|Year : 2015 | Volume
| Issue : 1 | Page : 132-135
A preliminary study of recombinant human interferon-α-2a activity against rabies virus in murine model
S Roy, D Patil, S Ghadigaonkar, R Roy, S Mukherjee, A Chowdhary, R Deshmukh
Department of Virology, Haffkine Institute, Department of Virology, Haffkine Institute for Training, research and Testing, Aacharya Donde Marg, Parel, Mumbai, Maharashtra - 400 012, India
|Date of Submission||05-Dec-2013|
|Date of Acceptance||28-Mar-2014|
|Date of Web Publication||5-Jan-2015|
Department of Virology, Haffkine Institute, Department of Virology, Haffkine Institute for Training, research and Testing, Aacharya Donde Marg, Parel, Mumbai, Maharashtra - 400 012
Source of Support: None, Conflict of Interest: None
Rabies remains an important public health problem in the world due to uncontrolled enzootic rabies. Although rabies associated fatalities may be prevented with timely immunoprophylaxis, but till date a therapeutic molecule has remained elusive. We investigated the role of rhuIFN α-2a in murine model challenged with rabies virus. Titre of 10 4.25 LD 50 /0.03 ml of 10% w/v RV CVS stock suspension were obtained. Based on 1LD 50 titre, challenge dose of 50 LD 50 was administered along with rhuIFN α-2a with pre-exposure (primed) and post-exposure with the rabies virus. Both showed increased survival time as compared with the virus controls. These findings suggest that the rhuIFN α-2a might have some anti-viral activity, which can be used for the treatment of rabies infection. Further research on the efficacy of interferon along with anti-viral drugs for the treatment will be helpful in designing combination therapy against the disease.
Keywords: LD 50 , mice, rabies, recombinant human interferon-α-2a
|How to cite this article:|
Roy S, Patil D, Ghadigaonkar S, Roy R, Mukherjee S, Chowdhary A, Deshmukh R. A preliminary study of recombinant human interferon-α-2a activity against rabies virus in murine model. Indian J Med Microbiol 2015;33:132-5
|How to cite this URL:|
Roy S, Patil D, Ghadigaonkar S, Roy R, Mukherjee S, Chowdhary A, Deshmukh R. A preliminary study of recombinant human interferon-α-2a activity against rabies virus in murine model. Indian J Med Microbiol [serial online] 2015 [cited 2019 Dec 15];33:132-5. Available from: http://www.ijmm.org/text.asp?2015/33/1/132/148412
| ~ Introduction|| |
Rabies causes about 55,000 human deaths annually worldwide of which 31,000 human deaths occur in Asia. India has the highest rate of human rabies in the world, primarily because of stray dogs, whose number has greatly increased since a 2001 law forbade the killing of dogs. An estimated 20,000 people die every year from rabies in India, which is more than a third of the global toll. ,
Rabies virus is a non-segmented negative-stranded RNA virus of the Rhabdoviridae family and induces a fatal neurological disease in humans and animals.  Rabies virus is unique in its mode of transmission via an animal bite and in its spread through the nervous system. After inoculation, the virus infects individual muscle cells and gains access to both sensory and motor nerve fibre endings. The neuromuscular junction is the major site of entry into neurons. The virus enters the central nervous system (CNS) by retrograde axonal transport, replicates within neuronal cell bodies and rapidly disseminates through the CNS by axonal pathways, including trans-synaptic spread. 
It is known that mice, hamster, or rabbits infected with rabies virus can be protected from disease by administering interferon or by inducing interferon by polyriboinosinic polyribocytidylic acid in animals around the time of infection. ,, It has been found that human interferon α-2a shows some antiviral effects when tested on monkeys, when challenged with rabies virus. ,, It was with this objective in mind that the present investigations were commenced.
| ~ Materials and Methods|| |
Interferon and rabies virus
Recombinant human interferon α-2a (rhuIFN α-2a) used in the study was cloned and expressed in Escherichia More Details coli.  The challenged virus standard strain of fixed rabies virus (RV CVS) was passaged by infecting 30 μl intra cerebrally (IC) in Swiss albino mice to increase the virus titre. The RV CVS stock suspension (10% w/v) was prepared by infecting mice brain and stored at −80ºC. LD 50 dose was calculated by Reed and Muench method and based on the 1LD 50 of the RV CVS; a challenge dose of 50 LD 50 RV CVS was used in pre-exposure and post-exposure treatments with rhuIFN α-2a.  The study was approved by the institutional animal ethics committee.
Swiss albino mice aged from 4-6 week old were purchased from Haffkine Institute (animal house) and kept in specially designed cages and kept in the laboratory under the normal light-dark rhythm, food and water supplement during the experiment. The RV CVS 10% (w/v) suspension was prepared from the infected brain specimen and inoculated in 30 Swiss albino mice, 6 groups of 5 mice each (10−1 to 10−6 dilution) and LD 50 was calculated by Reed and Muench method.
Pre-exposure and post-exposure treatments
Mice were divided into 3 groups of 5 mice each in both pre-exposure and post-exposure treatment. In pre-exposure treatment, mice was primed with rhuIFN α-2a (0.03ml ic/mice) 24 hour prior challenge with LD50 RV CVS. rhuIFN α-2a control mice was intracerebrally (ic) inoculated with rhuIFN α-2a (0.03 ml/mice). 50 LD 50 RV CVS (0.03 ml ic/mice) was inoculated intracerebrally and served as a virus control.
In a post-exposure treatment, 0.03 ml of 50 LD 50 RV CVS (ic route) was inoculated in Swiss albino mice 3 hours prior rhuIFN α-2a inoculation. The rhuIFN α-2a control mice was intracerebrally (ic) inoculated with rhuIFN α-2a(0.03 ml/mice). 50 LD 50 RV CVS (0.03 ml ic/mice) was inoculated intracerebrally and served as a virus control. The infected Swiss albino mice were observed for mortality upto 21 days. 
The pre-exposure and post-exposure treatments of rhuIFN α-2a challenged with RV CVS were repeated in triplicate. All the infectious work was carried out in Biosafety level-2 cabinet.
Statistical analysis was performed using two-way analysis of variance (ANOVA) test with GraphPad Prism 5.1 software.
| ~ Results|| |
The LD 50 titre was calculated by the Reed and Muench method. Titre of 10 4.25 LD 50 /0.03 ml of 10%w/v RV CVS stock suspension were obtained, and the challenged dose of 50 LD 50 was prepared. RV CVS challenged control mice showed characteristic rabies signs in the form of ruffling of hair, followed by hunch back from 3-days post infection (dpi) and toe walking, tremors and hind limb paralysis within 4- dpi of infection and subsequently succumb to death on 6 dpi [Table 1]. In rhuIFN α-2a administered control mice, slight ruffled hair was observed from 4 dpi but no mortality was seen up to 21 days. In the pre-exposure treatment, primed rhuIFN α-2a mice challenged with 50 LD 50 RV CVS showed ruffled hair and hunch back on 4 dpi, followed by toe walking, tremors and hind limb paralysis from 5 dpi, which subsequently succumbed to death on 10 dpi. In the post-exposure treatment, rhuIFN α-2a was administered in Swiss albino mice after 3 hours post infection with dose of 50 LD 50 RV CVS. Signs of ruffled hair and hunch back were observed on 4 dpi, followed by toe walking, tremors and hind limb paralysis from 5 dpi and mortality occurred on 8 dpi.
[Figure 1], represented the percentage of mice survival of pre- and post-exposure treatment of rhuIFN α-2a, challenged with dose of 50 LD 50 RV CVS. No survival was observed in mice challenged only with RV CVS on 6 days. In pre-exposure treatment the survival time was seen to be increased to 10 days; however, in post-exposure treatment the survival time was found to be 8 days. These findings and two- way analysis of variance (ANOVA) proves that there is significant increase in the survival time of the mice administered with rhuIFN α-2a in pre- and post-exposure studies when compared with the mice challenged only with RV CVS.
|Figure 1: Survival percentage of pre-exposure (primed) and post-exposure treatments with rhuIFN α-2a challenged with 50 LD50 RV CVS infection|
Click here to view
| ~ Discussion|| |
Rabies is a lethal disease caused by neurotropic virus. When exposure to a potentially rabid animal is recognised, prompt administration of virus-neutralising antibodies together with active immunisation can prevent development of the disease. However, once clinical symptoms occurs conventional post- exposure treatment becomes ineffective and unsuccessful to prevent death.  Previous studies have indicated protective role of human interferon in different animal models. , Sole administrations of interferon α or in combination with ribavirin have failed to provide survival of patients with clinical rabies encephalitis.  However, a group of physicians with expertise in rabies have made recommendations on therapies that could be considered for an aggressive approach. 
The present study was undertaken to elucidate the role of rhuIFN α-2a in mice challenged with rabies virus. In the pre-exposure treatment, primed rhuIFN α-2a was inoculated in Swiss albino mice and challenged with (50 LD 50 ) of RV CVS and observed up to 21 days along with the inoculated control mice (rhuIFN α-2a and RV CVS). In the primed mice, paralysis of hind limbs and tremors were observed from the 5-day post infection (pi) and succumb to death on the 10 dpi when challenge with 50 LD 50 RV CVS, whereas the RV CVS control mice died on the 6-day post infection whereas rhuIFN α-2a control mice survived up to 21 days. In the post-exposure treatment, hind limb paralysis and tremors occurred from the 5 dpi and the mortality occurred on the 8 dpi as compared to the RV CVS control, which occurred on the 6 dpi. There was significant increase in survival time of mice was observed in both pre- and post-exposure treatment with rhuIFN α-2a in comparison with RV CVS control group of mice.
A similar study conducted by Tohamy A. A et al., showed that administration of human Interferon (IFN α-2a) prior to Rabies Virus Infection increases survival in mice. The increased survival rate and reduced cytogenetic changes suggest that protection induced by interferon against rabies virus activity could be, at least partially, attributable to blockage of the replication of CVS strain of rabies virus. 
Similar observation was reported from study involving monkeys infected with rabies virus when administered by repeated intramuscular administration of human leukocyte interferon beginning 24 h after infection.  Bosko Postic et al., showed simultaneous inoculation of interferon and street rabies in rabbit in opposite hind limbs decreased the mortality but interferon in vitro failed to neutralise rabies virus. 
This is the preliminarily work done where we observed increased survival time of mice primed (pre-exposure) with rhuIFN α-2a challenged with 50 LD 50 RV CVS as compared to the virus control. Thus, the problem succinctly stated the activity of rhuIFN α-2a primed Swiss albino mice and post-exposure challenged rabies virus might show some biological/physiological activity on rabies induced infection. Both pre-exposure and post-exposure experiments showed increased survival rates as compared with the controls after known exposure to the virus. These findings suggest that recombinant human interferon-α-2a might help in providing protection against rabies virus.
Further research needs to be done on the mechanism of interferon and combination therapies with anti-viral drugs that should be explored to assess its protective role in rabies treatment.
| ~ Acknowledgement|| |
The authors would like to wish deep gratitude to Dr. DP. Chaudhari, Animal House In-charge, HBPCL, Mumbai, for providing animals and his guidance in veterinary field. The authors are also grateful to Dr. S. Muley, K. C. College, Mumbai for statistical analysis.
| ~ References|| |
Lafon M. Evasive strategies in rabies virus infection. In: Jackson AC, editor. Advances in Virus Research.
Elsevier; 2011. p. 33-53.
Wang ZW, Sarmento L, Wang Y, Li XQ, Dhingra V, Tseggai T, et al.
Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system. J Virol 2005;79:12554-65.
Fenje P, Postic B. Protection of rabbits against experimental rabies by poly I-poly C. Nature 1970;226:171-2.
Janis B, Habel K. Rabies in rabbits and mice: Protective effect of polyriboinosinic-polyribocytidylic acid. J Infect Dis 1972;125:345-52.
Wiktor TJ, Postic B, Ho M, Koprowski H. Role of interferon induction in the protective activity of rabies vaccines. J Infect Dis 1972;126:408-18.
Hilfenhaus J, Karges HE, Weinmann E, Barth R.
Effects of administered human interferon on experimental rabies in monkeys. Infect Immun 1975;11:1156-8.
Marcovistz R, Germano PM, Rivière Y, Tsiang H, Hovanessian AG. The effect of interferon treatment in rabies prophylaxis in immunocompetent, immunosuppressed, and immunodeficient mice. J Interferon Res 1987;7:17-27.
Weinmann E, Majer M, Hilfenhaus J. Intramuscular and/or intralumber postexposure treatment of rabies virus-infected cynomolgus monkeys with human interferon. Infect Immun 1979;24:24-31.
Roy R, Sapatnekar SM, Deshmukh RA. The Effect of heat shock on production of recombinant human interferon alpha 2a (rhIFN α-2a) by Escherichia coli
. Iran Biomed J 2005;9:155-62.
Koprowski H. Laboratory techniques in rabies. 4 th
ed. Geneva: WHO; 1996.
Tohamy AA, Fahmy AM, Dkhil MA, Diab MS, Protective role of interferon against cytotoxcicity induced by rabies virus in mice. Afr J Biotechnol 2010; 9:1097-105.
Roy A, Phares TW, Koprowski H, Hooper DC. Failure to open the blood-brain barrier and deliver immune effectors to the central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection. J Virol 2007;81:1110-8.
Baer GM, Moore SA, Shaddock JH, Levy HB. An effective rabies treatment in exposed monkeys: A single dose of interferon inducer and vaccine. Bull World Health Organ 1979;57:807-13.
Warrell MJ, White NJ, Looareesuwan S, Phillips RE, Suntharasamai P, Chanthavanich P, et al
. Failure of interferon alfa and tribavirin in rabies encephalitis. BMJ 1989;299:830-3.
Jackson AC. Update on Rabies. Res Rep Trop Med 2011;2:31-43.
Postic B, Fenje P. Effect of administered interferon on rabies in rabbits. Appl Microbiol 1971;22:428-31.