Bird Flu
Dr. Todkari Abhishek Mahendra, Livestock Development Officer, Grade I, Government of Maharashtra
Abstract:
Avian influenza, attributed to the Influenza A virus within the Orthomyxoviridae family, holds significant economic implications. Highly pathogenic strains, with mortality rates nearing 100%, pose substantial challenges for poultry farmers. Pathogenicity hinges on specific amino acids, namely arginine and cysteine, at the cleavage site. The implementation of culling policies in outbreak scenarios inevitably results in severe economic losses. In addition to seasonal epidemics, influenza viruses can unpredictably trigger pandemics, complicating containment efforts. Vaccination stands as a pivotal measure in curtailing influenza transmission. One promising approach to combat pandemics involves the development of a novel vaccine with extended and wide-ranging efficacy. The extracellular domain of the M2 protein (M2e) in influenza A viruses represents a conserved region and a promising candidate for a universal influenza vaccine.
Keywords: Avian Influenza, HPAI, Universal Influenza vaccine, epidemic, pandemic
Introduction:
The Orthomyxoviridae family encompasses crucial genera such as influenza A, B, and C viruses, along with thogotovirus (Cox et al., 2000), distinguished by antigenic variations in their nucleocapsid and matrix proteins. Influenza A viruses are further categorized into subtypes based on the antigenic properties of their hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins, exhibiting substantial variability with less than 30% amino acid conservation among subtypes (Fouchier et al., 2005). With 16 HA and 9 NA subtypes identified, influenza A viruses are the sole type among A, B, and C influenza viruses capable of infecting various animal species, including humans, birds, swine, and horses. Avian influenza (AI) viruses, classified under type A (Swayne et al., 2003), pose significant economic threats to poultry and potential health risks to humans.
Virion Properties:
The influenza virus presents a lipid envelope housing large glycoprotein spikes surrounding helically symmetrical nucleocapsid segments. Influenza A and B viruses exhibit two spike types: homotrimers of hemagglutinin glycoprotein (rod-shaped) and homotetramers of neuraminidase glycoprotein (mushroom-shaped). Influenza A virus, responsible for epizootics, epidemics, and pandemics, is an enveloped, negative-sense, single-stranded RNA virus with a helical nucleocapsid. Its release from host cells involves budding through the plasma membrane post-replication. Avian influenza virus comprises surface proteins HA, NA, and M2 (ion channel), inducing humoral immune responses. Additionally, nonstructural proteins, including PB2, PB1, PA, and NP, form the polymerase complex required for viral genome transcription.
Transmission:
Wild birds serve as natural hosts and reservoirs for avian influenza viruses, playing crucial roles in disease evolution, maintenance, and transmission. Migratory birds, particularly, contribute significantly to virus dissemination. Bird-to-bird transmission complexity varies with virus strain, bird species, and environmental factors. To some extent, the ability of virus to spread is related to quantity of the viral particles released by respiratory or intestinal route. Highly pathogenic viruses induce rapid avian deaths, potentially with a lower viral particle excretion and therefore poor transmission to susceptible hosts compared to low pathogenic strains. Virus shedding occurs in feces, contaminating water sources and facilitating transmission via fecal-oral or fecal-cloacal routes. For example, Webster et al. (1978) estimated up to 108.7 mean egg infectious doses per gram of faeces from infected ducks. This contaminates lake or pond water, and to the extent that virus may be isolated from untreated lake water where large numbers of waterfowl are found (Hinshaw et al., 1979). The virus may remain infective in lake water for up to 4 days at 22ºC and over 30 days at 0ºC (Hinshaw et al., 1980).
Pathology:
Avian influenza infections in poultry manifest diverse clinical signs, ranging from asymptomatic cases to severe respiratory diseases, decreased productivity, and occasionally, high morbidity and mortality rates. These viruses are categorized into mildly pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) based on intravenous pathogenicity tests. HPAI viruses cause severe disease with high mortality rates, while LPAI viruses vary in pathogenicity, often leading to secondary infections and lower mortality rates. The procedure for the pathogenicity testing includes the inoculation i.v. of 0.2 ml of a 1/10 dilution of allantoic fluid from embryonated egg passaged virus into 4-6-week-old specific pathogen free (SPF) chickens. If six or more of eight inoculated chickens die within 10 days, the virus is considered highly pathogenic, and if fewer die, it is considered to be mildly pathogenic or nonpathogenic. Typically, viruses classified as HPAI in the i.v. pathogenicity test cause severe disease with high mortality in the field. LPAI viruses can vary greatly in pathogenicity for poultry in the field, from no disease, respiratory disease with loss in production, and occasionally to high morbidity with high mortality. Although the range of disease symptoms of LPAI can overlap with the disease caused by HPAI, the high mortality in the field due to LPAI viruses is often related to secondary pathogens, and when the isolates are tested in the laboratory with SPF birds, mortality is usually much lower.
The primary difference between HPAI and LPAI virus is systemic versus local replication, respectively. This difference in the site of replication is related to differences in cleavage of the hemagglutinin (HA) protein into the HA1 and HA2 subunits which is necessary for the virus to be infectious. The HA protein from HPAI virus can be cleaved by endogenous proteases that are found in most cells of the body, but the HA protein of LPAI virus can only be cleaved by trypsin-like proteases that are primarily found in the respiratory and enteric tracts. HPAI viruses have multiple basic amino acids at the HA cleavage site which allows for the enhanced HA cleavability in many different cell types throughout the visceral organs (Steinhauer, 1999).
The haemagglutinin glycoprotein for influenza viruses is produced as a precursor, HA0, which requires post translational cleavage by host proteases before it is functional and virus particles are infectious (Rott, 1992). The HA0 precursor proteins of avian influenza viruses of low virulence for poultry have a single arginine at the cleavage site and another at position -4. These viruses are limited to cleavage by host proteases such as trypsin-like enzymes and thus restricted to replication at sites in the host where such enzymes are found, i.e. the respiratory and intestinal tracts. HPAI viruses possess multiple basic amino acids [arginine and lysine] at their HA0 cleavage sites either as a result of apparent insertion or apparent substitution (Vey et al., 1992; Wood et al., 1993; Senne et al., 1996) and appear to be cleavable by a ubiquitous protease(s), probably one or more protein-processing subtilisin-related endoproteases of which furin is the leading candidate (Stieneke-Grober et al., 1992). These viruses are able to replicate throughout the bird, damaging vital organs and tissues which results in disease and death (Rott, 1992).
Epidemiology:
Avian influenza outbreaks have occurred globally, with notable instances such as outbreaks in Pennsylvania in 1983 (Eckroade et al., 1984), USA in 1986 (Garnett, 1987), Pakistan in 1994 (Naeem, 1998; Alexander et al., 1996), Mexico in 1994 (Campos-Lopez et al., 1996), Italy in 1997-1998 (Fioretti et al., 1998), Hong Kong in 1997 (Claas et al., 1998), and Australia during the 1990s (Forsyth et al., 1993; Westbury, 1998). These outbreaks resulted in significant economic losses and necessitated various control measures, including depopulation, movement restrictions, and vaccination.
Economic Impact:
The poultry industry, experiencing rapid growth globally, faces substantial economic losses due to avian influenza outbreaks. HPAI viruses cause high mortality rates in poultry flocks, resulting in direct losses from bird culling, fall in value of birds, and production interruptions. Control measures, including biosecurity improvements and vaccination, aim to mitigate economic impacts. However, trade restrictions and decreased consumer demand further exacerbate losses across various sectors. From the perspective of poultry farmers, the losses due to HPAI are great, but the probability of infection in their flocks with the disease is rather low. A variety of measures to prevent and control HPAI are applied, either by poultry producers themselves or by the national animal health services. Preventive measures include improvements in biosecurity, both on farm and in markets, and vaccination. These are usually complemented by public investment in national surveillance systems and diagnostic capacity for early disease detection to limit the scale of outbreaks. None of the above measures alone is sufficient to effectively control HPAI, and thus measures need to be applied in concert.
Diagnosis:
In India, 5 regional disease diagnostic laboratories, viz. Bengaluru, Pune, Jalandhar, Kolkata, Guwahati, and a central disease diagnostic laboratory at IVRI Izatnagar are involved in testing, but ICAR-NISHAD Bhopal is the national reference laboratory for confirmation of avian influenza infection. Samples of oropharyngeal or cloacal swabs should be properly packed and labeled, and sent to ICAR-NISHAD for confirmation of outbreak.
Prevention and Control:
The procedure established by the Government of India in the event of suspected avian influenza outbreak entails several steps: immediate site visit by a Chief Veterinary Officer/District Animal Husbandry Officer (CVO/DAHO) upon receipt of initial information, provision of diagnostic kits to Veterinary Officers/Disease Investigation Officers (DIO), utilization of personal protective equipment (PPE), preliminary and clinical investigations conducted by DIO, collection and prompt dispatch of samples for confirmation, and timely reporting to all relevant stakeholders. An “Alert Zone” is identified, with corresponding restrictions imposed pending test results, and a designated veterinary officer appointed as “Designated Officer.”
Upon confirmation of avian influenza by the National Institute of High Security Animal Diseases (NIHSAD), Bhopal, the state government initiates control and containment measures. A containment operation ensues, followed by demarcation of surveillance and infected areas. The vicinity within a one-kilometer radius of the confirmed AI site is designated as the “Infected Zone,” while the area within a ten-kilometer radius forms the “Surveillance Zone.” Flexibility exists for the state government, in consultation with the Government of India, to adjust zone radii up to three kilometers based on infection/mortality dispersion. An absolute ban on poultry movement is enforced, along with closure of poultry and egg markets/shops and restriction of access to wild and stray birds. Measures include bird destruction/culling, disposal of carcasses and infected materials, premises and implement disinfection, implementation of the Post Operation Surveillance Plan (POSP), and declaration of disease freedom status followed by repopulation of infected zones. Farmers are compensated for culling losses, and restocking in culling zones occurs after three months upon sanitization certificate issuance post-POSP completion.
Vaccination:
Regarding vaccination, though it serves as a vital preventive measure for diseases, challenges arise due to seropositivity induced by vaccines, impacting the differentiation between vaccinated and infected individuals. Constraints on meat export discourage avian influenza vaccination. Novel approaches like reverse genetics and universal influenza vaccines are pivotal for averting potential pandemics.
Why Universal Influenza Vaccine?
The development of a universal influenza vaccine is imperative due to antigenic drift, rendering immunodominant regions of influenza viruses hypervariable and necessitating the production of antibodies against conserved epitopes. Existing vaccines targeting potential pandemic viruses exhibit narrow specificity and lack cross-reactivity within single subtypes. Traditional vaccination adversely affects international bird trade, mandating costly surveillance to detect infected birds among vaccinated populations. Universal vaccines inducing antibodies to conserved viral protein regions, such as the stalk domain of hemagglutinin and the ectodomain of M2, are under development to address these challenges.
Aspects of M2e making it universal vaccine candidate:
Key aspects of M2e making it a promising universal vaccine candidate include its ability to elicit both humoral (antibodies) and T-cell immune responses against conservative epitopes of the influenza virus. M2e, along with the HA stalk domain, represents widely utilized viral targets for universal influenza vaccine design due to their conserved nature and demonstrated efficacy in protecting against heterologous viral infections.
The extracellular domain of M2 (M2e) is particularly noteworthy as a potential antigen, given its high conservation across all influenza A viruses. M2 protein consists of three segments: the N-terminal or extracellular domain (23 amino acids), the hydrophobic transmembrane domain (19 amino acids), and the C-terminal domain (54 amino acids). M2e, comprising the first 23 amino acid residues, is highly conserved due to its genetic relationship with M1. Specifically, the encoding nucleotides for amino acids 1—9 of M2e overlap with those for amino acids 239—252 of M1, albeit in different reading frames. M1, as a highly conserved matrix protein, has minimal known mutations.
Evolutionarily, M2e sequences of influenza A viruses have diverged into several lineages primarily associated with host species. While the first nine amino acids exhibit the lowest variability within each analyzed group, the remaining residues show varying mutation percentages across different origins. This slight divergence among consensus sequences underscores the importance of selecting appropriate sequences for a universal vaccine. For instance, immunizing pigs with human M2e using different carriers failed to protect against the lethal swine subtype of influenza A virus. Thus, the development of an M2e-based universal influenza vaccine necessitates incorporating four different consensus M2e protein sequences to cover a wide spectrum of human, avian, and swine influenza A viruses.
M2e-based vaccines hold significant potential in mitigating highly pathogenic avian influenza (HPAI) outbreaks, which pose severe threats to the commercial poultry sector, resulting in substantial losses.
Conclusion:
In the present context, there is a significant threat posed by Avian Influenza to the poultry industry, stemming from its causative agent, the Influenza A virus of the Orthomyxoviridae family. The disease’s prevalence presents dire consequences for trade and has profound economic ramifications. Typically, strategies like stamping out or culling are employed for control, with vaccination often met with reluctance due to its adverse impact on trade dynamics. While vaccination remains crucial in preventing influenza spread, its efficacy against pandemic strains is limited, as it requires substantial time and effort to develop new vaccines tailored to specific viral variants. Addressing this challenge, the development of a universal influenza vaccine emerges as a pressing public health imperative. Such a vaccine would offer a comprehensive and long-lasting defense against a broad spectrum of influenza viruses, transcending seasonal variations and antigenic shifts. By providing robust protection against diverse strains, a universal vaccine holds the potential to revolutionize influenza control strategies and mitigate the socio-economic impacts of outbreaks.
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