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Genetics and pathogenesis of feline infectious peritonitis virus.

Brown MA, Troyer JL, Pecon-Slattery J, Roelke ME, O'Brien SJ - Emerging Infect. Dis. (2009)

Bottom Line: Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP.These data demonstrate distinctive circulating virulent and avirulent strains in natural populations.These findings may have potential as diagnostic markers for virulent FIP-associated FCoV.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland 21702, USA. brownmer@gmail.com

ABSTRACT
Feline coronavirus (FCoV) is endemic in feral cat populations and cat colonies, frequently preceding outbreaks of fatal feline infectious peritonitis (FIP). FCoV exhibits 2 biotypes: the pathogenic disease and a benign infection with feline enteric coronavirus (FECV). Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP. To resolve these alternative hypotheses, we isolated viral sequences from FCoV-infected clinically healthy and sick cats (8 FIP cases and 48 FECV-asymptomatic animals); 735 sequences from 4 gene segments were generated and subjected to phylogenetic analyses. Viral sequences from healthy cats were distinct from sick cats on the basis of genetic distances observed in the membrane and nonstructural protein 7b genes. These data demonstrate distinctive circulating virulent and avirulent strains in natural populations. In addition, 5 membrane protein amino acid residues with functional potential differentiated healthy cats from cats with FIP. These findings may have potential as diagnostic markers for virulent FIP-associated FCoV.

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Related in: MedlinePlus

A) Feline coronavirus genome indicating PCR products obtained (bars). Structural proteins are shaded in dark gray; nonstructural proteins are shaded in light gray. B) Forward and reverse primers used to amplify virus segments are listed in 5′ → 3′ orientation. The number of source cats and cloned sequences generated from feline infectious peritonitis (FIP) cases and feline enteric coronavirus (FECV) asymptomatic cats are presented. Pol, polymerase; NSP, nonstructural protein; FIPV, feline infectious peritonitis virus.
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Figure 3: A) Feline coronavirus genome indicating PCR products obtained (bars). Structural proteins are shaded in dark gray; nonstructural proteins are shaded in light gray. B) Forward and reverse primers used to amplify virus segments are listed in 5′ → 3′ orientation. The number of source cats and cloned sequences generated from feline infectious peritonitis (FIP) cases and feline enteric coronavirus (FECV) asymptomatic cats are presented. Pol, polymerase; NSP, nonstructural protein; FIPV, feline infectious peritonitis virus.

Mentions: Primers amplifying 7b (736 bp), membrane protein (575 bp), polymerase (386 bp), and spike-NSP3 (1,017 bp) (Figure 3) were designed based on available feline coronavirus sequence (1,12,13). PCR was performed by using 2 μL of cDNA in a 50-μL reaction containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 0.25 mmol/L concentrations of dNTPs (dATP, dCTP, dGTP, and dTTP), 2 mmol/L concentrations of each primer, and 2.5 units of Platinum Taq DNA polymerase (Invitrogen). PCR was conducted on a geneAmp PCR system 9700 thermocycler (Applied Biosystems, Foster City, CA, USA) with the following touchdown conditions: 2 min at 94ºC then a touchdown, always starting with 20 sec at 94ºC, then 30 sec at 60ºC (3 cycles), 58ºC (5 cycles), 56ºC (5 cycles), 54ºC (5 cycles), 52ºC (22 cycles), and then 1 min at 72ºC for extension, and with a final extension at 72ºC for 7 min and a hold at 4ºC. PCR products were visualized by electrophoresis on a 1% agarose gel and primers and unincorporated dNTPs were removed by using Microcon YM (Millipore, Billerica, MA, USA).


Genetics and pathogenesis of feline infectious peritonitis virus.

Brown MA, Troyer JL, Pecon-Slattery J, Roelke ME, O'Brien SJ - Emerging Infect. Dis. (2009)

A) Feline coronavirus genome indicating PCR products obtained (bars). Structural proteins are shaded in dark gray; nonstructural proteins are shaded in light gray. B) Forward and reverse primers used to amplify virus segments are listed in 5′ → 3′ orientation. The number of source cats and cloned sequences generated from feline infectious peritonitis (FIP) cases and feline enteric coronavirus (FECV) asymptomatic cats are presented. Pol, polymerase; NSP, nonstructural protein; FIPV, feline infectious peritonitis virus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2819880&req=5

Figure 3: A) Feline coronavirus genome indicating PCR products obtained (bars). Structural proteins are shaded in dark gray; nonstructural proteins are shaded in light gray. B) Forward and reverse primers used to amplify virus segments are listed in 5′ → 3′ orientation. The number of source cats and cloned sequences generated from feline infectious peritonitis (FIP) cases and feline enteric coronavirus (FECV) asymptomatic cats are presented. Pol, polymerase; NSP, nonstructural protein; FIPV, feline infectious peritonitis virus.
Mentions: Primers amplifying 7b (736 bp), membrane protein (575 bp), polymerase (386 bp), and spike-NSP3 (1,017 bp) (Figure 3) were designed based on available feline coronavirus sequence (1,12,13). PCR was performed by using 2 μL of cDNA in a 50-μL reaction containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 0.25 mmol/L concentrations of dNTPs (dATP, dCTP, dGTP, and dTTP), 2 mmol/L concentrations of each primer, and 2.5 units of Platinum Taq DNA polymerase (Invitrogen). PCR was conducted on a geneAmp PCR system 9700 thermocycler (Applied Biosystems, Foster City, CA, USA) with the following touchdown conditions: 2 min at 94ºC then a touchdown, always starting with 20 sec at 94ºC, then 30 sec at 60ºC (3 cycles), 58ºC (5 cycles), 56ºC (5 cycles), 54ºC (5 cycles), 52ºC (22 cycles), and then 1 min at 72ºC for extension, and with a final extension at 72ºC for 7 min and a hold at 4ºC. PCR products were visualized by electrophoresis on a 1% agarose gel and primers and unincorporated dNTPs were removed by using Microcon YM (Millipore, Billerica, MA, USA).

Bottom Line: Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP.These data demonstrate distinctive circulating virulent and avirulent strains in natural populations.These findings may have potential as diagnostic markers for virulent FIP-associated FCoV.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland 21702, USA. brownmer@gmail.com

ABSTRACT
Feline coronavirus (FCoV) is endemic in feral cat populations and cat colonies, frequently preceding outbreaks of fatal feline infectious peritonitis (FIP). FCoV exhibits 2 biotypes: the pathogenic disease and a benign infection with feline enteric coronavirus (FECV). Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP. To resolve these alternative hypotheses, we isolated viral sequences from FCoV-infected clinically healthy and sick cats (8 FIP cases and 48 FECV-asymptomatic animals); 735 sequences from 4 gene segments were generated and subjected to phylogenetic analyses. Viral sequences from healthy cats were distinct from sick cats on the basis of genetic distances observed in the membrane and nonstructural protein 7b genes. These data demonstrate distinctive circulating virulent and avirulent strains in natural populations. In addition, 5 membrane protein amino acid residues with functional potential differentiated healthy cats from cats with FIP. These findings may have potential as diagnostic markers for virulent FIP-associated FCoV.

Show MeSH
Related in: MedlinePlus