Journal of Postgraduate Medicine
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Year : 2000  |  Volume : 46  |  Issue : 3  |  Page : 164-71  

Singular anti-RNA virus-directed proteins.

RJ Rayanade, K Banerjee, MV Shirodkar 
 Department. of Immunology, Haffkine Institute for Training, Research and Testing, Parel, Mumbai - 400 012, India. , India

Correspondence Address:
R J Rayanade
Department. of Immunology, Haffkine Institute for Training, Research and Testing, Parel, Mumbai - 400 012, India.


AIMS: To additionally purify and characterise the anti-RNA virus-directed protein termed p14. MATERIALS AND METHODS: Antiviral assays of p14 against RNA and DNA viruses were carried out and its antigenic similarities with chicken interferon (CIFN) were studied. HPLC-Reverse Phase of p14 was performed to further purify p14. RESULTS: p14 showed antiviral activity against RNA viruses only and not against DNA viruses. It was antigenically distinct from CIFN. Purification of p14 yielded three proteins with antiviral activity, which had different physico-chemical properties than those described for interferons. CONCLUSIONS: The data presented on the antiviral, immunological and physico-chemical properties, establish the unique nature of p14 vis-á-vis those of interferons.

How to cite this article:
Rayanade R J, Banerjee K, Shirodkar M V. Singular anti-RNA virus-directed proteins. J Postgrad Med 2000;46:164-71

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Rayanade R J, Banerjee K, Shirodkar M V. Singular anti-RNA virus-directed proteins. J Postgrad Med [serial online] 2000 [cited 2023 Jan 28 ];46:164-71
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Early work demonstrated total blockade of Rous sarcoma virus (RSV) induced tumours in chickens, by West Nile virus (WNV), a flavivirus.[1],[2],[3] Other flaviviruses viz., Kyasnur Forest disease (KFD) and Japanese encephalitis (JEV), also blocked RSV in chickens.[4] Although interferon (IFN)- mediated mechanisms were, then, being mooted, almost universally, as underlying virtually all interference phenomena, preliminary in vivo and in ovo experiments failed to implicate IFN in the WNV-RSV system, since (a) the blockade was not reversible in chickens, even by prolonged corticosteroid treatment; (b) was not observed in ovo using WNV-free allantoic fluid from WNV-inoculated chick embryos and (c) was not demonstrable in ovo with UV-inactivated WNV.[1],[2],[3]

It was later found that the ability to block this tumour was not restricted to the flaviviruses. Rabies, a rhabdovirus, also blocked the same tumour in chickens.[5] Further investigations[6],[7],[8] provided evidence that, as hypothesised earlier[1],[2],[3] a substance could be detected in the serum/plasma of rabies virus-inoculated chicks, which might be responsible for the blockade of this tumour. This putative antiviral substance, induced maximally and contained in the heparinised plasma of White Leghorn chickens 48h following wing-web inoculation of rabies virus, was labelled crude plasma factor (CPF).[6],[7],[8]

Subsequent tests for characterisation of CPF, vis--vis the standard criteria prescribed for the establishment of an antiviral substance as an IFN, indicated that CPF selectively inhibited the replication of two RNA viruses (RSV and influenza), but not of two DNA ones (vaccinia and herpes simplex), even on simultaneous inoculation in ovo.[9] Additionally, the RNA virus-restricted antiviral activity of CPF was demonstrable against Newcastle Disease, WNV, Coxsackie, and rabies viruses, but not against DNA viruses such as buffalopox, inclusion body hepatitis, or chick embryo lethal orphan.[9] CPF protected the homologous chick embryo fibroblasts, as well as heterologous human amnion cells, against VSV.[9] In contrast, chick interferon (CIFN) could not protect the heterologous human amnion cells against VSV.[9] In this same overview, it was noted that CPF blocked viral replication, on simultaneous inoculation, with a number of viruses, whereas, priming (at least 18-24 h) was mandatory for demonstration of antiviral activity of CIFN.[9],[10]

Sequential purification of CPF yielded a partially purified substance labelled 14 kDa fraction (abbreviated to p14, hereafter).[11] Preliminary characterisation of p14, suggested that, it had RNA virus-restricted antiviral activity. It was pH- and heat-labile.[11] Preliminary attempt at sequencing, native-PAGE and isoelectric focussing studies of p14 (unpublished data) indicated that p14 was composed of more than one protein, with similar molecular mass (14 kDa).

The present paper sets forth pertinent data concerning the antiviral activity of p14 being selectively directed against three RNA viruses, endorsing the earlier observations.

  ::   Material and methodTop

Cells and viruses: Vero cells (ATCC-CCL-81) were maintained in Earles MEM (Sigma, St. Louis) supplemented with 5% goat serum, 2mM glutamine, 200 u/ml penicillin and 150 g/ml streptomycin (Sigma, St. Louis). Virus stock preparation (except for influenza) was in Vero cells. The influenza stock was prepared in 10-day White Leghorn embryonated eggs (Venkateshwara Hatcheries, Pune). The viral stock solutions were titrated, aliquoted, and stored at 700C. Primary chick embryo cells (CEC), from the same eggs, were grown in M-Hanks medium (Sigma, St. Louis) supplemented with 3% goat serum, 200 u/ml penicillin and 150 g/ml streptomycin. Vero and CEC were used for performing antiviral assays for p14 and CIFN. The RNA viruses studied were Chandipura (CHP-653703, a vesiculovirus), polio (PV1- Sabin, an enterovirus), Semliki Forest (SFV-718488, an alphavirus), Japanese encephalitis (JEV-P20778, a flavivirus) and influenza A (781882). The DNA viruses studied were vaccinia (VV-712696, an orthopoxvirus), herpes simplex (HSV1-753166, a human alpha herpes virus) and simian vacuolating virus 40 (SV40-782, a mastadenovirus).

p14: This antiviral fraction was obtained by partial purification of CPF, as described earlier.[11]

CIFN: Preparation was as described by Lampson et al.[12] The allantoic fluid, harvested 72-hr post-influenza virus inoculation, was treated with cold perchloric acid, and then centrifuged at 540 x g for 15 min at 40C; the supernatant constituted crude CIFN, which was stored at -700C. Assays for protein content and titre were similar to those used for CPF.[11] CIFN titre was expressed in terms of reference units/ml, calibrated against the reference standard for CIFN (MRC Research Standard A62/4 from NIAID, USA).

Gel-filtration of CIFN (at 40C): Sephadex G-100 (Pharmacia, NJ.) was equilibrated with 0.01M PBS (pH 7.2) in a column (30 x 2.5 cm), then calibrated (standard mol. wt. markers) at flow-rate 3 ml/min; fractionation was at 1 ml/min, the fractions collected (10M PMSF added) being assayed for protein content and titre. This semi-purified CIFN was used to raise murine polyclonal neutralising anti-CIFN.

Antiviral spectra of p14 and CIFN: Viral plaque-reduction assays[13] were performed in Vero, or CEC (in 24-well plates), depending upon the experimental protocol. The antiviral effect of p14 was tested against three RNA viruses viz. CHP, PV1 and SFV and three DNA ones viz. VV, HSV1 and SV40. CIFN was tested for antiviral effect against an RNA virus (CHP) and a DNA virus (VV).

To maintain strict objectivity, a number of double-blind experiments were conducted (n= 3 for p14 and n=6 for CIFN). In all antiviral studies, the overall differences among groups were tested by analysis of variance (ANOVA). If an overall difference among groups was shown significant by the F test, Dunnetts test was used to analyse which group (treatment) was significantly different from the control, at the 5 % level.

Raising and assay of murine polyclonal neutralising

antibodies against p14 and CIFN

a. Antigen: p14 (specific activity 8.8 x 103 u/mg) and partially purified CIFN (specific activity 1.6 x 102 Iu/mg) were used for immunisation of Swiss albino mice.

b. Immunisation procedure: Two groups of animals (12-14 g) (10/group) were bled (ophthalmic venous plexus), prior to commencement of immunization, and these pre-immunisation sera served as controls. Each mouse from a particular group was immunized with 50 u of p14, or 50 Iu CIFN (weekly intervals), subcutaneously. The first two injections were in Freunds complete adjuvant (Sigma, St. Louis), later ones in incomplete Freunds adjuvant. The mice were bled weekly (first week onwards), the yield being approximately 0.5 ml blood/mouse. After pooling the individual blood samples, the sera drawn were heat-inactivated (560C, for 30 min). Storage was at -200C, pending assay for anti-p14 and anti-CIFN neutralising antibodies.

c. Titration of neutralising antibodies: The titre of each antiserum was the reciprocal of its highest dilution which, when mixed with an equal volume of p14, or CIFN, neutralised 50% of its antiviral activity.

Serial 2-fold dilutions of anti-p14 serum were prepared, equivolumetric p14 (30 u/0.1 ml) added and these mixtures incubated (370C, 1h); equal volumes of these were next added to aliquots of 0.1 ml MEM containing 200 pfu of CHP virus. The medium from the preformed CEC monolayers (in 24-well plates) was aspirated and 0.1 ml from the mixture (containing virus and appropriate dilution of antibody) added, in triplicate, to the wells. The test p14 utilised in this neutralisation test was simultaneously assayed. The cultures were incubated (370C, 24h) and this was followed by viral plaque count and determination of neutralising titre.

Titration of anti-CIFN antibody was performed after Havell.[14] The titre of the antiserum was the reciprocal of its highest dilution which, when mixed with an equal volume of IFN (20 u/ml), neutralised 50% of the latters antiviral activity.

Specificities of anti-p14 and anti-CIFN neutralising antibodies: An equal volume of p14 (30 u/ml) was added to anti-CIFN serum (diluted 1:2) and the mixture incubated (370C, 1h); serial 2-fold dilutions of the mixture followed. Equal volumes of each dilution were added to 0.1 ml of medium containing 200 pfu of CHP virus; 0.1 ml from the above mixture (for each dilution) was added, in triplicate, to wells containing preformed CEC monolayers.

Equal volumes of CIFN (20 Iu/0.1 ml) and anti-p14 serum (diluted 1:2) were mixed, incubated (370C, 1hour) and followed by serial 2-fold dilutions of the mixture; 0.1 ml from each of the dilutions were added, in triplicate, to wells containing preformed CEC monolayers. After 24 hour incubation, the contents of each well were aspirated, followed by addition of 0.1 ml MEM containing 100 pfu of CHP virus.

The total units of p14, or CIFN, neutralised by anti-CIFN, or anti-p14 antibodies, respectively, were then computed.

Purification of p14 by HPLC-RP: p14 was further fractionated by HPLC-RP at room temperature. Lyophilised p14, dissolved in 0.1% trifluoroacetic acid (TFA, Sigma, St. Louis) was applied to a C18 column (Waters, USA), which had been equilibrated previously and also eluted with the same gradient program: Initial, 100% acetonitrile 0% TFA; 5 min, 75% acetonitrile 25% TFA; 35 min, 50% acetonitrile 50% TFA; 40 min, 25% acetonitrile 75% TFA; 45 min, 100% acetonitrile 0% TFA, at a flow rate of 1 ml/min (0.05 AUFS; 280 nm). The collected fractions were lyophilised to remove the organic solvents, redissolved in chilled PBS (0.01M, pH 7.2) and assayed for protein content and titre as described previously, for p14.[11] The three fractions possessing antiviral activity were designated RT-13.75, RT-16.04, and RT-41.60, respectively, based on their retention times on the column.

Physicochemical studies with p14 antiviral fractions: RT-13.75, RT-16.04, and RT-41.60

The protocols followed in each case were similar to our earlier ones, for p14.[11]

a. pH and temperature sensitivity: Ten units of each fraction were exposed to pH 2/ pH 10 at 40C for 20 hour and the treated samples then assayed for antiviral activity. Ten units of each fraction were incubated at 650C and 800C for hour, then assayed for antiviral activity.

b. Sensitivity to proteinase K (PK-Bethesda Research Laboratory, USA): Ten units of each fraction were treated with PK at 220C for 3 minute, then assayed for antiviral activity.

  ::   ResultsTop

Preparation and partial purification of CIFN in our laboratory were necessitated not only for raising polyclonal antiserum and for preliminary characterization, but, especially, to confirm its well known broad-spectrum antiviral activity, which constitutes a sine qua non, by definition, for any IFN. Thus, this CIFN baseline was deemed mandatory for the experiments designed, hereunder, to test the validity of the hypothesis that p14 differed radically from CIFN, since its antiviral activity was restricted exclusively to the RNA variety, of the different viruses studied.

Titration of our CIFN preparation by the plaque-reduction method yielded a titre of 274.7 Iu/ml; its estimated specific activity was 6.6 Iu/mg [Table:1]. Purification of this crude CIFN, by gel-filtration on Sephadex G-100, yielded an antiviral fraction with molecular mass range 40-45 kDa. The specific activity of this fraction was 1.6 x 102 Iu/mg, per cent recovery of the antiviral activity being 39.5 [Table:1]. This 25-fold purified CIFN was used in raising anti-CIFN specific polyclonal antibody.

p14 showed a significant inhibitory activity against the RNA viruses CHP, PV1 and SFV, in an antiviral assay using the plaque-reduction method [Figure:1], wherein mixtures of appropriate p14 unitages with 100pfu of each virus were simultaneously inoculated on to Vero (heterologous) cells. Although graded antiviral responses were observed with all three viruses, PV1 and SFV, required higher doses of p14 for inhibition of replication, as compared to CHP. Priming of Vero cells, for 24h with p14, also resulted in inhibition of PV1.

Next, studies testing for possible antiviral activity of p14 against VV, HSV1 and SV40 were performed. Simultaneous inoculation of p14 with these viruses, on to Vero cells, failed to exhibit any antiviral effect and no antiviral effect against VV was noted, even on pre-treatment of Vero cells with p14 (data not shown here). Thus p14, like its parent, CPF, manifested RNA virus-restricted antiviral activity. This could be demonstrated in heterologous cells (Vero), either on pre-treatment, or upon simultaneous inoculation with PV1 [Figure:1].

Contrariwise, 24h pre-treatment of CEC with CIFN, showed significant inhibition of both CHP and VV, no inhibition being noted when CIFN was mixed with CHP, or VV, and inoculated simultaneously on to CEC [Figure:2]. Simultaneous inoculation of CIFN with CHP on Vero cells, or pre-treatment of Vero cells with CIFN, showed no protection against CHP. Thus, CIFN inhibited both RNA and a DNA virus in homologous cells [Figure:2], but failed to exhibit such an effect in heterologous cells. Hence, as anticipated, the antiviral activity of CIFN was manifested only on pre-incubation and only in homologous cells, these results with CIFN being, therefore, at complete variance with those, described above, for p14.

p14 and semi-purified CIFN were injected subcutaneously, at weekly intervals, into different groups of mice. The development of neutralising antibodies against p14 and CIFN was monitored, the mice being bled, at weekly intervals, and antibody titres estimated on the basis of the capacity of the specific immune sera raised to neutralise the antiviral activities of p14, or CIFN [Figure:3]. In addition, the specific immune sera were used in cross-neutralisation experiments. [Figure:3] shows that anti-p14 antibodies failed to neutralise the antiviral activity of CIFN; likewise, anti-CIFN antibodies failed to neutralise the antiviral activity of p14.

Since preliminary studies on microsequencing, native-PAGE and IEF of p14 (unpublished data) had suggested that it might be composed of more than one protein, further purification of p14 using HPLC-RP was deemed mandatory. Indeed, HPLC-RP of p14 yielded nine fractions, each typified by its characteristic retention time on the column [Figure:4]. However, when assayed for antiviral activity by the plaque reduction method, only three fractions, designated RT-13.75, RT-16.04 and RT-41.60, exhibited significant antiviral activity [Figure:4], the specific activities being 6.2 x 105, 2.1 x 105 and 1.8 x 106 u/mg, respectively [Table:2]. [Table:2] also summarises the purification process of p14, which resulted in increases in the specific activities of the antiviral fractions, as compared to the specific activity of p14.

[Table:3] sets forth the physicochemical properties of the three fractions. Exposure to pH 2 and pH 10, of 10u of each fraction, for 20 hr at 40C, resulted in near complete loss of its antiviral activity [Table:3]. Exposure to temperatures of 650C and 800C, for hour, resulted in complete loss of antiviral activity of RT-13.75 and RT-41.60. Some residual antiviral activity was noted with RT-16.04, both on exposure to 650C and 800C, which could indicate that RT-16.04 was somewhat more heat-stable than the other antiviral fractions. Complete loss of antiviral activity, of the three antiviral fractions, was observed on treatment with PK (220C, 3 min) [Table:3].

  ::   DiscussionTop

Though CIFN was discovered more than four decades ago and, ironically, in the ensuing years, the genes of many mammalian IFNs were cloned, the IFNs of the chickens are poorly characterised.[15] Some homologues of mammalian cytokines have been cloned in the chicken these include type I IFN, type II IFN (17), IL-8 and the TGF- cytokine family.[16],[18],[19],[20],[21],[22],[23],[24] Even so, currently, the field of cytokine research in chickens remains largely unexplored.

A family of intron-less genes coding for two distinct serotypes of CIFN has been characterised.[15] While Type 1 IFNs are potent antiviral agents, the Type 2 IFNs exhibit both antiviral and macrophage activating properties.[25],[26] However, if one accepts the current definition of IFNs, as inducible proteins which stimulate antiviral activity in vertebrate cells[10] and the essential criteria of the definition[27] that (a) they posses broad-spectrum antiviral activity, (b) exhibit higher antiviral activity in homologous cells than in heterologous ones and (c) priming is essential for this activity, one can then analyse the findings of the present authors, particularly vis--vis the data obtained with p14 and its three antiviral fractions, RT-13.75, RT-16.04 and RT-41.60, so as to highlight the differences in the properties of these fractions from those postulated, above, for the IFNs.

IFNs inhibit replication of both, RNA and DNA viruses.[10] RNA viruses such as influenza, Mengo and VSV are inhibited by IFNs. Equally, DNA viruses like VV, HSV1 and SV40 are, also, inhibited.[27] In the present investigation, results comparable to those from the above reports were obtained. CIFN showed antiviral activity against both, an RNA (CHP) as well as a DNA (VV) virus [Figure:2]. In contrast, an earlier overview[9] had noted that CPF showed protection against RNA viruses (Newcastle disease, WNV, Coxsackie and rabies), but not against any DNA ones (buffalopox, inclusion body hepatitis and chick embryo lethal orphan). In the present investigation, p14 showed antiviral activity against RNA viruses (CHP, PV1 and SFV) (Fig. 1). However, p14 showed no antiviral activity against DNA viruses (VV, HSV1 and SV40, data not shown here), bolstering the earlier hypothesis that CPF displayed RNA virus-restricted antiviral activity.[9] PV1, especially, and SFV were inhibited at higher doses of p14 than those used for demonstration of anti-CHP activity [Figure:1]. At the moment, the present investigators are unable to explain this observation.

The antiviral activities of some IFN-proteins, such as 2-5 A synthetase and Mx proteins, show a high degree of specificity for certain viruses. For example, 2-5 A synthetase induces an antiviral state with high specificity towards picornaviruses, while the Mx proteins inhibit only influenza.[27] IFN-induced human proteins with homology to the mouse Mx protein inhibited influenza and VSV.[27] No Mx protein has yet been identified in the chicken;[28] the finding of the present authors, that p14 exhibits RNA virus-restricted activity, therefore, assumes significance.

It has been clearly established that, in the majority of investigations, IFN activity in heterologous cell cultures is much lower than in homologous cell cultures.[29] Buckler and Baron found that mouse IFN exerted only 5 per cent of its homologous antiviral activity in closely related rat, or hamster cells.[29] CIFN failed to protect duck embryo fibroblasts against viral infection.[30] However, human leukocyte IFN exhibited similar levels of activity in either human, or bovine cells.[31] In the present investigation, CIFN showed antiviral activity only in homologous (CEC) [Figure:2], but not in heterologous (Vero) cells. In contrast, CPF protected both, homologous (CEC) and heterologous (human amnion) cells[9] and p14 showed antiviral activity in heterlogous (Vero) cells [Figure:1].

In case of CIFN, previous reports state that pre-incubating of homologous cells with CIFN is a sine qua non for demonstration of its antiviral effect.[32],[33] Appreciable protection against SV40 was achieved in permissive monkey cells, only when IFN treatment preceded SV40 injection, by at least 10 hr.[27] In the present investigation, results comparable to those from the above reports were obtained. CIFN showed antiviral activity on priming homologous cells (CEC) [Figure:2], but failed to show any activity on simultaneous inoculation with the viruses (CHP, VV) on to homologous cells (CEC).

Furthermore, no antiviral activity was demonstrable, either on pre-incubation, or on simultaneous inoculation with CHP, on to heterologous (Vero) cells. Contrariwise, p14 showed antiviral activity against three RNA viruses (CHP, PV1, and SFV), on simultaneous inoculation, on to heterologous (Vero) cells [Figure:1], and against PV1 on pretreatment (24h) of heterologous Vero cells.

IFNs of varying purity have been used to elicit neutralising antibodies in different animals.[14] In the present investigation, p14 and partially purified CIFN were used for immunizing mice. The p14 used to raise anti-p14 neutralising antibody had been obtained from our earlier studies.[11] CIFN was partially purified; it had a molecular mass of 40-45 kDa, which is in agreement with a molecular mass of 40-45 kDa, as determined by gel-filtration using Sephadex G-100.[34] The estimated degree of purification of CIFN, in the present case, was 25-fold [Table:1], which permitted use of this partially purified material in raising anti-CIFN neutralising antibody. The use of partially purified CIFN to raise antibodies has been reported previously by other investigators, as well.[35]

Antibodies to cloned CIFN neutralised the bulk of antiviral activity in preparations of partially purified CIFN from various natural sources,[25] suggesting that a single serotype of IFN is predominantly induced under experimental conditions. Detailed analyses showed that the chicken genome contains a family of at least 10 IFN genes, now designated IFN1, which all appear to code for one serotype of CIFN. A second serotype IFN 2, shows limited sequence homology to IFN 1.[15] It was, thus, abundantly clear that key evidence needed to demonstrate that p14 was not CIFN lay in the direction of absence of cross-reactivity between neutralising antibodies raised against each of these two antigens. Indeed, specific polyclonal, neutralising antibodies against p14 (which developed somewhat later than anti-CIFN, [Figure:3]) failed to neutralise the antiviral activity of CIFN and, conversely, anti-CIFN could not neutralise the antiviral activity of p14 against CHP virus (Fig. 3), in CEC. In future studies, it will be necessary to analyse the cross-reactivity of all the three antiviral fractions obtained from p14 with the two serotypes of CIFN.

HPLC-RP has been used for purification of proteins that are stable in organic solvents.[36] Hobbs et al[37] purified human IFNs by HPLC-RP without loss of biological activity. Similarly, purification of p14 by HPLC-RP was believed by us to be possible, without loss of antiviral activity. Certainly, HPLC-RP enhanced the degree of purification of the three antiviral fractions derived from p14 to the 7.2 x 104- 6.1 x 105 range, in comparison with that of p14 [Table:2].[11] The typical conditions used in HPLC-RP of proteins low pH and high concentration of organic solvents favour denaturation, so that a protein dissolved in the mobile phase will consist of a mixture of native, partly-folded and aggregated structures. For example, two isomers of proline peptide eluted as two separate peaks.[38] In the present study, the three antiviral fractions require further characterization to establish: (a) whether they constitute a single protein, eluting as three separate peaks, or, (b) they are, indeed, distinct antiviral protein fractions, and (c) how much homology if any, do they exhibit, have (if any) with Type 1 and Type 2 CIFNs?

The existence of an acid-labile type of CIFN was reported in 1990,[39] a finding contradicting reports of absence of ?,?,? CIFNs,[40],[41] as well as that, of an earlier finding, that partially-purified CIFN was stable at pH values between 1 to 10.[35] Contrariwise, p14 lost 98-99% of its activity at pH 2 and pH 10, respectively.[11] In the present investigation, the three antiviral fractions obtained, following HPLC-RP of p14, lost most of their antiviral activities on exposure to pH 2 and pH 10 for 20 hour at 40C [Table:3], as did the parent p14,[11] yet showed activity after elution from a C18 column under acidic conditions (0.1% TFA) [Figure:4]. Acid and alkaline denaturation/reactivation of proteins is common and the mechanism of this inactivation can vary with individual proteins and specific environmental conditions.[42] Differences in the specific environmental conditions, existing in the above two situations, might, likely, explain this observation.

CIFN lost no activity at 660C for 1 hour; at 760C and 840C for 1 hour, 50% and 80% destruction, respectively, was observed.[12],[35] These results are diametrically opposed to those of Desai et al[6], who found CPF to be heat-labile at 56C, 1 hour. p14, too, is heat-labile, 99% of its activity being lost at 600C for hr and 50% at 40C for hr.[11] The results of the present investigation confirm those reported earlier for CPF and p14.[6],[11] The three antiviral fractions were heat-labile (65C and 80C, hour), though RT-16.04, was marginally more stable than RT-13.75 and RT-41.60 [Table:3].

CIFN activity was destroyed by proteolytic enzymes (trypsin, chymotrypsin, pepsin and papain).[12] In the present study, the proteolytic enzyme, PK, destroyed the antiviral activity of all three antiviral fractions [Table:3], their proteinaceous nature having thus being confirmed.

Thus, in the light of the present data on its antiviral, immunological and biochemical properties, p14 clearly stands out as a novel group of proteins, distinct from CIFNs.

Investigations initially carried out at the National Institute of Virology, Pune. Further work is in progress at the Haffkine Institute, Mumbai.

  ::   AcknowledgmentsTop

The authors gratefully acknowledge the help of Dr. AS Paintal, DST Centre for Visceral Mechanisms, VP Chest Institute, University of Delhi, for constant encouragement and Prof. AS Arekar of the Haffkine Institute, Mumbai, for guidance in the biostatistical evaluation.


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