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Peste des Petits Ruminants Virus (PPRV) causes a severe contagious disease of sheep and goats and has spread extensively in last years through Asia and Africa. PPRV, known to infect exclusively small ruminants, has been recently reported in camels in Iran and Sudan. Reported clinical symptoms are similar to those observed in small ruminants, fatality rate still unknown. However most of the authors reported seropositive camels without clinical signs. Camel sensitivity to PPRV is still controversial and more investigation need to be performed. In this study, we tested camel susceptibility by an experimental infection using a virulent PPRV strain belonging to lineage IV. Young dromedary camels were infected intravenously and observed one month for clinical symptoms. Viraemia and virus secretion charge in swabs were evaluated by PCR. Seroconversion was assessed by ELISA and virus neutralisation test. Infected animals did not manifest any clinical symptoms of the disease and no virus was detected in secretions. Seroconversion was observed from day 14 post infection.

Control of lumpy skin disease in cattle is based on vaccination with live attenuated vaccines. The Kenyan strain KSGP 0240 is commonly used to vaccinate ruminants against capripox infections, but the conferred protection is still controversial. In this study, we report the draft genome sequence of the vaccine strain KSGP 0240, reisolated from cattle.

Lumpy skin disease virus (LSDV) causes an economically important disease in cattle. The only method for successful control is early diagnosis and efficient vaccination. Adverse effects of vaccination such as local inflammation at the injection site and localized or generalized skin lesions in some vaccinated animals have been reported with live vaccines. The aim of this work was to compare the safety of two lumpy skin disease (LSD) vaccine strains, Kenyan (Kn) Sheep and Goat Pox (KSGP O-240) and LSDV Neethling (Nt) strain, and to determine the etiology of the post-vaccination (pv) reactions observed in cattle. Experimental cattle were vaccinated under controlled conditions with Nt- and KSGP O-240-based vaccines, using two different doses, and animals were observed for 3 months for any adverse reactions. Three out of 45 cattle vaccinated with LSDV Nt strain (6.7%) and three out of 24 cattle vaccinated with Kn strain (12.5%) presented LSD-like skin nodules, providing evidence that the post-vaccination lesions may not be strain-dependent. Lesions appeared 1–3 weeks after vaccination and were localized in the neck or covering the whole body. Animals recovered after 3 weeks. There is a positive correlation between the vaccine dose and the appearance of skin lesions in vaccinated animals, at the 105 dose, 12% of the animals reacted versus 3.7% at the 104 dose. Both strains induced solid immunity when protection was measured by neutralizing antibody seroconversion.

Peste des Petits Ruminants (PPR) is a highly contagious and often fatal disease of sheep and goats. Conventional live vaccines have been successfully used in endemic countries however, there are not completely safe and not allowing differentiation between vaccinated and infected animals (DIVA). In this study, a recombinant Newcastle disease virus (NDV) expressing the hemagglutinin of PPRV (NDV-PPRVH) was evaluated on small ruminants by serology response in sheep and goats, experimental infection in goats and immunity duration in sheep. The NDV-PPRVH vaccine injected twice at 28 days' interval, provided full protection against challenge with a virulent PPR strain in the most sensitive species and induced significant neutralizing antibodies. Immunological response in goats was slightly higher than sheep and the vaccine injected at 108.0 50 % egg infective dose/mL allowed anti-PPRV antibodies that lasted at least 12 months as shown by antibody response monitoring in sheep. The NDV vector presented a limited replication in the host and vaccinated animals remained negative when tested by cELISA based on PPRV nucleoprotein allowing DIVA. This recombinant vaccine appears to be a promising candidate in a free at risk countries and may be an important component of the global strategy for PPR eradication.

Background Animal vaccination is an important way to stop the spread of diseases causing immense damage to livestock and economic losses and the potential transmission to humans. Therefore effective method for vaccine production using simple and inexpensive bioprocessing solutions is very essential. Conventional culture systems currently in use, tend to be uneconomic in terms of labor and time involved. Besides, they offer a limited surface area for growth of cells. In this study, the CelCradle™-500A was evaluated as an alternative to replace conventional culture systems in use such as Cell factories for the production of viral vaccines against small ruminant morbillivirus (PPR), rift valley fever virus (RVF) and lumpy skin disease virus (LSD). Results Two types of cells Vero and primary Lamb Testis cells were used to produce these viruses. The study was done in 2 phases as a) optimization of cell growth and b) virus cultivation. Vero cells could be grown to significantly higher cell densities of 3.04 × 109 using the CelCradle™-500A with a shorter doubling time as compared to 9.45 × 108 cells in Cell factories. This represents a 19 fold increase in cell numbers as compared to seeding vs only 3.7 fold in Cell factories. LT cells achieved modestly higher cell densities of 6.7 × 108 as compared to 6.3 × 108 in Cell factories. The fold change in densities for these cells was 3 fold in the CelCradle™-500A vs 2.5 fold in Cell factories. The titers in the conventional system and the bioreactor were not significantly different. However, the Cell-specific virus yield for rift valley fever virus and lumpy skin disease virus are higher (25 virions/cell for rift valley fever virus, and 21.9 virions/cell for lumpy skin disease virus versus 19.9 virions/cell for rift valley fever virus and 10 virions/cell for lumpy skin disease virus). Conclusions This work represents a novel study for primary lamb testis cell culture in CellCradle™-500A bioreactors. In addition, on account of the high cell densities obtained and the linear scalability the titers could be further optimized using other culture process such us perfusion.

Sheep and goat pox (SGP) with peste des petits ruminants (PPR) are transboundary viral diseases of small ruminants that cause huge economic losses. Recombinant vaccines that can protect from both infections have been reported as a promising solution for the future. SGP was used as a vector to express two structural proteins hemagglutinin or the fusion protein of PPRV. We compared immunity conferred by recombinant capripoxvirus vaccines expressing H or F or both HF. Safety and efficacy were evaluated in goats and sheep. Two vaccine doses were tested in sheep, 104.5TCDI50 in 1 ml dose was retained for the further experiment. Results showed that the recombinant HF confers an earlier and stronger immunity against both SGP and PPR. This recombinant vaccine protect also against the disease in exposed and unexposed sheep. The potential Differentiating Infected from Vaccinated Animals of recombinant vaccines is of great advantage in any eradication program.

A new inactivated vaccine against Bluetongue virus (BTV) serotypes 1 and 4, was developed from field isolates. Safety and efficacy of the vaccine were evaluated in sheep by serological monitoring and virus nucleic acid detection after experimental infection of vaccinated animals. Seroconversion was observed in vaccinated animals at day 14 post vaccination (pv) with neutralizing antibody titer of 1.9 and 1.8 for serotypes 1 and 4, respectively. The titer increase significantly after the booster reaching 2.7 and persist one year >1.5 for both serotypes. After challenge with virulent isolates, vireamia was recorded in control animals, as evident by q-PCR with threshold cycles (Ct) ranging from 24 to 31 and peaked at day 10 post challenge, while no vireamia was detected in vaccinated animals. Vaccinated sheep were fully protected against the disease and infection.

Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries. In this study, we expressed the modified antigen, EG95NC-GST, in Escherichia coli for use as a vaccine against Echinococcus granulosus in ruminants. We tested three different media formulations for E. coli culture and established for each culture conditions for optimal levels of soluble EG95 expression. The results demonstrate that SOC and TB media provided high yields in cell density and EG95 protein expression. Purification of the recombinant protein with affinity chromatography (using FPLC) was also performed to increase the purity of the EG95NC−-GST antigen.

Lumpy skin disease virus (LSDV), sheeppox virus (SPPV) and goatpox (GTPV) virus have been usually grown on primary cells for diagnosis, production and titration purposes. The use of primary cells present several inconvenient, heavy preparation, heterogeneous cell population, non-reproducible viral titration and presence of potential endogenous contaminants. Therefore investigating sensitivity of candidate continuous cell lines is needed. In this study, we compared the above Capripox viruses (CaPVs) sensitivity of primary cells of four origin (heart, skin, testis and kidney), with three cell lines (Vero, OA3.Ts and ESH-L). We tested sensitivity for virus isolation, replication cycle and titration, revealed by cytopathic effect (CPE), immunoenzymatic staining and immunofluorescence. Our results show that ESH-L cells and primary fetal heart cells present the highest sensitivity for CaPVs growth and detection. Vero cells can replicate those viruses but without showing any CPE while the titer obtained on OA3.Ts is lower than primary and ESH-L cells. ESH-L cells are an effective alternative to primary cells use for growing Capripoxviruses and their diagnosis.

Cystic echinococcosis (CE) is a zoonotic disease caused by the cestode parasite Echinococcus granulosus. The disease has an important impact on human health as well as economic costs including the cost of treatment as well as loss of productivity for the livestock industry. In many parts of the world where the disease is endemic, sheep and other livestock play an important role in the parasite's transmission. A vaccine to protect livestock against CE can be effective in reducing transmission and economic costs of the disease. A recombinant antigen vaccine has been developed against infection with E. granulosus (EG95) which could potentially be used to reduce the level of E. granulosus transmission and decrease the incidence of human infections. Further development of the EG95 recombinant vaccine as a combined product with clostridial vaccine antigens is one potential strategy which could improve application of the hydatid vaccine by providing an indirect economic incentive to livestock owners to vaccinate against CE. In this study we investigated the efficacy of the EG95 recombinant vaccine produced in Morocco by vaccination of sheep, including a combined vaccine incorporating EG95 and clostridia antigens. Vaccination with EG95 either as a monovalent vaccine or combined with clostridia antigens, protected sheep against a challenge infection with E. granulosus eggs and induced a strong, long lasting, and specific antibody response against the EG95 antigen.

Rift Valley fever (RVF) causes serious health and economic losses to the livestock industry as well as a significant cause of human disease. The prevention of RVF in Africa is a global priority, however, available vaccines have only been partially effective. Therefore, the objective of this study was to evaluate the safety and immunogenicity of a live, attenuated recombinant RVFV arMP-12ΔNSm21/384 nucleotide deletion vaccine candidate in domestic ruminants. Evaluation involved testing to determine the infectivity titer of the vaccine virus in Vero cells for industrial scale up vaccine production. Safety experiments were conducted to determine the potential of the vaccine virus to revert to virulence by serial passages in sheep, the possibility of virus spread from vaccinated sheep and calves to unvaccinated animals, and the potential health effects of administering overdoses of the vaccine to sheep, goats and calves. The immunogenicity of 3 doses of 104, 105 and 106 Tissue Culture Infectious Doses50% (TCID50) of the vaccine was assessed in 3 groups of 10 sheep and 3 groups of 10 goats, and doses of 105, 106 and 107 TCID50 was evaluated in 3 groups of 10 calves subcutaenous vaccintation. The results showed that the infectivity titer of the vaccine virus was 108.4 TCID50/ml, that the vaccine did not spread from vaccinated to un-vaccinated animals, there was no evidence of reversion to virulence in sheep and the vaccine overdoses did not cause any adverse effects. The immunogenicity among sheep, goats and calves indicated that doses of 104-106 TCID50 elicited detectable antibody by day 7 post-vaccination (PV) with antibody titers ranging from 0.6 log to 2.1 log on day 14 PV with sustained titers through day 28 PV. Overall, these findings indicated that the RVFV arMP-12ΔNSm21/384 vaccine is a promising candidate for the prevention of RVF among domestic ruminants.

Peste des Petits Ruminants virus (PPRV) is a member of the Morbillivirus subgroup of the family Paramyxoviridae, and is one of the most contagious diseases of small ruminants throughout Africa and the rest of the world. Different cell lines have previously been used to isolate PPRV but with limited success. Thus, to improve the isolation of Morbilliviruses, human, canine, and goat homologues of the lymphocyte receptor signaling lymphocyte activation molecule (SLAM) have been introduced into cells that can support virus replication. However, the amino acid sequence of SLAM varies between species, and often requires adaptation of a particular virus to different versions of the receptor. The protein sequence of Nectin-4 is highly conserved between different mammals, which eliminate the need for receptor adaptation by the virus. Cell lines expressing Nectin-4 have previously been used to propagate measles and canine distemper viruses. In this study, we compared infections in Vero cells expressing canine SLAM (VeroDogSLAM) to those in Vero cells expressing Nectin-4 (VeroNectin-4), following inoculations with wild-type strains of PPRV. Virus isolation using VeroNectin-4 cells was successful with 23% of swabbed samples obtained from live infected animals, and was 89% effective using post-mortem tissues of infected sheep. By contrast, only 4.5% efficiency was observed from swab samples and 67% efficiency was obtained in virus isolation from post-mortem tissues using VeroDogSLAM cells. The average incubation period for virus recovery from post-mortem tissues was 3.4 days using VeroNectin-4 cells, compared with 5.5 days when using VeroDogSLAM cells. The virus titers of PPRV obtained from VeroNectin-4 cells were also higher than those derived from VeroDogSLAM cells. A comparison of the growth kinetics for PPRV in the two cell lines confirmed the superiority of VeroNectin-4 cells for PPR diagnostic purposes and vaccine virus titration.

Background Peste des petits ruminants (PPR) is a highly contagious viral disease of small ruminants in Asia and Africa. In 2008, a PPR outbreak was reported for the first time in Morocco and a mass vaccination campaign allowed control of the disease. In this study, the susceptibility of four Moroccan local breeds of small ruminants to PPR virus was investigated by experimental infections. The objective was to make recommendations for improved epidemiological surveillance in Morocco by evaluating the susceptibility of the dominant Moroccan small ruminant breeds. Three parameters were studied: hyperthermia, clinical scoring and virus excretion. The outcome was compared to Alpine goats, which are considered one of the most sensitive breeds. Results The study showed that the local goat breed was the most sensitive breed with a susceptibility rate of 67%, followed by Timahdit, Beni Guil and Sardi sheep with 48, 29 and 26%, respectively. Serological testing including enzyme-linked immunosorbent assay and viral neutralization showed that the Timahdit breed developed a stronger antibody response compared to the other breeds. Although the clinical signs observed in the sheep were mild, evidence of viral excretion was detected by means of a polymerase chain reaction assay. Conclusions It is recommended that effective surveillance should focus on susceptible breeds complemented with serological surveillance of the sheep population.

Equine influenza is an infectious and contagious disease of horses. Studies on this topic are rare in the Maghreb countries. Therefore, the aim of this work is to present the various studies conducted on serological and molecular equine influenza virus since 1972 in the Maghreb region in particular in Morocco, Algeria and Tunisia. A total of four equine influenza strains were isolated in the Maghreb Arab region. A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004(H3N8), A/equine/Essaouira/3/2004(H3N8) and A/equine/Algiers/1/1972(H3N8). The highest homology of HA nucleotide sequences of A/equine/Nador/1/1997(H3N8) with European strains: A/equine/Italy/1199/1992(H3N8) and A/equine/Brescia/1999(H3N8) clearly clustered A/equine/Nador/1/1997(H3N8) with the strains belonging to the European lineage. However, A/equine/Algiers/1/1972(H3N8), A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) were placed in the predivergent lineage indicating that like-Miami/63 strains infected equids in Morocco in 2004. This finding does not corroborate the recent studies of the H3N8 subtype of equine influenza viruses which have demonstrated that the oldest equine H3N8 strains, circulating before 1990 apparently went extinct.

Background Three equine influenza viruses, A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004(H3N8), and A/equine/Essaouira/3/2004(H3N8), were isolated from different Equidae during local respiratory disease outbreaks in Morocco in 1997 and 2004. Their non-structural (NS) genes were amplified and sequenced. Results The results show high homology of NS nucleotide sequences of A/equine/Nador/1/1997 with European strains (i.e., A/equine/newmarket/2/93 and A/equine/Grobois/1/1998) and clustered into the European lineage. However, NS gene of A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) strains indicated high homology with equine influenza strains that had circulated before 1990 (A/equine/Fontainbleu/1/1979(H3N8), which belonged to a pre-divergent phase Amino acid sequence comparison of the NS1 protein with reference strain A/equine/Miami/1963(H3N8) shows that the A/equine/Nador/1/1997(H3N8) strain has 12 substitutions at the residues D/24/N, R/44/K, S/48/I, R/67/Q, A/86/V, E/139/K, A/112/T, E/186/K, L/185/F, A/223/E, S/213/T and S/228/P. In both A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8) strains, the NS1 sequences present one common mutation at the residue: S/228/P. Conclusion It seems that all of these substitutions are not produced at the key residues of the RNA-binding domain (RBD) and the effector domain (ED). Consequently, we can suppose that they will not affect the potency of inhibition of cellular defences, and the virulence of the Moroccan equine strains will be maintained.

Rift valley fever (RVF) is a highly significant vector-borne disease causing huge economic loses in livestock (ruminants and camels) and also human fatalities. The disease is endemic in most Sub-Saharan African countries, including West Africa, and has been present in the Middle East since 2010. Vaccination is considered to be the most effective way to prevent and control the expansion of the disease. Currently available attenuated live vaccines for RVF have significant limitations in that they are either thermolabile (CL13 strain vaccine) or causes abortion and teratogenic effects (Smithburn strain vaccine). This study therefore set out to develop a safe and effective thermostable live attenuated RVF vaccine. The existing CL13 vaccine, which is a naturally attenuated strain, was made thermostable through three cycles of heating (56°C) and selection. The resulting candidate vaccine (CL13T) was stable at 4°C for 20 months and shows significantly improved levels of thermostability over the existing CL13 vaccine. A pilot batch of the CL13T vaccine was produced and tested for safety and efficacy in cattle, sheep and goats. The vaccine was found to be safe, with no clinical signs or side effects observed in vaccinated animals, and there was no evidence for circulation of the virus in the blood of animals post-vaccination. On testing for efficacy in cattle, sheep and goats, through the detection of neutralizing antibodies post-vaccination, good levels of neutralizing antibodies were detected for a minimum of one year in sheep and goats, and neutralizing antibodies were detected for least 4 months in cattle. This new thermostable vaccine could represent an efficient tool for the control of rift valley fever in endemic countries. The vaccine also has the potential to be used, along with an appropriate diagnostic test, to differentiate vaccinated from infected animals (DIVA).

Background The equine influenza (EI) is an infectious and contagious disease of the upper respiratory tract of horses. Two outbreaks were notified in Morocco during 1997 and 2004 respectively in Nador and Essaouira. The aims of the present study concern the amino acids sequences comparison with reference strain A/equine/Miami/1963(H3N8) of the HA2 subunit including the cleavage site of three equine influenza viruses (H3N8) isolated in Morocco: A/equine/Nador/1/1997(H3N8), A/equine/Essaouira/2/2004 (H3N8) and A/equine/Essaouira/3/2004 (H3N8). Results The obtained results demonstrated that the substitutions were located at Ectodomain (ED) and transmembrane domain (TD), and they have only one arginine in cleavage site (HA1-PEKQI-R329-GI-HA2). In the Ectodomain, the mutation N/154 2 /T deleted the NGT glycosylation site at position 154 for both strains A/equine/Essaouira/2/2004(H3N8) and A/equine/Essaouira/3/2004(H3N8). Except for mutation D/1602/Y of the A/equine/Nador/1/1997(H3N8) strain, the other mutations were involved in non conserved sites. While the transmembrane domain (TM) of the strain A/equine/Essaouira/3/2004(H3N8) exhibits a substitution at residue C/199 2 /F. For the A/equine/Nador/1/1997(H3N8) strain the HA2 shows a mutation at residue M/207 2 /L. Three Moroccan strains reveals a common substitution at the residue E/211 2 /Q located between transmembrane domain TM and the cytoplasmic domain (CD). Conclusion The given nature virulence of three Moroccan strains, the identified and reported mutations certainly played a permissive role of infection viral process.

SUMMARYPrior to the recent outbreak of equine encephalosis in Israel in 2009, equine encephalosis virus (EEV) had only been isolated from equids in South Africa. In this study we show the first evidence for the circulation of EEV beyond South Africa in Ethiopia, Ghana and The Gambia, indicating that EEV is likely to be freely circulating and endemic in East and West Africa. Sequence analysis revealed that the EEV isolate circulating in The Gambia was closely related to an EEV isolate that was isolated from a horse from Israel during the EEV outbreak in 2009, indicating that the two viruses have a common ancestry. Interestingly horses in Morocco tested negative for EEV antibodies indicating that the Sahara desert may be acting as a geographical barrier to the spread to the virus to North African countries. This evidence for EEV circulation in countries in East and West Africa sheds light on how the virus may have reached Israel to cause the recent outbreak in 2009.

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Understanding the role of humans in the dispersal of predominately animal pathogens is essential for their control. We used newly developed Bayesian phylogeographic methods to unravel the dynamics and determinants of the spread of dog rabies virus (RABV) in North Africa. Each of the countries studied exhibited largely disconnected spatial dynamics with major geo-political boundaries acting as barriers to gene flow. Road distances proved to be better predictors of the movement of dog RABV than accessibility or raw geographical distance, with occasional long distance and rapid spread within each of these countries. Using simulations that bridge phylodynamics and spatial epidemiology, we demonstrate that the contemporary viral distribution extends beyond that expected for RABV transmission in African dog populations. These results are strongly supportive of human-mediated dispersal, and demonstrate how an integrated phylogeographic approach will turn viral genetic data into a powerful asset for characterizing, predicting, and potentially controlling the spatial spread of pathogens., Author Summary At least 15 million doses of anti-rabies post-exposure prophylaxis are administered annually worldwide, and an estimated 55,000 people die of rabies every year. Over 99% of these deaths occur in developing countries, predominantly in Asia and in Africa where rabies is endemic in domestic dogs. Despite the global health burden due to rabies, little is known about the patterns of the spread of dog rabies in these endemic regions. We used recently developed Bayesian analytical methods to unravel the dynamics and determinants of the spatial diffusion of dog rabies viruses in North Africa based on viral genetic data. Our analysis reveals a combination of restricted spread across administrative borders, the occasional long-distance movement of rabies viruses, and a strong fit between spatial spread of the virus and road distances between localities. Together, these data indicate that by transporting dogs, humans have played a key role in the dispersal of a major animal pathogen. Our studies therefore provide essential new information on the transmission dynamics of rabies in Africa, and in doing so will greatly assist in future intervention strategies.

Rift Valley Fever (RVF) is an emerging zoonotic disease present in sub-Saharan Africa and the Arabian Peninsula. Vaccination of cattle against RVF with a RVF virus clone 13 (CL13) strain has proven to be efficacious, and avoids the side effects caused by other available live vaccines. In order to determine the temperature stability of the CL13 vaccine, lyophilized and liquid forms were tested and titrated for the presence of live virus after storage for various time periods at various temperatures. Results showed that the virus could be stored lyophilized at 4 °C for more than 12 months, with no reduction of infectivity. However, the vaccine was shown to be unstable at room temperature and at 37 °C in both lyophilized and liquid forms. This data shows that the CL13 vaccine is highly reliant on a cold chain, emphasizing the need for the vaccine to be made thermostable in order to allow for efficient vaccine storage and delivery in endemic tropical countries.

The equine influenza (EI) is a highly contagious respiratory viral disease of equines. The aim of the present study was to determine the amino acid mutation sequences of the partial nucleoprotein (NP), which includes four epitopes of the equine leucocyte antigen (ELA) of A/equine/Nador/1/1997 (H3N8). These epitopes are critical for their binding to major histocompatibility molecule complex (MHC) class I and recognition by specific Cytotoxic T Lymphocytes (CTLs). The isolate was subjected to RT-PCR amplifications followed by sequencing analysis. Phylogenetic analysis showed that Moroccan isolate belongs to equine host-specific lineage and more closely related to Italian strains A/equine/Rome/5/1991 and A/equine/Italy/1062/1991. Amino acid sequence comparison of the NP showed that the strain A/equine/Nador/1/1997 has twelve substitutions at the residues T/284/A, A/286/T, R/293/K, I/299/V, V/312/I, N/319/K, S/344/L, V/353/I, M/374/I, C/377/N, N/397/S and R/452/K. All substitutions concerned both the interaction domains NP–NP and NP–PB2. However, the mutation N319 K enhances the NP–PB2 interaction and polymerase activity in mammalian infected cells. S/344/L mutation was located on the FEDLRVSNFI epitope (aa 338–347), this substitution is likely to help the virus to overcome the barrier of cell-mediated immunity of the host. The identified mutations were grouped into two groups, one included residues that facilitate the adaptation and evolution of influenza viruses within the equine lineage such as A/286/T, R/293/K, S/344/L, V/353/I and R/452/K, while the second contained the substitutions which enhance the virulence as polymerase activity (N319 K) and mutations that affect CTL epitopes, resulting in an escape from immune surveillance by specific CTLs (S/344/L).