Sanjay Kapoor

Rabies – epidemiology, pathogenesis, public health concerns and advances in diagnosis and control: a comprehensive review

ABSTRACT Rabies is a zoonotic, fatal and progressive neurological infection caused by rabies virus of the genus Lyssavirus and family Rhabdoviridae. It affects all warm-blooded animals and the disease is prevalent throughout the world and endemic in many countries except in Islands like Australia and Antarctica. Over 60,000 peoples die every year due to rabies, while approximately 15 million people receive rabies post-exposure prophylaxis (PEP) annually. Bite of rabid animals and saliva of infected host are mainly responsible for transmission and wildlife like raccoons, skunks, bats and foxes are main reservoirs for rabies. The incubation period is highly variable from 2 weeks to 6 years (avg. 2–3 months). Though severe neurologic signs and fatal outcome, neuropathological lesions are relatively mild. Rabies virus exploits various mechanisms to evade the host immune responses. Being a major zoonosis, precise and rapid diagnosis is important for early treatment and effective prevention and control measures. Traditional rapid Sellers staining and histopathological methods are still in use for diagnosis of rabies. Direct immunofluoroscent test (dFAT) is gold standard test and most commonly recommended for diagnosis of rabies in fresh brain tissues of dogs by both OIE and WHO. Mouse inoculation test (MIT) and polymerase chain reaction (PCR) are superior and used for routine diagnosis. Vaccination with live attenuated or inactivated viruses, DNA and recombinant vaccines can be done in endemic areas. This review describes in detail about epidemiology, transmission, pathogenesis, advances in diagnosis, vaccination and therapeutic approaches along with appropriate prevention and control strategies.

Evolution of Influenza Viruses

The rate of evolution in influenza A viruses is the fastest followed by influenza B, C viruses. The key factor in the evolution of influenza B and C viruses is prolonged co-circulation of antigenically and genetically distinct lineages. However, predominantly clonal selection, and to a very limited extent co-circulation of sublineages, is responsible for the evolution of influenza A viruses. Studies on phylogenetic analysis have identified several host-specific virus lineages for various viral proteins, except HA and NA genes. The evolution of influenza A viruses particularly is influenced by several factors such as origin and evolution of HA gene, receptor specificity, antigenic drift and shift, recombination, mixing vessels, host species jumping, etc. Phylogenetic analysis has helped to compare past and present influenza viruses as well as the determination of the common ancestor of the virus. Considerable genetic diversity, divergence and antigenic drift observed in the H5N1 virus during the last 16 years of its circulation in poultry have led to the development of unified nomenclature system in which these viruses were classified into various virus clades. This is required to understand the evolutionary mechanism of the development of pandemic H5N1 strains. The discovery of new subtypes, H17N10 and H18N11, from bats has increased the repertoire of known subtypes of influenza viruses, and the known range of mammals that can be infected by these viruses. Multiple reassortments were responsible for the generation of the novel H7N9 isolates that caused disease and death in humans in 2013.

Properties of Influenza Viruses

The three genera, viz. Influenzavirus A, Influenzavirus B, and Influenzavirus C are classified as separate genera, out of the five genera in the family Orthomyxoviridae (the other two genera being Thogotovirus and Isavirus). The genome of influenza viruses is made up of single-stranded RNA of negative polarity, containing 7–8 segments. The eight genomic segments of influenza A virus code for nine structural proteins are PB1, PB1-F2, PB2, PA, HA, NA, NP, M1 and M2, and for two non-structural proteins, they are NS1 and NS2. The functions of various proteins of influenza viruses are described. The viruses are pleomorphic and can occur as spherical or filamentous forms, having a size of 80–120 nm in spherical form and >300 nm in filamentous form. The influenza viruses are comparatively unstable in the environment and are susceptible to heat, extremes of pH and dryness, organic solvents and detergents such as sodium deoxy cholate (SDC) and sodium dodecyl sulphate (SDS). Although the virus gets inactivated with various concentrations of formalin, binary ethylenimine and beta propiolactone, surprisingly the haemagglutinating and neuraminidase activities are retained. Incomplete influenza virus particles are formed in the cells infected at high multiplicity of infection (M.O.I.). These are also called Defective Interfering (D.I.) particles as their genome is defective, and they interfere with the replication of the complete influenza virus particles.

Role of Migratory Birds in Spreading Influenza Viruses

The major source of several influenza viruses in other species are aquatic birds. Long distances travel is carried out by many migratory bird species between their breeding grounds and non-breeding areas. These migratory birds as well as wild birds are considered as reservoirs of majority of influenza A viruses. The geospatial analysis of the pathways of migratory birds present in different geographical locations will throw further light on their role in influenza virus epidemiology. The influenza virus dynamics among migratory wild birds and mammals including humans are closely linked as is evident from H1N1 spread. It is important to note that the migratory water fowls play a negative role as far as the economic benefit out of poultry industry is concerned and imposes threat to lives of human as well, because of their capability to transmit the highly pathogenic avian influenza (HPAI) virus across the continents. Interestingly, several species of familiar songbirds or perching birds act as bridge species and has a possible role in transmitting the H5N1 AI to or from wild habitat. Surveillance and tracking of migratory and resident wild birds, utilisation of ornithological expertise, and analysis of the H5N1 ecology are needed for increasing our knowledge about strain- or host-specific pathogenecity, degree of shedding of virus and the routes of transmission between wild birds. In this aspect, it is quiet noteworthy that 13 membered International Scientific Task Force including UN bodies, wildlife treaties and specialist intergovernmental as well as non-governmental organizations have been created on the ground of various scientific studies concerning the role of migratory birds as potential transmitter of H5N1 subtype of Highly Pathogenic Avian Influenza (HPAI) virus.

Public Health Importance and Pandemic Potentials/Threats of Influenza Viruses

Influenza A viruses of different subtypes, infect a variety of animal species and with their ability to undergo reassortments and mutations readily, are a potential public health risk. The significant carriers as well as sources of viruses are poultry birds (ducks, wild/migratory birds, chickens) and pigs, and sharing of ponds having discharged household wastewater with the excreta of humans, pigs and birds, contribute to the development of a reassortant virus through evolutionary mechanisms within ‘mixing vessels’. Pandemic threat to humans in case of bird flu is limited to 4 HA types, viz. H5, H7, H9 and H10 (AIV subtypes H5N1, H7N2, H7N3, H7N7, H7N9, H9N2, H10N8 and H10N7). Handling of infected birds or infected eggs/meat causes serious trouble in relation to transmission of bird flu rather than eating poultry products. Few authenticated cases of human-to-human transmission of avian influenza (bird flu) have been documented. However, the bird flu virus has not yet learnt the capability to be spread in a rapid and vicious manner from human-to-human in a pandemic way. This kind of human-to-human transmission of bird flu virus can trigger a human pandemic claiming millions of lives, as happened during the earlier pandemics of the twentieth century. The chance of H5N1 human pandemic virus may arise some time in the near future because of mixed infection with a bird flu (H5N1) virus and a currently circulating H3 or H1 subtype human influenza virus. If a severe pandemic occurs with a pandemic flu virus having a lethal killing weapon like that of bird flu (H5N1) virus and rapid spread like that of recent/current swine flu (H1N1) virus, then this deadly evolving influenza virus could cause serious socio-economic and public health consequences. More than 208 countries have been affected with swine flu during the last 4 years taking lives of nearly 13,600 people. Pigs act as a ‘mixing vessel’ and have played an important role in the evolution of a novel subtype of Swine flu (H1N1 subtype) virus that has enormous pandemic potential. Interestingly, transmission of swine origin influenza A viruses (H1N1, H1N2 and H3N2) can occur between humans and animals, especially in children.

Conclusions and Future Perspectives

The influenza A virus is most important among the three types of influenza viruses (types A, B and C) that normally cause respiratory disease in human, animals and poultry. The avian and swine influenza A viruses are zoonotic in nature. The genome of influenza viruses is segmented single-stranded RNA of negative polarity. The matrix and nucleoprotein proteins determine type specificity. Influenza A viruses have further been classified into 18 haemagglutinin subtypes and 11 neuraminidase subtypes. Many different factors affect the evolution of influenza viruses which may have a direct effect on their virulence, pathogenicity, immunity, drug resistance etc. The influenza A virus H5N1, Swine flu (H1N1 subtype) virus [SO-IV; H1N1pdm], and H7N9 subtypes have in recent years emerged as a dangerous flu strains that have caused a large number of human deaths. The influenza viruses in human cause seasonal flu every year or sometimes may cause pandemics. The pathogenicity and transmissibility are the main determinants of potential of an emerging viral strain to become a pandemic influenza virus. Virus replication in the endothelium including proteolytic activation of the haemagglutinin, polarity of virus budding, and tissue specific expression of virus receptors appears to play a pivotal role in pathogenesis. The expression of virus pathogenicity is dependent upon the functional integrity of each gene, and on a gene constellation optimal for infection of a given host. Commercial antiviral drugs are available for treatment of the human flu. Commercial vaccines are available in the market against human, equines, swine and avian influenza viruses. The DIVA vaccine strategies for avian influenza viruses are being developed and refined. The current focus is to develop universal vaccine against influenza viruses which should provide broad protective immunity against conserved antigens present in many different subtypes of influenza viruses. Besides the use of antivirals and vaccines, the legislative measures, stringent biosecurity measures, strict quarantine and trade limitations play an important role for the prevention and control of influenza viruses.

Prevention and Control of Influenza Viruses

The 2003–2004 outbreaks of highly pathogenic avian influenza (HPAI) have proven to be disastrous to the regional poultry industry in Asia, and have raised serious worldwide public health apprehension regarding the steps that should be taken to urgently control HPAI. Control measures must be taken based on the principles of biosecurity and disease management and at the same time making public aware of the precautionary measures at the verge of outbreak. Creation of protection and surveillance zones, various vaccination strategies viz. routine, preventive, emergency, mass and targeted vaccination programmes using live, inactivated and recombinant vaccines are the common strategies adopted in different parts of the globe. The new generation vaccines include recombinant vaccines and recombinant fusion vaccine. The pro-poor disease control programmes, giving compensation and subsidies to the farmers along with effective and efficient Veterinary Services forms integral part of control of HPAI. Following biosecurity principles and vaccination forms integral part of control programme against swine and equine influenza as well. Use of neuraminidase (NA) inhibitors (Zanamivir and Oseltamivir) for the treatment of human influenza has been widely accepted worldwide. The threat of increasing resistance of the flu viruses to these antivirals has evoked interest in the development of novel antiviral drugs for influenza virus such as inhibitors of cellular factors and host signalling cascades, cellular miRNAs, siRNA and innate immune peptides (defensins and cathelicidins). Commercial licensed inactivated vaccines for humans against influenza A and B viruses are available consisting of three influenza viruses: influenza type A subtype H3N2, influenza type A subtype H1N1 (seasonal) virus strain and influenza type B virus strain. As per WHO, use of tetravaccine consisting of antigens of influenza virus serotypes H3N2, H1N1, B and H5 is the most promising method to control influenza pandemic. All healthy children in many countries are required to be vaccinated between 6 and 59 months of age. The seasonal vaccines currently used in humans induce strain-specific humoral immunity as the antibodies. Universal influenza virus vaccines containing the relatively conserved ectodomain of M2 (M2e), M1, HA fusion peptide and stalk domains, NA, NP alone or in combination have been developed which have been shown to induce cross-protection. The T cell-based vaccines are another recent experimental approach that has been shown to elicit broad-spectrum heterosubtypic immunity in the host. As far as HPAI is concerned, various pandemic preparedness strategies have been documented.

Pathogenesis and Pathogenicity of Influenza Viruses

Influenza viruses can produce a wide variety of pathological lesions in different organs of poultry and mammals. Based on morphological, cellular and biochemical evidences in various animal models, the virus exerts pathological effect by two mechanisms, viz. necrosis and apoptosis. The tissue tropism of the influenza virus and tissue specific expression of virus receptors appears to play a pivotal role in pathogenesis. The mortality may be due to systemic viral spread, cytokine storm, or alveolar flooding due to inhibition of cellular sodium channels. The levels and functional potential of alveolar macrophages, various cytokines have a role in the influenza virus-induced pathology. Although all gene product of influenza virus contribute to pathogenicity, however, haemagglutinin plays a key role. The structure of the HA cleavage site is different in low pathogenic avian influenza viruses (LPAI) and highly pathogenic avian influenza viruses (HPAI). Also, the location of host proteases causing cleavage of these two type of the HA is different. The other important virulence determinant is the presence of a carbohydrate side chain nearby the cleavage site that interferes with the protease accessibility. The emergence of H5N1 in 1997, H1N1pdm in 2009 and now H7N9 influenza A viruses in 2013 provide lot of lessons to be learnt by understanding their pathogenesis and pathogenicity. Pathogenicity and transmissibility play a central role in the possibility and probability of a viral strain to emerge as a new influenza subtype in humans and act as a potentially pandemic influenza virus.

Epidemiology of Influenza Viruses

Influenza A viruses, in comparison to B and C group of viruses possess a broader host range, infecting many different mammalian and avian species including humans, fowl, pigs, horses, dogs, cats, tiger, and other mammals such as mink, seals and whales. Influenza A viruses, based on the haemagglutinin (HA) and neuraminidase (NA) proteins, are further classified into subtypes. There are 18 HA subtypes and 11 NA subtypes for influenza A viruses. Transmission of Influenza viruses may occur either directly, through airborne route or indirectly from infected host or contaminated surfaces. Pigs exhibit a unique role as the mixing vessel for the genetic reassortment of different influenza viruses. Avian influenza (Avian flu/Fowl plague) is among the most fearful viral diseases of birds, particularly affecting domesticated birds with very high flock mortality, resulting in enormous economic losses to poultry industry worldwide. The disease affects a wide range of feral migratory birds subclinically and these birds are crucial for the spread of the disease. The flu virus is becoming more and more dangerous especially in the last 10 years. Equine influenza, canine and feline influenza are of less significance compared to avian and swine flu. Influenza A, B, and C viruses are capable of infecting man and the incidence of human flu is more during winters in temperate countries, whereas it is more common during winters and rainy seasons in tropical and subtropical countries. Influenza viruses are always imposing a constant threat to mankind because of its perpetual evolving and reemerging nature, extremely high range of hosts, speedy transmission, lack and limitation of effective control and vaccination strategies and fatal consequences.

Replication Cycle of Influenza Viruses

Each of the genomic segments and the encoded proteins of influenza virus have some role in its replication. Various host factors have been described that are involved at various stages of influenza virus replication. The influenza A virus enters into the host cell by receptor mediated endocytosis. The HA of human and avian influenza viruses bind with sialic acid receptors with α-2, 6 linkage and α-2, 3 linkage, respectively. Both types of receptors are present in pigs. The fusion of the viral envelope with endosomal membrane leads to release of the genomic segments of influenza virus into the cytoplasm of the host cell. ‘Cap snatching’ and ‘stuttering’ are some of the unique phenomena in the replication cycle of influenza virus. The virus produces mRNAs, cRNAs and vRNAs during the course of its replication. The nuclear localization signals (NLSs) located on all the protein constituents, viz. PA, PB1, PB2, NP of the RNP complex are required for the entry of RNPs into the nucleus. The export of the newly formed vRNPs from the nucleus, for further virus assembly at the host cell membrane, is mediated by nuclear export signal (NES) carrying M1 and NEP proteins of influenza virus. The M2 has a pivotal role in the formation of viral particles while the M1 is required during assembly and budding off of the viral particle. The NA and NP proteins also play a role in assembly and release.