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A CASE STUDY OF LETHAL CPV NEW VARIANTS INFECTION

Mirela Popa, Virgilia Popa, Miliana Petrof, Nicolae Alexandru, Marian Culcescu, Mihai Visan National Society Pasteur Institute, Bucharest, Romania

Introduction. Canine parvoviruses (CPV) include four - evolutionary closely related - generations: genotype 2 (vaccinal -1978), old variants - 2a/b (after 1980), new 2a/b variants (and simultaneously 2c - 2000) and the latest ones 2c(a/b). These viruses cause different serious diseases, especially in young dogs, since they prefer replication in rapidly dividing cells. Generally, CPV recombination and high genetic heterogeneity is the result of super infection or co-infection with multiple strains. In the light of the importance of multi-infections as potentially source of CPV genetic diversity the present study has explored the molecular characteristics of parvovirus strains circulating in a Romanian kennel. Materials and methods. 1. necropsy and histopathological examination (Masson) of Limier breed dogs; 2. total DNA extraction by silica-gel membrane binding samples ante- and post-mortem; 3. CPV genomes classification (isolate vs. vaccinal strain) by TaqMan reaction (VP2 ORF); 4. CPV genomes characterization (type of variant) by RFLP of the partial VP2 and integral NS1 ORFs. Results. 1. Macroscopic level - strong dehydration, hemorrhagic enteritis, large mesenteric lymph nodes and gallbladder. Histhopathological observations - catarrhal enteritis, lyses of intestinal villi, rare intranuclear inclusions of epithelial cells and hemorrhagic pneumonia.

Catarrhal enteritis, lyses of intestinal villi. HEAx 400 Cathar with rare intranuclear inclusions of oustanding Hemorrhagic pneumonia. Lung. HEA x 400 intestinal epithelial cells. HEA 400x

3. Ubiquitous CPV2 variant detection - beyond CPV genotype 2 vaccination.


A1, NFW+mix Fam, FAM

A4, PPV+mix Fam, FAM

A8, 7770int.+mix Fam, FAM

A10, 7771int.+mix Fam, FAM

A12, CPV2/PRV-S2 1/10.000, FAM

B8, 7770dil.1/10+mix Fam, FAM


5.6

Real Time PCR Taqman. Canine parvovirus type 2 (CPV2). 3.5 months dogs faeces. Thermal cycler Mx3005P, Stratagene. Primers, probes , cycling program - Decaro 2006. Brilliant II Multiplex QPCR Master Mix, Agilent.
1.93 1.73 1.53

A1, NFW, HEX

A1, NFW, FAM

A3, PPV-neg.fam, HEX

A3, PPV-neg.fam, FAM

A4, PPV-neg.hex, HEX

Real Time PCR Taqman. Canine parvovirus type 2 (CPV2). 3 months dog lung, spleen, intestine. Thermal cycler Mx3005P, Stratagene. Primers, probes sequences, cycling program - Decaro 2006. Brilliant II Multiplex QPCR Master Mix, Agilent.

A4, PPV-neg.hex, FAM

B10, 7771 dil.1/10+mix Fa, FAM

A8, CPV2-PRV-S2 dil.1/10, HEX

D8, 7770int.+mix Hex, HEX

A8, CPV2-PRV-S2 dil.1/10, FAM

Fluorescence (dRn)

4.6

D10, 7771int.+mix Hex, HEX

A10, CPV2-PRV-S2 dil.1/10, HEX

Fluorescence (dRn)

E1, NFW+mix Hex, HEX


3.6

A10, CPV2-PRV-S2 dil.1/10, FAM

E4, PPV+mix Hex, HEX

1.33 1.13 0.93 0.73 0.53 0.33 0.13

A12, CPV2-PRV-S2 dil.1/1., HEX

E8, 7770 dil.1/10+mix He, HEX


2.6

A12, CPV2-PRV-S2 dil.1/1., FAM

E10, 7771 dil.1/10+mix He, HEX

C8, 1697-pulmon, HEX

E12, CPV2/PRV-S2 1/10.000, HEX


1.6

C8, 1697-pulmon, FAM

C10, 1697-splina, HEX

C10, 1697-splina, FAM

0.6

C12, 1697-intestin, HEX

C12, 1697-intestin, FAM

-0.4 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45

-0.07 1 11 21 31 41

Cycles

Cycles

4. RFLP analysis allowed identification of a new CPV2 genotype - an intermediate of the 2a/b new variants.

Vaccinal CPV2 VP2 680bp CPV2 NS1 2007bp L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 L3 1 2 3 4 5 6 7 8 9 10 11 12 13 14

AluI digestion of 2007bp CPV2 NS1 CPV2 VP2 510bp 724bp 450bp AlwI digestion of 510bp CPV2 VP2 attenuated 466bp 289bp 177bp L1 1 357bp 221bp no 523/201bp no 2b new no 499/225bp no 2c(a/b) 2 4 6 7 8 9 10 11 12 165bp 90bp 13 L3

virulent

RsaI digestion of 2007bp CPV2 NS1 L1 1 2 3 4 5 6 7 8 9 10 11 44bp 12 no 534bp no 2c(a/b) no 312bp no 2a new, no 2c 427bp 368bp 266bp 216bp no 534bp 168/166bp no 2c(a/b) no 312bp no 2a new, no 2c 132bp 96bp 1

AluI digestion of 510bp CPV2 VP2

384bp old variant 154bp new variant 93bp 61bp 6 7 8 9 genotype 2 84bp 70bp 10 11 12 L2

* * 7

* *

150bp 111bp

L1

L1

57bp 9 * 10 11 12 * 13 L3 Legend: L, ladder; L1, 50bp; L2, low range; L3, 1kbp Plus; 1, faeces; 2, intestine; 3, gallbladder; 4, spleen; 5, heart; 6, faeces; 7, intestine; 8, spleen; 9, lung; 10, attenuated strain; 11/12, old variants; 13, attenuated strain, passage 3. Dog I samples 1-5; dog II samples 6-9. L1 2 4 6

Conclusions. (a) CPV mixed infection with a true member of the new generations and (b) lack of protection after genotype 2 vaccinations. This study adds new data about the evolutionary dynamics of CPV in vaccinated dogs, confirming the CPV re-adaptation in its canine hosts.

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