Canine Parvovirus: The Need for Effective Antiviral Therapies

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Canine Parvovirus The Need for Effective
Antiviral Therapies

• Josh Lizer

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Background

• Family Parvoviridae
• Genus parvovirus
• Non-enveloped, DNA virus
• Mutated from Feline Panleukopenia Virus
• Mostly species specific--Canidae

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Clinical Disease

• Hemorrhagic diarrhea
• Vomiting
• Pyrexia
• Dehydration
• Fecal Viral Shedding
• Leukopenia

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The Problem

• Specific antiviral treatments are lacking
• Currently only treat symptoms
• Antibiotics, fluids, antiemetics

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Example of CPV treatment
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Research of New Treatments

• Passive Immunity
• Ie. convalescent dog serum
• Antibody currently one of the only actual
  specific antiviral treatments
• Septi-Serum
• Treats septicemia caused by Gram negatives
• Dosage 2-4ml/lb bw
• Decrease mortality and length of
  hospitalization(4)

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Oral Antibody

• Originally used as superior protein supplement
  for pig diets
• Spray dried porcine plasma
• Also had therapeutic effects
• E. coli, Vibrio, Clostridium, Crypto, rotavirus

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Why Oral Antibody?

• Demonstrated to protect against disease
• Decrease fecal shedding
• Completely treat or alleviate disease
• Survives passage through GI
• Active Ab has been recovered in feces
• Targets virus in gut
• Main area of viral destruction

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The Example of anti-E. coli Oral Ab in pigs

• Piglets fed egg yolk Ab (EYA) protected against
  E. coli challenge (Yokoyama et al 1992 Zuniga et
  al 1997)
• Significant reduction in challenge strain fecal
  excretion, better clinical protection (Imberechts
  et al 1997)
• E. coli-induced diarrhea alleviated in 24 hrs
  after treatment with EYA controls continued to
  have diarrhea and 60 mortality (Marquardt et al
  1999)

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Why Oral Antibody?

• Nguyen et al. (2006) challenged 2 mo. puppies
• Chicken egg yolk IgY 6 hrs post challenge
  administered orally
• Groups 2g Ab, 0.5gcontrol powder, control
  powder
• 3x dayfirst 5 days 2x daynext 2 days

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DRUM ROLL.
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Great Success!

• Group 1 NO CLINICAL SIGNS shedding for 7 d
• Group 2 3
• 2/3 and 4/4 with clinical signs
• Shedding for 12 d

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Clinical Scoring System
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Results
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This summer at ISU

• I will be testing the effects of oral Ab in
  challenged puppies
• Showing clinical signs before treating
• Is oral Ab conjunct with standard treatment
  effective after dog already showing clinical
  signs?

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Recombinant Feline Interferon Omega (rFeIFN-?)

• Interferons have potent antiviral and anti-tumor
  activity
• Antiviral activity against feline calicivirus,
  herpesvirus, and entereopathogenic virus
• Produced in silkworm larvae using baculovirus
  vector

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Producing IFN in the silkworm

• cDNA library created using Feline cells
  (LSA-D4-K17) as a poly(A) RNA donor, pcDV1
  plasmid primer, and E. coli host
• cDNA coding for FeIFN is screened by transfecting
  simian established cells COS1 or COS7 and
  selecting plasmid which provides these cells
  ability to express anti-viral activity by
  transient expression.
• E. coli transformed with selected plasmid
  (pFeIFN1)

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Create recombinant silkworm nuclear polyhedrosis
virus (SNPV)

• Extract pFeIFN1 from E. coli
• Remove DNA coding for FeIFN protein from the
  plasmid and insert into cloning vector for
  silkworm to prepare recombinant plasmid
• Recombinant plasmid is transfected with SNPV DNA
  into silkworm cells to construct recombinant virus

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Infect silkworms with SNPV

• 1.rSNPV cultured in established silkworm cells or
  in body of silkworm (by injection)
• 2. Body fluid collected from silkworm, FeIFN
  recovered from supernatant. If grown in cells,
  FeIFN recovered in supernate of centrifuged cell
  culture.
• 3. FeIFN purified by column chromatography or
  affinity chromatography

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rFeIFN-?

• Found to bind to IFN receptors in dogs
• Results in increased 2,5-oligoadenylate
  synthetase production, which blocks viral protein
  synthesis
• Application in CPV.?

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How Interferon Works

• http//www.uic.edu/depts/accc/seminars/flashintro/
  interferon.html

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How Interferon Works

• Source http//pathmicro.med.sc.edu/mhunt/interfero
  n.jpg

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rFeIFN-? clinical studies

• Ishiwata et al. (1998)
• 1 mega unit (MU)/kg IV beginning 4 days after
  challenge, for 3 days
• 24 dogs
• Saline Placebo

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Clinical Scoring Table
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Results
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Results (significance)
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Results

• Estimating onset of treatment effect (IFN group
  only)
• Ratio of dogs with enteritis in morning compared
  to those in evening. Treatment reduced ratio of
  dogs with clinical enteritis in evening on d4
  after virus inoculation
• (Chi-squared test, p0.0104)
• Tendency observed on day 6 as well
• (p0.0513)
• Suggests short half life of the interferon!

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Conclusions

• Treatment with rFeIFN-? ameliorates severe
  enteritis caused by CPV-2.
• Dosage was enough to switch enteritis from
  serious to mild form and improve symptoms such as
  anorexia and vomiting during days of IFN
  administration

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rFeIFN-? Clinical Study 2

• Martin et al (2002)
• 10 pups
• 2.5 MU/kg IFN beginning 4 days after challenge,
  for 3 days
• Placebo

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Clinical Scoring Table
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Results
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Weight Differences
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Conclusions

• rFeIFN-? at dosage of 2.5 MU/kg exerts
  significant therapeutic and life-saving effects
  on pups with parvoviral enteritis

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rFeIFN-? Clinical Study 3

• De Mari et al. (2003)
• Field trial
• 92 dogs in 19 veterinary clinics
• 2.5 MU/kg IFN for 3 days, standard parvo
  treatment
• Placebo

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Results
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Conclusion

• Vaccinated and unvaccinated pups could benefit
  from the therapeutic life-saving effects of
  rFeIFN-?

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rFeIFN-? Clinical Study 4

• Kuwabara et al. (2006)
• 1 MU/kg IFN for 3 days
• Placebo
• Observed in healthy (n18) and naturally
  CPV-infected dogs (n13)
• 31 dogs

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ResultsNormal Dogs

• Normal Dogs
• Phagocytic activity of whole blood cells and
  macrophage activity 2x baseline after 3 hr
  (plt0.05)
• Peripheral lymphocyte activity also increased
  (plt0.01)

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ResultsInfected Dogs
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Conclusion

• Continuous immunoenhancement after IFN
  administration and WBC counts gt5x10e3/ul may be
  essential for improving severe clinical syptoms

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Final Remarks

• Oral antibody rFeIFN-? ?
• Larger dose sizes and increased duration of
  administration for rFeIFN-? ?

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Additional References

• Yokoyama, H., R. C. Peralta, R. Diaz, S. Sendo,
  Y. Ikemori, and Y. Kodama. 1992. Passive
  protective effect of chicken egg yolk
  immunoglobulins against experimental
  enterotoxigenic Escherichia coli infection in
  neonatal piglets. Infect. Immun. 60998-1007.
• Zuniga, A., H. Yokiyama, P. Albicker-Rippinger,
  E. Eggenberger, and H. U. Bertschinger. 1997.
  Reduced intestinal colonisation with F18-positive
  enterotoxigenic Escherichia coli in weaned pigs
  fed chicken egg antibody against the fimbriae.
  FEMS Immunol. Med. Microbiol. 18153-161.
• 13. Imberechts, H., P. Deprez, E. Van Driessche,
  and P. Pohl. 1997. Chicken egg yolk antibodies
  against F18ab fimbriae of Escherichia coli
  inhibit shedding of F18 positive E. coli by
  experimentally infected pigs. Vet. Microbiol.
  54329-341.
• Marquardt, R. R., L. Z. Jin, J. W. Kim, L. Fang,
  A. A. Frohlich, S. K. Baidoo. 1999. Passive
  protective effect of egg-yolk antibodies against
  enterotoxigenic Escherichia coli K88 infection
  in neonatal and early-weaned piglets. FEMS (Fed.
  Eur. Microbiol. Soc.) Immunol. Med. Microbiol.
  23283-288.