06/23/2009

Punta toro virus (PTV)

Single Dose Therapeutic Treatment of Phlebovirus (Punta Toro Virus )Infection in Mice with P-MAPA

 

Phleboviruses of the Bunyaviridae family are considered as one of the species causing serious disease in animals and humans producing significant hepatic disease that resembles severe disease caused by the related human and livestock pathogen, Rift Valley fever virus (RVFV) (Anderson et al., 1990; Fisher et al., 2003 ; Pifat and Smith, 1987). Among these are the Chagres, Alenquer, Candiru and Punta Toro viruses (PTV).

 

The Punta Toro  virus (PTV) is endemic to Central and South America, and appears to be infectious for man. Intensive antiviral studies using Punta Toro virus in a murine system have indicated that immunomodulators also appear to be viable alternatives for therapy of phlebovirus infections (Sidwell et al., 1992).

 

Punta Toro virus (PTV), has been adapted to induce a Rift Valley fever virus-like disease in C57BL/6 mice.  This virus has been used as a model to study the potential role of immunomodulating substances in therapies (Sidwell et al., 1992), since Punta Toro infection and disease in mice usually responds to immunomodulators that result in the induction of type I interferons and/or interferon-gamma.

 

In view of this the PTV mouse disease model has facilitated the discovery of antivirals  and other compounds that are active against more biohazardous phleboviruses, such as Rift Valley Fever and Sandfly Fever viruses.

 

 Rationale for the use of P-MAPA in phleboviruses

 

 The general approach to the treatment of infectious diseases has been changing in recent years. Instead of focusing primarily on the search for better antimicrobials, researchers are now concerned about potential drawbacks such as the emergence of resistant varieties of parasites, and have turned their attention to improving immunological response.

 

Research into cytokines has been particularly successful, providing insights into their role in host defense against pathogens. This has accelerated the development of new compounds that can act effectively on the immunological system. 

 

 Extensive studies showed that P-MAPA-induced proliferation of lymphocyte T, increases cytokine production (mainly interferon-gamma and interleukin-2), NK cell activity and stimulate NO release by macrophages.

 

These data suggest immunomodulation by the induction of Th-1 type response and indicate that P-MAPA may be broadly active, including infections caused by intracellular pathogens such as viruses, including phleboviruses, since Punta Toro infection and disease in mice is  often associated with an impaired immune function (Romani et al., 1997; Samuel 2001; Gowen et al.2006).

 

 Material and methods 

 

Animals: Female 7-week C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and held for 5 days prior to experimentation.They were fed standard mouse chow and tap water ad libitum.

 

Virus: Adames strain PTV was provided by Dr. Dominique Pifat of the U. S. Army Medical Research Institute for Infectious Diseases, Ft. Detrick (Frederick, MD). The virus used was from a stock prepared after four passages of the original virus stock through LLC-MK2 rhesus monkey kidney cells and one passage in hamsters. The virus (0.2 mL) was inoculated via the subcutaneous (s.c.) route.

 

Compounds: P-MAPA was provided by Farmabrasilis, (Brazil). Ribavirin was supplied by ICN Pharmaceuticals, Inc. (Costa Mesa, CA, USA). Both drugs were prepared in sterile saline for intraperitoneal (i.p.) administration.

 

Liver and serum virus titers: Virus titers were assayed using an infectious cell culture assay as previously described (Sidwell et al., 1988). Briefly, a specific volume of liver homogenate or serum was serially diluted and added to triplicate wells of LLC-MK2 cell monolayers in 96-well microplates. The viral cytopathic effect (CPE) was determined 6 to 7 days post-virus exposure and the 50% endpoints were calculated as described (Reed and Muench, 1938).

 

The detection limit for the virus titer assays was 2.8 log10 cell culture 50% infectious doses (CCID50)/0.1 g of liver tissue or ml of serum. In samples presenting no detectable liver or serum virus, a value of < 2.8 log10 was assigned.

 

Therefore, a mean virus titer value preceded by "<" (Table A) indicates that at least one of the samples had undetectable levels of virus and is likely an overestimate of the actual mean viral load. For statistical analysis, a value of 1.8 log10 was assigned for samples with undetectable levels of virus. (Table A).

 

Serum Alanine aminotransferase (ALT) determinations: Serum ALT levels were measured using the ALT (SGPT) Reagent Set purchased from Pointe Scientific, Inc. (Lincoln Park, MI) following the manufacturers recommendations. Reagent volumes were adjusted for analysis with 96-well microplates.

 

 Statistical analysis: The log-rank test for survival analysis was performed using JMP statistical software (SAS, Cary, North Carolina). The Fishers exact test (two-tailed) was used for evaluating increases in total survivors. The Mann-Whitney test (two-tailed) was performed to analyze the differences in mean day of death (MDD), virus titers, serum ALT levels and liver score comparisons.

 

Acknowledgement : Support by National Institute of Allergy and Infectious Diseases-USA (NIAID), Virology Branch, is acknowledged.

 

 Experimental design

 

A total of 15-25 mice from each group were treated with several doses of P-MAPA (1, 10, 100 mg/kg-single dose), Ribavirin (50 mg/kg, twice a day x 5 days), or saline.

 

Animals were challenged with 1.3 ×  10 ⁴  CCID-50 of PTV.

 

P-MAPA was administered as a single dose 24 h post-infectious challenge.

 

Ribavirin (50 mg/kg) was given twice a day for 5 days beginning 4 h pre-virus challenge.

 

Five mice from each group were sacrificed on day 3 of infection and their livers were removed, weighed, and scored on a scale of 0-4 for hepatic icterus; 0 being normal and 4 being maximal yellow coloration. Serum was collected for ALT determinations and infectious virus titers were determined for both liver homogenates and serum samples. (Table A).

 

The remaining animals in each group were observed for death out to 21 days. For comparison, three sham-infected animals were included as normal controls in order to establish baselines for all parameters
tested.

 

The amounts of P-MAPA tested for antiviral activity (1, 10, 100 mg/kg) were also evaluated for signs of over toxicity in uninfected mice.

 

Results

 

Treatment with a single dose (100 mg/kg) of P-MAPA, administered i.p. 24 h post-infectious challenge, was remarkably effective at preventing death due to PTV infection (100% survival), providing complete protection from an infectious dose that killed 65% of the mice in the saline placebo-treated group. 

 

This dose also reduced systemic viral burden and liver discoloration assayed on day 3 of infection. Ribavirin, as expected also protected 100% of challenged mice. Overall, the 10 and 1 mg/kg doses of P-MAPA did not have a significant impact on the outcome of infection (Table A).

 

 

Table A . Effect of single dose i.p. P-MAPA treatment on PTV infection in mice.   

 

 

 LEGENDS

 

^aMean day of death of mice dying prior to day 14.^bDetermined on day 3 of infection; 5 mice per treatment group (4 mice for the ribavirin group). ^cLog₁₀ cell culture 50% infectious dose (CCID₅₀)/0.1 g of liver or ml of serum.  Percentage of animals presenting with detectable virus levels are indicated in parenthesis.

^dAlanine aminotransferase; measured in international units per liter. Score of 0 (normal liver) to 4 (maximal discoloration).

*P< 0.05; **P< 0.01; ***P< 0.001 compared to saline placebo-treated mice.

 

Ribavirin (50 mg/kg) was given i.p. twice a day for 5 days beginning 4 h pre-virus challenge.

P-MAPA, (1,10 and 100 mg/kg-single dose)  i.p. 24 h post-infectious challenge

 

 

CONCLUSIONS

 

The study examining the antiviral activity of a single dose of P-MAPA revealed that the 100 mg/kg dose, administered i.p. 24 h post-infection, was remarkably effective at preventing death due to PTV infection.

 

This dose also reduced systemic viral burden and liver discoloration assayed on day 3 of infection. Overall, the 10 and 1 mg/kg doses did not have a significant impact on the outcome of infection.

 

As expected  for uninfected mice, there was no apparent over toxicity at the tested doses of P-MAPA as the animals appeared healthy and maintained group weights comparable to the saline treated animals .These findings agree with previous toxicology data of the compound

 

Taken together, our findings indicate that non-specific immunotherapy with P-MAPA appears to be an effective treatment for blocking Punta Toro virus-induced disease and indicates that further exploration in other viral disease models may be warranted.

 

By comparing P-MAPA with other immunomodulatory compounds with high efficacy against on Punta Toro virus, it is possible to conclude that P-MAPA also appears as a promising immunomodulator.

 

Further work is needed in the development of compound for use in treating viral infections. Use of P-MAPA as adjuvant immunotherapy with antivirals is a possibility.

 

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