Avian viral diseases outbreak due to Avian Influenza (AI), Infectious Bursal Disease (IBD), Newcastle Disease (ND) and Infectious Bronchitis (IB) viruses remain the main cause for huge loss to poultry industry across the world. There are many predisposing factors for the viral disease outbreaks, but some instances like emerging new viral strain, changing weather, rearing of waterfowl along with poultry farms, migratory birds and intense farming make the situation more critical. Some viruses which are endemic in some regionsare difficult to eliminate by conventional vaccination methods. Despite maintaining good preventive measures like healthy nutrition, biosecurity and vaccination program, viral outbreakoccurs frequently than earlier. Since viral outbreaks causes high mortality within few days, there is a need for strategies to induce early innate immune response against these viruses. Even though extensive research has been conducted in the last several decades to exploit innate immune response against viral outbreaks, the molecular mechanism of avian innate immune response is not fully understood. Hence, it’s vital to understand the mechanism at molecular basis and its action against viral pathogens. This review outlines the recent advances in antiviral strategies (novel polysaccharides,probiotics,toll like receptor agonist,cytokines and interferon stimulating gene) focusing on early activation of innate immunity components.
Table-1: First occurrence of different poultry viral outbreaks worldwide.
Figure 1: Common poultry virus versus organs affected in poultry.
Avian Influenza Viruses(AIVs)
AIVsare enveloped, single stranded ribonucleic acid (RNA) viruses belonging to the family Orthomyxoviridae. AIVs are typically classified according to their pathogenicity; as a low pathogenic avian influenza (LPAI) virus or a highly pathogenic avian influenza virus (HPAI). Although HPAI viruses, such as H5N1 are of great concern to the poultry industry and public health, outbreaks of LPAI viruses had become problematic. HPAI viruses cause severe outbreaks and mortality in chickens, whereas, LPAI viruses usually cause mild respiratory disease.
Infectious Bursal Disease Virus (IBDV)
IBDV, a bi-segmented, double-stranded RNA Birna virus, is an important naturally occurring viral diseases of commercial chickens worldwide. IBD viruses have recently been classified into 7 genogroups based on the molecular structure of hypervariable region of Viral Protein2, and include the vital strains: classical (cl), very virulent (vv), antigenic variant (av) and other strains circulating worldwide. Infectious bursal disease virus is an endemic agent in poultry which causes a highly contagious immunosuppressive disease in chickens and destroys dividing Immunoglobulin M (IgM) bearing B-lymphocytes in Bursa of Fabricius.Immunosuppression due to IBDV increases susceptibility of chickens to opportunistic pathogens which may result in vaccine failure. IBDV-infected birds may become a transmitter for other viral pathogens. For example, low pathogenic duck adapted avian influenza virus becomes more virulent when serially passaged in IBDV-infected chickens. This might be one of the reasons for LPAI outbreaks in West Bengal,North East regions and other parts of India.
Newcastle Disease Virus (NDV)
Newcastle diseaseis a widely present avian viral disease, caused by the virulent forms of avian paramyxovirus of serotype 1 (APMV-1) and has global economic importance. The virus infects wide range of birds (around 300 different species), both domestic and wild population. Strains of NDV are classified into two classes(Class-I and Class-II). Use of D. G. Diel.,et. al. (2012a) system and criteria led to the classification of Class-I NDV isolates into a single genotype (genotype-1), while Class-II encompassed viruses delineated into 15 genotypes (I to XV). Recently, few more genotypes (XVI, XVII, XVIII and XXI)has been identified.Waterfowland pigeons are natural reservoirs of NDV pathotype, except viscerotropic velogenic viruses for which natural reservoirs areunidentified. NDV has three pathotypes: lentogenic, mesogenic and velogenic. Viruses of low pathogenicity or lentogens result in subclinical diseases, whereas, moderate pathogenicity or mesogens causes clinical signs of disease, but typically result in non-lethal outcomes in chickens. Viruses of high pathogenicity or velogens cause serious disease and mortality among affected birds. NDV affects neurological, gastrointestinal and respiratory systems of birds. Clinical signs of infection with APMV-1 may include decreased egg production, depression, diarrhea, respiratory distress, neurologic signs, torticollis, and death. According to Office International des Epizooties(OIE), NDV is regarded as the fourth most devastating disease in poultry after highly pathogenic avian influenza, avian infectious bronchitis and low pathogenic influenza.Studies have reported the presence of class-II genotype-XIII in Northeast, Central and other part of Indiapoultry fields.Recently, Dimitrov Kiril M.,et.al.has shown the presence of genotype VI,VII,XII and XVIII across the world.
Avian Infectious Bronchitis Virus (IBV)
Avian IBV is ahighly contagious pathogen with geographically distinct strains.In chickens, the virus transmits mainly through aerosols and ingestion of feces, or contaminated water and feed. The resulting disease, infectious bronchitis (IB), is predominantly mild to severe respiratory disease. Besides, the virus can infect other part of respiratory system, including lungs and air sacs. Depending on virus strain, the virus can be found in other epithelial cells, such as oviduct and kidney epithelial cells, and causes infection in other tissues. Some of these strains, including variant D388, known as QX genotype, or the Massachusetts (Mass) serotype cause pathological lesions in chicken oviducts, which leads to cystic oviduct formation in young pullets, and subsequently false layer syndrome in the peak of production. In broilers, pathognomic changes were the presence of mucoid secretion,congestion in trachea, mild focal areas of lung consolidation, swelling and congestion of kidney(mottled kidney), and some time pallor of ureters that contain urate deposits.
Inclusion Body Hepatitis(IBH)
IBHwith the hydropericardium syndrome (HPS) (IBH-HPS), popularly known as “Litchi heart disease”,is an acute, viral disease in young, 2–7 week old broiler chickens. Outbreaks of IBH are characterized by a sudden onset of mortality that can exceed 30% with a short clinical course of 4–5 days. Clinical signs include severe depression, ruffled feathers and a crouching position. On necropsy, the affected birds have pale, swollen, friable, hemorrhagic livers with focal to extensive necrosis,accumulation of a clear or straw-colored fluid of aqueous or gelatinous consistency in the pericardial sac.Kidneys were swollen and congested, and basophilic intranuclear inclusion bodies in hepatocytes.Fowl adenoviruses (FAdV) are classified into five species (FAdV-A to FAdV-E) with 12 serotypes (FAdV-1 to 8a, 8b thru 11) based on restriction enzyme digestion patterns and serum cross-neutralization tests.The disease is caused by a non-enveloped, icosahedral fowl adenovirus serotype-4 (FAdV-4) belonging to Adenovirus C species of the family Adenoviridae.
Immunesystem is a multifaceted arrangement of membranes (skin, epithelial and mucus), cells, and molecules whose function is to eradicate the invading pathogens or cancer cells from a host. Defense against viral infections in poultry consists of innate and adaptive mechanisms. Immune response may result in either eliminating or neutralization of pathogen/ antigen.
Innate defense is mainly formed by natural killer cells, granulocytes and macrophages, and their secreted products such as nitric oxide and various cytokines. Innate defense is of crucial importance early in viral infections. Adaptive defense can be divided into humoral and cellular immunity. Natural killer cells and dendritic cells are connecting link between the innate and adaptive immunity. Induction of the innate immune pathways is important for early anti-viral defense.Stimulation of innate immunity could be an alternative therapeutic approach to current treatments, as it induces local production of antimicrobial molecules. The main attractive features of innate immunity are rapid response after stimulation, cross protection against un-related pathogens and potential for therapeutic protection against viral outbreaks. The innate immunity features itself make it more demandable in present scenario as there is aneed of rapid response, cross and therapeutic protection against viral outbreaks. Priming of innate immunity becomes crucial as future activation of adaptive immune systemdepends on itas shown in Figure-2.
Figure-2: Possible immune responses by activated macrophage.
PathogenAssociated Molecular Patterns and Pattern Recognition Receptors
Pathogen associated molecular patterns(PAMPs) are unchanging molecules produced by the invading pathogens as alarm signals.PAMPs are highly distinct molecular structures present in pathogens and microbes, including lipopolysaccharides (LPS), microbial lipoprotein and peptidoglycans of microorganisms. Pattern recognition receptors (PRRs) are sensor like receptors present in innate immune cells which detect molecule typical for the pathogen.Recognition of pathogen depends on PRRs ability to identify PAMPs specific for each pathogen. Early recognition of pathogen is essential for optimum innate immune response. Three main families of PRRs include toll-like receptors (TLRs),retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and a nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs).These families act alone or together to induce intracellular signaling-inflammatory cytokines, anti-apoptotic factors, type I interferons (IFNs) and host defense peptide (HDP) production as shown in Figure-3.
Figure-3: Activation pathway of macrophage by viral PAMP- host PRR/ TLR recognition.
Abbreviations: MDA5- Melanoma differentiation associated gene5,MyD88-Myeloid differentiation primary response88,IRF7- Interferon regulatory factor7,NFkB- Nuclear factor kappa B,IFN- Interferon,ISG- Interferon stimulated gene,IL- Interleukin,NO- Nitric oxide,JAS-Janus kinase,STAT- Signal transducers and activators of transcription,PKR- Protein kinase R.
Toll-like Receptors (TLRs)
Toll-like receptors are innate immune molecules that can induce anti-viral responses after interacting PAMP. Overall, TLRs are the most studied member of PRRs in chickens. There are 10 chicken TLRs. Among multiple types of TLRs, TLR3 and TLR7 recognise double-stranded RNA and single-stranded RNA virus respectively.For viral diseases, role of cytoplasmic PRRs are vital as viral nucleotides are recognized inside the cell by intracellular or cytoplasmic PRRsas shown in Figure-3.
Recent Advances in Antiviral Strategies
Novel Polysaccharides: Algal beta glucan
Use of immune modulators such as beta-1,3-glucan strengthen an animals’ innate immunity. In poultry, supplementing feeds with beta-1,3-glucan has shown to increase immune cell activation and immune cell recruitment to the gut.Studies showed that supplementation ofalgal beta-1,3-glucan product improves the immune response to NDV and IBD. Recently, paramylon, an algal beta-glucan from the microalgae Euglena gracilis has gained attraction by leading industry manufacturer. In contrast to yeast, this microalga has more than 90% intracellular beta glucan. Macrophages isolated from birds fed β-glucans demonstrated enhanced interleukin (IL)-1, IL-2 and interferon (IFN)-γ levels. Dietary β-glucan increases the size of primary and secondary lymphoid organs, providing further evidence of their immunomodulating capabilities.Studies have shown that linear beta-1,3-glucan get dissolved in gastrointestinal tract and release small(1-3 micron) beta glucan granules which stimulate innate immunity.Due to anti-inflammatory properties of linear beta-1,3-glucan,it provides optimum immune response without consuming energy of host. Aleta™, mainly composed of linear1-3 algal beta glucan is a latest generation natural feed additive for poultry and it stimulates immune system and improves host defense mechanism,consequently reducing viral disease outbreaks.
In addition to balancing the intestinal microbiota population, probiotics enhance the immune responses to infectious agents in humans and chickens.In poultry, about 70% immunity resides in gut,and mucosal immunity development becomes of utmost importance.Studies have shown that Bacillus subtilis PB6 (CLOSTAT™) improve immunity by improving phagocytosis index and intestinal cytokines interleukin – 6, IL- 10 significantly(P≤0.05).Bacillus subtilistype probiotics contributed positively for better growth performance, improved immune system and modulated morphology of lymphoid organs and gut mucosa in broilers.Treatment of macrophages with certain Lactobacillus strains of probioticssignificantly(P≤0.05) enhanced the antiviral functions. Probiotic lactobacilli can induce expression of certain antimicrobial peptides and stimulate production of natural antibodies in chickens. Probiotic effects are not just limited to intestine,but also exert effects on other organs such as respiratory or reproductive system. Studies haveshown immunomodulation of alveolar macrophages byBacillus subtilis against some viral disease.
TLR Ligand or Agonist
TLR Agonists for TLR2, TLR3, TLR7, TLR8 and TLR9, either singly or in combination stimulate the innate immune responses to protect against influenza A virus (IAV) infection in mammals and poultry. Increased expression of IL-1β in macrophages induced with Pam3CSk4,TLR2 ligand coincide with the reduced AIV replication.TLR7 binds to viral single-stranded ribonucleic acid (ssRNA) or synthetic analogs like resiquimod, which activates the myeloid differentiation primary response gene 88 (MyD88)-mediated pathway. Activation of this pathway leads to the production of pro-inflammatory cytokines interleukin (IL)1β and IL-6as shown in Figure-3.
Interferon gamma (IFN-γ) is a proinflammatory cytokine released by T cells. Studies indicated the relationship between late expression of IFN-γ and more viral replication.However, study done by Leonardo Susta.,et. al.,2013, had suggested that early expression of IFN-γ had a significant protective role against the effects of highly virulent NDV infection in chickens.Diet with graded levels of chromium(Cr) either through feed or drinking water modulates the expression of IFN-γ.Chromium supplementation through water produced immediate and higher IFN-γ mRNA expression in broiler chickens but sustained higher IFN-γ mRNA expression in response to NDV through feed.
Interferon Stimulating Genes (ISGs)
Interferon stimulating gene expressions are stimulated by type-I interferon. Host cells produce many ISGsthrough Janus kinase/signal transducers and activators of transcription(JAK-STAT) signaling pathway to combat viral infection(Figure-3). Viperin is an antiviral ISG with enzymatic activity that prevents the release of newly-synthesized influenza virion by unsettling lipid rafts during virus assembly.Protein kinase R(PKR) is an interferon induced kinase with antiviral, antiproliferative and pro-apoptotic activities. Binding of dsRNA in cytoplasm activates the kinase activity of PKR(Figure-3). Then it interferes with viral replication and cell proliferation via inhibiting protein translation.
Recognition of pathogen is a prerequisite for activation ofimmune response. Balanced expression of different cytokines is the key for successful immune response. Out of the strategies mentioned, novel polysaccharides (Aleta™,Algal Beta-Glucan),probiotics (CLOSTAT™) and chromium induced early IFN-γ expression may be practical and suitable approach for treatment and prevention against viral outbreaks in current situation. Activation of viral nucleic acid induced mechanisms involve the production of IFN and other pro-inflammatory cytokines, which could be considered as the therapeutic candidates against poultry viruses. Thus, understanding the molecular mechanism involved in virus-host interaction,it is vital for antiviral immunity and development of new antiviral strategies in poultry industry.
Note: References available on request.
by Dr. Ajay Kumar, Dr. Venket Shelke and Dr. Partha Das, Kemin Industries South Asia Pvt. Ltd.