3′ ITRs contains ORFs which are predicted to encode NS1 (viral replicase), VP1 (capsid protein), accessory proteins p10, p15 and NP-L/I embodied in NS1. NS-2 is formed via alternative slicing which joins the N-terminal ORF (NS-2n in green) and the partial aa sequence of NP-L/I (in green) [5]. This gene organization is conserved among known MKPV/MuCPV strains [10]. There is a dinucleotide deletion at nucleotide 495 in the MIT-WI1 as well as MKPV MSKCC and MuCPV NYC compared with MKPV CI [10].

Table 2. Nucleotide sequence similarities between MIT-WI1 and known MKPV/MuCPV genomes.

Strain	Origin	Host	% nt Sequence similarity^a	Accession #
MKPV MIT-WI1	MIT and WI MA	Mice	100 (4440 nt. complete)^b	MT093738^d
MKPV MSKCC	Memorial Sloan Kettering Cancer Center NY	Mice	99.9 (4315 nt, partial)	NC_04083.1^e
MKPV CI	Centenary Institute Sydney Australia	Mice	98.2 (4442 nt, complete)	MH670588.2^e
MuCPV NYC	New York City	Wild Mus musculus	98.2 (4174 nt)	MF175078.1^f
MuCPV XJC	Xingjiang China	Rodents^c	94.5 (1165 nt, partial)	MG679365.1^g
TdCPV2	Tasmania Australia	Tasmanian devil	74.4 (4262 nt, partial)	MK513529.1^g

Notes: ^acompared with the MIT-WI1 genome.

^bNumber of nucleotides (nt) in each of either complete or partial genomes.

^cLee et al. decribed as wlid Mus musculus mice (10).

^dThis study.

^eRef. [5].

^fRef. [6].

^gUnpublished.

Table 3. Amino acid sequences (aa) similarities of the corresponing proteins between MIT-WI1 and other known MKPV/MuCPV.

Strain	% p10 (98 aa)^a	% p15 (130 aa)^a	% NS1 (659 aa)^a	% VP1 (496 aa)^a	% NS2 (295 aa)^a
MKPV MSKCC	100	100	99.7	99.8	100
MKPV CI	98.9	100	98.8	99.8	99.2
MuCPV NYC	N.A.	100	98.5	99.8	99.2
MuCPV XJC	N.A.	N.A.	98.4 (387aa, partial)^b	N.A.	N.A.
TdCPV2 Aus	N.A.	78.6 (132 aa)^b	72.1	60.7 (494 aa)^b	81.2 (294 aa)^b

Note: aa: amino acids; N.A.: not appliable.

^aParenthesis, numbers of aa deduced in the corresponding genes of the MKPV MIT-WI1 genome

^bParenthesis, numbers of deduced aa in the respective genes different from the MKPV MIT-WI1.

Figure 3. Development of PCR assays for detection of MKPV/MuPCV. A, NS1 regions of 331 nt among known MKPV/MuCPV strains were aligned with the DNASTAR Lasergene 13 package. The nucleotide sequences of conserved PCR primers and qPCR primers/probe were identified. Letters in red represent degenerate nucleotides distinct from the conserved ones. Number in parenthesis corresponds to the genomic position of MKPV CI. B, Validation of the MKPV qPCR assay. Values of the linear regression R² and interception demonstrates optimal amplification efficiency for the assay; its detection limit is ≤ 10 copies.

Table 4. MKPV status in selected mouse strains at MIT-WI animal facilities.

Location	Genotype	Age (months)	Application	Sample type	MKPV ^+ %^a
WI-A	UTX^flox	18–28	Experimental	Frozen Kidney	66.6% (4/7)^c
WI-B	Crl:CD1(ICR)	3.5	Sentinal	faecal	66%(2/3)^c
WI-B	C57BL/6 c-Kit^W–sh	4–10	Experimental	Frozen Kidney	33% (1/3)^c
				faecal, pooled	5.5% (1/18^b)
WI-C					21.4% (3/14^b)
WI-C	NSG	2.5–6		Frozen Kidney	71.4% (10/14)^c
WI-D	B6.Il2rRag2W	3.5–22		FFPE Kidney	95.5% (23/24)
WI-E	Rederived B6.Il2rRag2W	8–10		Frozen Kidney	0 (0/14)

Notes: ^anumbers in parenthesis represent number of MKPV⁺ per total sample numbers.

^bPer cage.

^cSanger sequencing of select PCR products.

Figure 4. Abundance of MKPV in kidney of immunodeficient mouse strains was significantly correlated with severity of IBN. (A) B6.Il2rRag2W, (B) NSG and (C) UTX^flox. Abundance of renal MKPV in mice was significantly correlated with severity of IBN in immunocompromised mice (A) and (B), but not with non-IBN renal pathology in immunocompetent mice (C). Pearson correlation was performed with a two-tailed test.

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