Limits...
Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

Kim Y, Liu H, Galasiti Kankanamalage AC, Weerasekara S, Hua DH, Groutas WC, Chang KO, Pedersen NC - PLoS Pathog. (2016)

Bottom Line: The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available.Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent.These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, United States of America.

ABSTRACT
Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.

Show MeSH

Related in: MedlinePlus

Changes in the viral RNA levels in P15 and P16 before and during antiviral treatment.(A and B) The viral RNA fold changes in the macrophages from the ascites of P15 (A) and P16 (B) over time are shown. The Ct values from viral RNA real-time qRT-PCR were normalized to β-actin and the 2-ΔΔCt method was used to calculate the relative change in viral RNA level, compared to the pre-treatment value. (C) The viral RNA level (2-ΔCt) in the omentum of P15 and P16 which are collected after 4 and 7 days of antiviral treatment, respectively. The bar graph shows the 2-ΔCt values calculated by normalizing the Ct values from viral RNA real-time qRT-PCR to β-actin. (D) The 2-ΔCt values for each viral RNA in the macrophages from the ascites of P15 and P16 at pre-treatment and during treatment are listed in the table. N/A, not available.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814111&req=5

ppat.1005531.g004: Changes in the viral RNA levels in P15 and P16 before and during antiviral treatment.(A and B) The viral RNA fold changes in the macrophages from the ascites of P15 (A) and P16 (B) over time are shown. The Ct values from viral RNA real-time qRT-PCR were normalized to β-actin and the 2-ΔΔCt method was used to calculate the relative change in viral RNA level, compared to the pre-treatment value. (C) The viral RNA level (2-ΔCt) in the omentum of P15 and P16 which are collected after 4 and 7 days of antiviral treatment, respectively. The bar graph shows the 2-ΔCt values calculated by normalizing the Ct values from viral RNA real-time qRT-PCR to β-actin. (D) The 2-ΔCt values for each viral RNA in the macrophages from the ascites of P15 and P16 at pre-treatment and during treatment are listed in the table. N/A, not available.

Mentions: Since FIPV is highly associated with tissues and is not reliably detected in blood at high levels in cats with FIP [17], assessment of the efficacy of antiviral drugs in reducing the viral load poses a difficulty in live animals. Although measuring virus titers of the exudate macrophages from the ascites allows to determine the effects of antiviral drug against the replication of FIPV, ascites rapidly decreased with antiviral treatment and we were not able to collect ascites in the recovered cats. However, we determined the viral load in two cats from the second study (P15 and P16) prior to and during antiviral treatment. These cats were euthanized after 4 and 7 days of antiviral treatment. On necropsy, both cats had severe pancreatitis, a possible complication of meloxicam treatment, but no lesions (P16) or mild lesions (P15) typical of FIP were found. Virus titers in the macrophages from the ascites were determined by real-time quantitative RT-PCR and the Ct values were analyzed by the comparative Ct method using the β-actin as a reference gene [39]. The results showed that viral RNA level in the macrophages from the ascites decreased commensurately with the duration of antiviral treatment in these cats. The fold reduction of viral RNA level determined using the delta delta Ct method was 1,595.7 in P15 that received 4 day-antiviral treatment (Fig 4A) and 171,755.9 in P16 that received 7 day-antiviral treatment (Fig 4B), compared to the pre-treatment viral RNA level in the macrophages of each cat. The viral RNA levels (2-ΔCt) in the macrophages from the ascites are summarized in Fig 4D. The viral RNA level in the omentum of P15 and P16 is also shown in Fig 4C. Based on these results, the reduction in virus titers in P15 and P16 seems to correlate with the necropsy findings of mild or no FIP lesions in those cats. These results on viral titers show that FIPV 3CLpro is a valid target for FIPV antiviral drugs and GC376 can effectively reduce the virus load in the macrophages from the ascites and the omentum of cats with FIP.


Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

Kim Y, Liu H, Galasiti Kankanamalage AC, Weerasekara S, Hua DH, Groutas WC, Chang KO, Pedersen NC - PLoS Pathog. (2016)

Changes in the viral RNA levels in P15 and P16 before and during antiviral treatment.(A and B) The viral RNA fold changes in the macrophages from the ascites of P15 (A) and P16 (B) over time are shown. The Ct values from viral RNA real-time qRT-PCR were normalized to β-actin and the 2-ΔΔCt method was used to calculate the relative change in viral RNA level, compared to the pre-treatment value. (C) The viral RNA level (2-ΔCt) in the omentum of P15 and P16 which are collected after 4 and 7 days of antiviral treatment, respectively. The bar graph shows the 2-ΔCt values calculated by normalizing the Ct values from viral RNA real-time qRT-PCR to β-actin. (D) The 2-ΔCt values for each viral RNA in the macrophages from the ascites of P15 and P16 at pre-treatment and during treatment are listed in the table. N/A, not available.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814111&req=5

ppat.1005531.g004: Changes in the viral RNA levels in P15 and P16 before and during antiviral treatment.(A and B) The viral RNA fold changes in the macrophages from the ascites of P15 (A) and P16 (B) over time are shown. The Ct values from viral RNA real-time qRT-PCR were normalized to β-actin and the 2-ΔΔCt method was used to calculate the relative change in viral RNA level, compared to the pre-treatment value. (C) The viral RNA level (2-ΔCt) in the omentum of P15 and P16 which are collected after 4 and 7 days of antiviral treatment, respectively. The bar graph shows the 2-ΔCt values calculated by normalizing the Ct values from viral RNA real-time qRT-PCR to β-actin. (D) The 2-ΔCt values for each viral RNA in the macrophages from the ascites of P15 and P16 at pre-treatment and during treatment are listed in the table. N/A, not available.
Mentions: Since FIPV is highly associated with tissues and is not reliably detected in blood at high levels in cats with FIP [17], assessment of the efficacy of antiviral drugs in reducing the viral load poses a difficulty in live animals. Although measuring virus titers of the exudate macrophages from the ascites allows to determine the effects of antiviral drug against the replication of FIPV, ascites rapidly decreased with antiviral treatment and we were not able to collect ascites in the recovered cats. However, we determined the viral load in two cats from the second study (P15 and P16) prior to and during antiviral treatment. These cats were euthanized after 4 and 7 days of antiviral treatment. On necropsy, both cats had severe pancreatitis, a possible complication of meloxicam treatment, but no lesions (P16) or mild lesions (P15) typical of FIP were found. Virus titers in the macrophages from the ascites were determined by real-time quantitative RT-PCR and the Ct values were analyzed by the comparative Ct method using the β-actin as a reference gene [39]. The results showed that viral RNA level in the macrophages from the ascites decreased commensurately with the duration of antiviral treatment in these cats. The fold reduction of viral RNA level determined using the delta delta Ct method was 1,595.7 in P15 that received 4 day-antiviral treatment (Fig 4A) and 171,755.9 in P16 that received 7 day-antiviral treatment (Fig 4B), compared to the pre-treatment viral RNA level in the macrophages of each cat. The viral RNA levels (2-ΔCt) in the macrophages from the ascites are summarized in Fig 4D. The viral RNA level in the omentum of P15 and P16 is also shown in Fig 4C. Based on these results, the reduction in virus titers in P15 and P16 seems to correlate with the necropsy findings of mild or no FIP lesions in those cats. These results on viral titers show that FIPV 3CLpro is a valid target for FIPV antiviral drugs and GC376 can effectively reduce the virus load in the macrophages from the ascites and the omentum of cats with FIP.

Bottom Line: The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available.Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent.These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, United States of America.

ABSTRACT
Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.

Show MeSH
Related in: MedlinePlus