Evaluation of antiviral activity of Ocimum sanctum and Acacia arabica leaves extracts against H9N2 virus using embryonated chicken egg model

Influenza A viruses (IAVs), members of the family Orthomyxoviridae are characterized by a single stranded, segmented negative-sense RNA genome. Among the IAVs, avian influenza (AI) H9N2 has become endemic in terrestrial poultry in several countries of the Eurasian continent including India in recent years [1‐4]. The spread of AI H9N2 has resulted in significant economic losses in poultry mainly because of reduced egg production and high mortality associated with co-infection with other respiratory pathogens [5]. Although AI H9N2 does not fall under the definition of highly pathogenic avian influenza (HPAI) viruses, there has been ever increasing speculation about pandemic potential of H9N2 viruses [6].

So far, a total of 28 laboratory-confirmed cases of human infection with avian influenza A (H9N2) viruses, however none fatal, have been detected globally (http://​www.​who.​int/​influenza/​human_​animal_​interface/​Influenza_​Summary_​IRA_​HA_​interface_​25_​02_​2016.​pdf). Among them, the recent one includes from China [7]. In addition, serological evidences of AI H9N2 virus exposure to human have been reported on several occasions from Iran, China and India [8‐11]. The higher human infection capability of these viruses was provided by the fact that H9N2 binds to α-2,6 sialic acid receptors that are abundant in the human upper respiratory tract while H5N1 chiefly bind to human receptors in the lower respiratory tract [12]. The recently emerged influenza A (H7N9) and (H10N8) infecting humans had acquired gene segments from H9N2 virus [13, 14]. The potential of genetic reassortment of IAVs, due to segmented genome, from different animal species is thought to be a mechanism for the emergence of influenza viruses with pandemic potential [14]. Endemicity of H9N2 circulation in poultry especially in India could further aggravate the current situation. Among the control measures, existing vaccines are unable to keep up with the mutation rates of viruses. New vaccine development takes a long time and at the same time, viruses are also developing resistance to the currently used drugs [15]. Hence, there is no immediate response drug to the newly emerging virus infections/outbreaks. To address this problem, there is an exigent need for the development of a new paradigm preventive and therapeutic agent to control the immediate spread of viral outbreaks. In this scenario, traditional herbal medicines have been postulated to prove effective due to fewer side effects, relatively low cost and easy availability [16].

Two plants, Ocimum sanctum and Acacia arabica are widely distributed and easily available throughout various geographical locations in India. The efficacy of O. sanctum as inhibitory compound has been documented against several viruses like Newcastle Disease virus, Vaccinia virus and Infectious Bursal Disease virus [17]. Similarly, A. arabica has been explored for its virucidal properties against Peste des petits ruminants (PPR) virus [18], along with inhibition of Goatpox virus replication [19]. However, antiviral H9N2 influenza activity of different extracts derived from the leaves of these two plants has not been studied.

For evaluation of the antiviral properties of medicinal plants against IAVs, three methods viz. tissue culture [20, 21], laboratory/experimental animals [22] and in ovo model [23] have been used frequently. Each method has its pros and cons. We preferred in ovo model, which is at the borderline of in vitro and in vivo studies and thus does not conflict with either ethical or legal aspects of animal protection [23, 24]. Therefore, keeping in view the endemicity and pandemic potential of H9N2, this study was taken up to assess the H9N2 inhibitory potential of various extracts derived from leaves of O. sanctum and A. arabica using in ovo model.

Spread of H9N2 subtype has demonstrated an increased theoretical threat to humans because of the potential emergence of novel subtypes of avian influenza [13]. It is being considered as an emerging pandemic threat in view of the clinical and confirmed cases of H9N2 in China, Hong Kong, Bangladesh and Egypt [14]. This reaffirms the need for search of new compounds with antiviral activity. With increase in drug resistance to synthetic antivirals, natural products remain an important alternative for the control infectious diseases. In the present study, the in ovo model has been used for studying antiviral activities of different leaves extracts of Ocimum sanctum and Acacia arabica against low pathogenic avian influenza (LPAI) H9N2.

In our previous work we were able to demonstrate 100% virucidal activity of cold aqueous extract of bark of A. arabica at a concentration of 0.625 mg/ml against HPAI H5N1 using in vitro model. Mild therapeutic activity was also demonstrated up to 48 h in cold aqueous extract of Ocimum tenuiflorum when tested against HPAI H5N1 [20].

In the present work, we had selected two plants, Ocimum sanctum and Acacia arabica for studying their efficacy against avian influenza H9N2 virus because these plants are widely distributed and easily available throughout various geographical locations in India and have strong record in the literature for their antiviral activity against different viruses [16, 17]. Three different approaches viz. virucidal (dose dependent), therapeutic (time dependent) and prophylactic (dose dependent) were employed to systematically explore the potential of extracts of leaves of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) against LPAI H9N2 using in ovo model.

In dose dependent virucidal activity, all the extracts of both the plants in doses (135, 67 and 33 mg/0.1 ml) displayed absence of HA titer indicating that all the extracts had inhibitory potential. On further reduction of dose rate in double dilution, polyphenol_acasia and polyphenol _Ocimum only maintained virucidal activity up to a low dose rate of 8.75 mg/0.1 ml which was confirmed by absence of HA in the harvested allantoic fluid of ECEs in the next two passages also. This virucidal effect could be due to masking/blocking of HA protein of the virus by the herbal extracts during the incubation period, which might have inhibited the H9N2 virus replication. Catechin compounds in polyphenols are known to have antiviral activity mediated by preventing adsorption of viruses to cells [34, 35]. Highly significant to significant decrease in the viral genome copy numbers was recorded in all the extracts at the MNTC dose (135 mg/0.1 ml) except crude extract _acacia (P < 0.05–0.001). Although the virucidal effect was seen more prominently in the HA test where the virus HA titer was absent in comparison to the virus control (mean HA titer 2^7.3) and vehicle control (mean HA titer 2^5.8), the quantification of the virus by real time RT-qPCR also corroborated with the HA results especially at higher extract doses. The results of both the assays clearly indicated that crude extract_ocimum in particular is a potent inhibitor of H9N2 virus replication.

Our study is further supported by the fact that individual plant extracts were less effective compared to crude extracts which may due to synergistic effect of individual component and some of the uncharacterised components [36]. Earlier studies for evaluation of virucidal activity against H9N2 virus by crude extract _echinecea and crude extract _sambucus against H9N2 virus has also been ascribed to blocking of the influenza virus entry via at least two virion targets, HA and NA on the surface of virus [37]. Mentofin® (combination of eucalyptus oil and peppermint) in another study has been reported to have complete virucidal activity against H9N2 virus in the presence of organic matter (skimmed milk) [38].

On assessing the therapeutic potential, we were able to demonstrate that all the extracts of O. sanctum (crude extract _ocimum , terpenoid _ocimum and polyphenol _ocimum ) significantly reduced the viral genome copy numbers (P < 0.0001) at all the tested time intervals of 24, 48 and 72 h post-inoculation. However, crude extract _ocimum and terpenoid _ocimum had higher therapeutic potential at 48 and 72 h post-inoculation, respectively. Although terpenoid _ocimum and polyphenol _ocimum treated groups showed a mean HA titer of 2^5.1, 2^4.8, respectively at 48 h post-inoculation and a slightly decreased mean HA titer of 2^4.8, 2^3.8, respectively at 72 h post-inoculation, the genome copy numbers in these treated groups were significantly reduced in comparison to virus control (P < 0.0001). The possible reason for this could be due to the decrease in concentration of the active ingredient which might have allowed the virus to replicate because of single dose regimen followed in the experiment. In addition, other possible reason might be the high sensitivity of real time PCR assay. Surprisingly, in A. arabica treated groups, crude extract _acacia and polyphenol _acacia showed significant decrease in viral genome copy numbers at a later time interval of 72 h post-inoculation (P < 0.01). Since the route of administration was via albumen, slow movement/distribution of A. arabica extracts from albumen to allantoic cavity might be the reason for delayed effect. Variable bioavailability of drugs, oseltamivir and ribavirin via albumin route has been established previously also [39].

In the case of dose dependent prophylactic activity assessment, only the high doses of crude extract _ocimum , polyphenol _ocimum and polyphenol _acacia were effective as observed by the absence of HA titer and significant decrease in viral genome copy numbers. Lower doses of these extracts failed to provide sufficient prophylactic activity. Activity of different polyphenolic compounds in plants worked their best when added to the cells just around or before the time of virus adsorption using in vitro model. They do possess high binding affinities with viral HA and NA which might be a reason for their prophylactic activity [34, 40‐42]. Thus, the therapeutic or prophylactic efficacy testing using in ovo model could be improved by considering the multiple dose regimens and monitoring the active ingredients diffusion of plant extracts through suitable methods. Moreover, since these extracts shows no adverse effect, a clinical pilot study would give further information on the potency of these extracts in protecting against H9N2 virus infection in chickens.

The treatment with the crude extract derived from the leaves of Ocimum sanctum leads to the significant H9N2 virus reduction in assessing the all the three; virucidal, therapeutic and prophylactic activities using in ovo model. At this stage, we conclude that crude extract _ocimum could be a promising extract for developing safe and efficacious antiviral compound against H9N2 virus. The protective efficacy of crude extract _ocimum might be attributed to multiple mechanisms of action, e.g. specific inhibition of a stage in viral intracellular multiplication and non-specific interference with virus-cell interactions like masking/blocking the HA glycoprotein. Furthermore, terpenoid _ocimum was also effective for virucidal and therapeutic activity and polyphenol _acacia for prophylactic activity. Nevertheless, it would of great importance to study further which particular active ingredient or a combination of these three compounds (crude extract _ocimum , terpenoid _ocimum and polyphenol _acacia ) could provide broader antiviral activity against H9N2 virus. This study also established the use of influenza virus-infected chick embryos for evaluation of new antiviral substances which could reduce the use of small laboratory animals required for such studies.