Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1):24-41 Review article DOI: https://doi.org/10.3126/njb.v9i1.38647 ©NJB, BSN 24 Avian/Bird flu: A review: H5N1 outbreaks in Nepal Dhiraj Shrestha1, Balkrishna Bhattachan2, Hiramani Parajuli3, and Sujata Shrestha4 * 1Department of Microbiology, Shi-Gan International College of Science and Technology, Kathmandu, Nepal. 2Biotechnology Society Nepal, BSN Bulletin, Bhaktapur, Nepal. 3Department of Microbiology, Tri-Chandra Multiple College, Kathmandu, Nepal. 4Department of Laboratory, Central Jail Hospital, Kathmandu, Nepal. Received: 11 Dec 2019; Revised: 03 Feb 2021; Accepted: 26 Feb 2021; Published online: 31 Jul 2021 Abstract Avian/Bird flu is a viral disease of birds, caused by avian influenza virus (AIV). A highly pathogenic avian influenza (HPAI) H5N1 has breached the barrier of species to humans and other animals escalating the pandemic threat. If the H5N1 evolves to a human-to-human transmissible virus retaining its pathogenicity, it can trigger an influenza pandemic. H5N1 has a mortality rate of about 60%, varying with strains. Meaningful antigenic alteration in hemagglutinin (HA) and/or neuraminidase (NA) results in recurring pandemics. The HPAI H5N1 subtype alone has outreached more than 77 nations around the world since the first human case and death was reported in 1997. Wild and migratory birds are the AIV reservoirs. Poultry is primarily impacted by incidents and outbreaks of the disease. A wide range of serological and molecular methods have substantially aided in the identification of bird flu in humans. Candidate vaccines have been developed, yet are not ready for widespread use. Oseltamivir (brand name: Tamiflu) is the preferred drug for the management of human Influenza- like illness (ILI). Surveillance, mass awareness, and pandemic preparedness abiding WHO recommendations are of paramount importance for the prevention of bird flu outbreaks. Keywords: Avian Influenza Virus (AIV), Avian Flu, Bird Flu, H5N1, Nepal Corresponding author, email: ssuju.046@gmail.com Introduction The word ‘influenza’ has Italian origin meaning ‘influence’; from the belief that epidemics were due to the influence of the stars [1]. It became a term for a particular disease after the 1743 epidemic. But, the virus was isolated from a human only in 1933 [2, 3]. Avian influenza virus (AIV), better known as bird flu, is a type-A influenza virus of the Orthomyxoviridae family. Theoretically, owing to the combination of antigens, hemagglutinin (HA) and neuraminidase (NA), type-A influenza comprises thousands of distinct antigenic subtypes [4]. To date, 18 subtypes of HA and 11 subtypes of NA have been identified, while two extra subtypes of HA and NA have been identified in bats [5, 6]. World Organisation for Animal Health (OIE) defines AIV as “an infection of poultry by any influenza A virus, including by subtypes H5 and H7” [7]. OIE requires notification for all low-pathogenic avian influenza (LPAI) virus outbreaks, i.e. H7 and H5 subtypes as they can mutate into highly pathogenic avian influenza (HPAI) viruses, as documented in some poultry outbreaks. Non-H5 and non-H7 LPAI are not deemed notifiable [8]. In certain poultry, such as ducks, some HPAI virus (e.g., H5N1) have been shown to cause no illness. HPAI has been associated with AIV subtypes H5 and H7, including the viruses H5N1, H7N7, and H7N3. Human infections have varied from moderate (H7N3, H7N7) to severe and lethal (H5N1) infections [9]. The HPAI H5N1 virus has raised concerns around the world as it threatens poultry, especially chickens; also it has shown the potential to pass from poultry to humans and caused severe infection and death [10]. The first reported human infection of H5N1 occurred in 1997 in Hong Kong [11]. The virus has been endemic in several countries after the re-emergence of H5N1 in Asia, Africa, the Pacific region, Europe, and the Middle East in 2003, and continues to cause poultry outbreaks [12]. In Dhaka, the presence of the H5N1 virus was confirmed in March 2007 [13]. The first case of AIV was identified at a remote non-commercial poultry farm in Kakarvitta, Eastern Nepal, on January 16, 2009 in Nepal [14, 15]. More than 256 H5N1 outbreaks has been witnessed in poultry since 2009 [16]. Incidence and Prevalence Climate change can bring a quick ecosystem shift which alters the evolution and ecology of infectious diseases including AIV. This ecosystem shifts had played a Nepal Journal of Biotechnology Publisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 Journal Homepage: www.nepjol.info/index.php/njb ISSN (Print): 2091-1130 https://orcid.org/0000-0002-3521-2279 mailto:ssuju.046@gmail.com Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 25 cumulative effect in the outbreak of the influenza pandemic in the past century [17]. Furthermore, studies show an increase in temperature of freshwater in temperate and Arctic regions had a persisting impact altering the suitable habitat variation in prevalence rate and viral spreading [18]. Several studies have posited agricultural system, poultry trade, availability of water temperature, salinity as factors resulting in an alteration of AIV ecology [19, 20]. Studies have shown that the occurrence of new AIVs is supported by either point mutation, partial genes recombination, or genetic re- assortment of the whole genome. Most new strains of AIV evolved due to point mutations while genetic re- assortment performs a key task in the genesis of H5N1 and H7N9 strains [21]. The pathogenicity of AIV is associated with efficient virus replication. Host immune responses and the genetic markers are determinants for efficient virus replication [22]. The occurrence of human influenza A (H5N1) often parallels with massive outbreaks of avian H5N1 influenza A, while avian outbreaks in 2004 and 2005 have seldom led to human infection [23]. Different types of studies based on geographical distribution and outbreak of AIVs revealed that prevalence is related to the migratory route and stages of the itinerary of migrating birds [24, 25]. Studies have concluded that Asia is the prevalent continent for the avian influenza virus due to a unique ecosystem of various lakes, wetlands, creeks, and rivers that constitute migratory bird wintering areas [26]. Due to consequence and the risk represented to human health, AIV is of great concern and are widely studied in recent times. Wild birds of genera Antidae (ducks, geese, and swans) and Laridae (gulls, terns, and kittiwakes) have been in focus for decoding the epidemiological links between hosts and transmissions of AIV [27]. Etiology The pandemic influenza virus has its origins in avian influenza [28]. Its genomic material is composed of eight segmented negative strand RNAs. The influenza A virus shows external spikes when examined with an electron microscope. Various techniques can distinguish two kinds of spikes. The HA molecules made up one type and the NA molecules the other. There are roughly 5 times as many HA spikes as NA spikes [29] (Figure 1). The 18 different subtypes of HA and 11 different subtypes of NA exist allowing for 198 potential different viral strains. As of 2019, only 131 subtypes have been detected in nature [30]. Figure 1. Schematic diagram of the Influenza A virus with the virus components. Note: NA=neuraminidase, HA=hemagglutinin, M1=matrix protein-1, M2=matrix protein- 2, ss RNA=single stranded RNA, PB2=polymerase basic-2, PB1=polymerase basic-1, PA=polymerase acidic, NP=nucleoprotein, NS1=non-structural protein-1, NS2=non- structural protein-2. Genomic variation Genetic analyses have indicated that since 1997 the H5N1 virus has evolved into multiple genotypes [31]. Whether the genetic modifications in the HA protein are the result of immune escape or are related to host adaptation is not clear [32]. Hence, the best H5N1 epitopes is difficult to predict to target with vaccines without understanding the antigenicity of emerging strains. As H5N1 viruses have expanded their geographical and host ranges, it has become increasingly important to determine acquired features that permit successful human-to-human transmission. The transmission of H5N1 viruses to humans has been ineffective, arising either by direct interaction with infected poultry or through ingestion of undercooked meat or blood of infected birds [33]. In influenza A viruses, antigenic drift is a continuous mechanism where point mutations occurs during replication of the viral genome. This mechanism is apparent in all the influenza A virus gene products; however, it is most pronounced in the HA and NA glycoproteins’ antibody-binding sites. These mutations cannot be repaired because of the lack of a proofreading mechanism, and the resulting aggregation of variations in the amino acid sequence transforms these antigenic sites in such a way that they are less detectable by the host antibody response. Thus, these virus strains are naturally selected as host antibodies no longer no longer neutralized them, and they lead to increased viral fitness [34, 35]. Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 26 Antigenic Variation Both the HA and the NA undergo antigenic variation [36]. Antigenic shift is a major change in antigenicity, while antigenic drift is a minor change sufficient to spark a new outbreak. In the human population, pandemics happened in 1957 (Asian influenza) and 1968 (Hong Kong influenza); both are associated with the presence of antigenically transformed HA molecules. The HA antigenic variation is known to be more important quantitatively than that of the NA. The human pandemic virus of 1918 (Spanish influenza) was the common ancestor of human and classical swine H1N1 influenza virus [37, 38]. Epidemiology Avian flu or bird flu was initially known as Fowl Plague, and later Fowl Plague virus was discovered as an influenza virus. The outbreak of avian flu was first documented in 1878 in Italy. Later on, in 1924 and 1929 two massive outbreaks in poultry were recorded in the United States [39]. Since the first reported human case and death by the influenza virus in 1997 in Hong Kong, the eruption of the disease has been documented from wild and domestic birds including humans. HPAI H5N1 alone has been reported from over 77 countries [40, 41]. Till the date, a total of 860 human cases have been reported since 2003 with more than 50% deaths by H5N1[42]. Since 2013, 1,568 human cases and 616 deaths were reported worldwide attributed to the novel H7N9 [43]. Geographically the disease is globally prevalent dominating the Asia continent showing the higher outbreaks in China, Vietnam, India, Taiwan, Israel, Japan, and South Korea [26]. Host range, transmission, and spread AIV is found distributed in a wide variety of hosts. Predominantly free-flying water birds, like geese, ducks, shorebirds, and gulls, are major reservoirs of AIVs. Besides this, AIV can infect both wild and domestic birds like chicken, turkeys, partridges, pheasants, quails, pigeons, and ostriches [44]. However, the AIVs primarily associated with transmission in chickens but not in ducks were found to be adapted to ducks by acquiring genes from duck influenza viruses [45]. AIV is essentially a disease of birds, but evidence has shown that the infection can be transmitted in cats, dogs, eagles, ferrets, hamsters, horses, humans, macaques, marine mammals, mice, minks, pigs, and tigers; but the zoonotic infection had been only reported in China [46]. The transmission and spread of disease were primarily related to poultry contact, yet human-to-human transmission is also possible [47]. Moreover, the outbreak of avian flu is also associated with the vicinity of water source and 97.5% of reported cases showed the proximity of water source [26]. Virus replication The influenza A virus genome consists of segmented RNA encoding 10 proteins [48]. These 10 viral proteins are necessary for a successful infection cycle within immuno-competent hosts. On the surface of the host cells, the HA protein binds to sialic acid, enabling the virus to enter [49]. The host cell endocytoses the virus particles. The endosome maturation lowers the pH triggering a HA conformational change. This results in the membrane fusion of the endosome and the virion. The viral matrix protein-2 (M2) acts as an ion channel that further lowers the pH of the virus particle. This aids in the dissociation of the matrix protein-1 (M1). The virus ribonucleoproteins (vRNPs), including polymerase basic- 1 (PB1), polymerase acidic (PA), and polymerase basic-2 (PB2), are released into the cytosol [50]. For viral replication and transcription, the vRNPs are then moved into the host cell nucleus. The nuclear export protein (NEP) and M1 traffic out freshly produced vRNPs into the cytoplasm and then to the plasma membrane after replication, transcription, and protein synthesis. Several viral proteins, including M1 and M2, contribute to budding. Finally, in both the viral and host cell membrane, NA removes sialic acid from glycoproteins. This stops HA and host cells from interacting, releasing new infective virus particles [1]. Nonstructural protein 1 (NS1) counteracts the innate host-cell defense within the infected cell, including interferon, for efficient virus replication [51]. New proteins generated by co- transcription or co-translation with the host are being continuously identified [52]. Pathogenesis Virulence factors comprise the highly cleavable HA that several cellular proteases can activate, a specific substitution in the polymerase basic protein-2 enhances viral replication [53]. Non-structural protein-1 substitution confers improved resistance to interferons and tumor necrosis factor-alpha inhibition [54]. The H5N1 viruses potently induce proinflammatory cytokines in macrophages, the most notable being tumor necrosis factor-alpha, which may contribute to the unusual severity in humans [55]. With antigenicity change [56], constellation of internal gene, expanded range of avian species [57], enhanced pathogenicity [58], and increased environmental stability, these viruses keep Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 27 evolving. Despite extensive exposure to infected poultry, comparatively low frequencies of H5N1 infection in humans suggest that the species barrier to acquisition of this avian virus is important [33]. Disease in birds Avian flu has been reported in many wild and domestic birds. Chickens, ducks, swans, gooses, quails, crows, storks, etc. have been known to have been infected with AIV. It is understood that H5N1 infections show distinct clinical presentations in chickens and ducks. The infection is characterized by clinical signs and high mortality in chickens, whereas the infection is generally asymptomatic in ducks. This leads to an underestimation of the prevalence of the disease [59]. Infected ducks can thus help to sustain and spread the virus “silently” to other vulnerable hosts [60]. The H5N1 infection in ducks has been considered a threat to the poultry flock and public health due to its silent nature [57]. Thus, monitoring and surveillance based on ducks is substantial for AIV control [61]. Since ducks may become infected and co-infected with multiple AIVs, re- assortment of the viral genes is likely [62]. Spatial evidence on HPAI outbreaks in Southeast Asia has shown that scavenging ducks lead to HPAI outbreaks in domestic poultry [63]. Evidence shows that ducks play a major role in the transmission of HPAI since they do not exhibit the disease symptoms. Characterization of virus In 1996, the HPAI H5N1 virus was found in Gundong province, China which was thought to have originated from the H5 virus in migratory birds through both drift and shift mutation mechanisms [64]. Since then the influenza virus in human cases has evolved and is classified in a different list based on hemagglutinin phylogeny known as a clade. This list of the clade is updated for continuous change [65]. There are 13 major clades in the list distinguished by the hemagglutinin gene of the H5N1 representative subtype of HPAI. These clades are determined by sequencing and measuring the average pairwise nucleotide distance (APD) [66]. These clades were further split by measuring the within-clade average pairwise distance to determine the sub-clade resulting in more than 32 clades or sub-clades [67]. Clade 0 comprises viruses that were first identified to cause human infections in Gundong province, China 1996 (A/goose/Guandong/1/96 linage). In the early phase of epidemic (2004-2005), clade 1 viruses predominated in Vietnam, Thailand, and Cambodia, and clade 2.1 viruses are endemic in Indonesia. The clade 2.1.3.2b was found to be restricted to a certain geographical area i.e. Java, Indonesia [68]. A major outbreak of H5N1 disease in migratory birds has been associated with clade 2.2 and the virus is distinct from Z genotype virus and was first detected in Russia, Kazakhstan and later spread, causing avian disease in Africa, Central Asia, Europe, the Middle East, South Asia, and human disease in western Africa, Asia, and the Middle East resulting pandemic in 77 countries [69]. In southern China, clade 2.3 has been dominant and has also been reported in adjacent countries. Later on, clade 2.3.2.1c was detected in Canada and Austria. Clade 7 was restricted to China and Vietnam in the year 2006-2009 [70]. The pattern of viral replication The characterization of virologic course of AIV H5N1 is yet to be done. Many studies reported prolonged viral replication. AIV can be detected in nasopharyngeal or lower respiratory sites ranging from 1 to 16 days. Nasopharyngeal replication has been lower in humans as compared to lower respiratory tract replication [71]. AIV has also been documented to replicate in the gastrointestinal tract [72, 73]. The invasive infection has been documented in humans [72]. The HA cleavage site’s polybasic amino acid sequence is linked with visceral dissemination in birds [33]. Host immune response (Innate type) Despite widespread exposure to infected poultry, the comparatively low disease frequencies in humans illustrate the species barrier of AIV [33]. H5N1’s ability to infect birds or humans tends to be partially determined by the HA binding specificity. Generally, HAs of human strains of influenza virus preferentially bind sialic acids bound to the terminal galactose of the oligosaccharides on the cell surface by α 2,6 linkage (SA α 2,6), which is abundant in human respiratory epithelia [74]. The HA of avian strains, on the other hand, binds preferentially to α 2,3-linked sialic acids (SA α 2,3). These linkages are common in the avian intestinal tract [75]. For host cell infection and the dissemination and virulence of influenza viruses, For host cell infection and the dissemination and virulence of influenza viruses, HA interaction with sialylated glycans on the cell surface is important [76]. Mutations altering the specificity of the receptor binding of avian viruses may be essential for avian to human crossover, and for enabling direct human-to-human transmission [77]. Pathogenesis of disease is contributed by the innate immune response [33]. Elevated blood Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 28 levels of interleukin-6, interleukin-8, interleukin-1 b, soluble interleukin-2 receptor, tumor necrosis factor (TNF), TNF-a, interferon-g, monokines induced by chemokines interferon-inducible protein 10, interferon-g, and monocyte chemoattractant protein 1 were observed in the fatal cases [71, 78]. Such responses can be attributed to the sepsis syndrome, acute respiratory distress syndrome (ARDS), and multiorgan failure. Corticosteroids are used to minimize such responses. Individuals developing specific humoral immune responses can survive [33]. Transmission Transmission of AIV occurs through direct contact via the inhalation of droplets and droplet nuclei. Transmission also occurs perhaps through indirect (fomite) contact and self-inoculation onto the upper respiratory tract or conjunctival mucosa [79]. The data is consistent with bird-to-human, potentially environmental-to-human, and limited human-to-human transmission to date. Animal to human transmission is mainly attributed to exposure to live ill poultry and butchering of birds [80]. In addition to plucking and preparing infected birds, handling of fighting cocks, handling poultry notably asymptomatic infected ducks, and eating of undercooked poultry or duck’s blood has also been proposed for transmission of AIV. Also, feeding tigers and leopards in zoos with raw, infected chickens has been proposed for transmission of AIV [81]. Human-to-human transmission has also been manifested in recent years. But no case of human-to-human transmission through aerosols of small particles or through social contact has been reported. Still, severe illness has been reported in clinicians exposed to an infected patient. The survival of H5N1 in the environment outlines the possible mode of transmission. Direct intranasal or conjunctival inoculation, and oral intake of contaminated water during swimming is the possible mode of transmission. Also, contamination of hand and subsequent self- inoculation is the possible mode of transmission. The use of untreated poultry feces as fertilizer has increased the risk of environmental transmission [82]. Clinical features Incubation: The incubation period of H5N1 infection is 2–4 days after exposure to infected, sick, or dead poultry [83]. But incubation time can be prolonged up to 8 days [33, 84]. The degree of virus shedding during this time is still unknown [85-87]. Initial symptoms The most common and initial symptoms that appear after H5N1 infection is respiratory distress. Milder cases show uncomplicated flu-like illness [88]. Pneumonia is often of viral origin with no bacterial superinfection, but may differ from cases to cases [78]. Crackles on examination of the chest are also seen [89]. Other frequently occurring symptoms include a high fever with >38°C, sore throat, cough, shortness of breath, rhinorrhoea, etc. [33]. Some cases suffer from diarrhea, vomiting, and abdominal pain apart from typical clinical manifestation [90]. Conjunctivitis or upper respiratory infections are not common [87]. Further complication includes multi-organ failure like renal disorder, cardiac malfunction, and pulmonary hemorrhage. Reye’s syndrome, pneumothorax, ventilator-associated pneumonia, and ARDS are also seen in some cases [33]. Though central nervous system involvement is rare there are cases where convulsion and progressive coma are reported leading to the death if not treated on time [90, 91]. Two patients in Vietnam were detected with encephalitis only [92]. Clinical course A patient infected with H5N1 has a rapid clinical course, developing progressive lower respiratory tract disease and viral pneumonia. Mechanical ventilation may be needed depending upon the severity of the case [33]. ARDS following the multi-organ failure develops in 68% of patients within six days of disease onset [91]. The average duration from onset of disease to hospital admission is four days and from onset to death in critical cases is nine days [93]. Few cases of seropositive patients but without typical manifestation are also reported [94]. Mortality/Morbidity The mortality rate of H5N1 is significantly high and is at an alarming rate accounting for about 60% [42]. But the rate may increase up to 73% in the youth of the age group 10-19 years followed by 18% in the age group 50 years [95]. The mortality rate was even 90% in severe cases [96]. Most of the death cases are frequent with late admission [97, 98]. Diagnosis Specimen for upper respiratory tract infections Nasal swab/nasal wash Polyester or Dacron swab is recommended with an aluminum or plastic shaft. The dry swab is inserted in the internal nares below the inferior turbinate. It is then allowed to absorb secretion for some time and is rotated Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 29 around the area before the withdrawal. The tip is then broken and dipped in viral transport media (VTM) vial. For nasal wash, physiological saline is used [99]. The patient is advised to close the pharynx by saying the letter “K” and the saline is introduced in nostrils one at a time with the head tilted backward and later the saline is collected in a cup or dish by tilting the head forward. The obtained wash is then mixed with the VTM [100]. Throat swabs A throat swab is considered to be the high yielding specimen for upper respiratory tract infections and should be obtained within three days after symptoms appeared for the best result [100]. By using the tongue depressor or blade, polyester or Dacron swab is gently rubbed several times in the posterior pharynx. The tip is then broken and dipped in a VTM vial [99]. Nasopharyngeal secretions A catheter with a mucus trap is inserted in nostrils. By use of a vacuum, the catheter is withdrawn with a rotating motion. After that, the catheter is flushed with a transport medium in a 1:2 ratio [99]. Specimen for lower respiratory tract infections An endotracheal aspirate or bronchoalveolar lavage is the best specimen for lower respiratory tract illness. For increasing the potential of viral isolation, multiple specimens can be taken from multiple respiratory sites for at least two consecutive days [72]. Blood samples Both acute and convalescent serum samples are recommended if feasible. Within the first 3-5 days after the onset of symptoms, acute serum samples should be taken. Convalescent serum samples collected after 3-4 weeks would be of great use if collected in conjunction with acute samples [99, 101]. Other specimens It may be either plasma or rectal swab in diarrhea for the detection of viral. Spinal fluid/ tap is also preferred in the case of meningitis [100]. Autopsy specimens along with para-mortem biopsies are also recommended [28]. Transportation and storage of specimens VTM contains balanced salt solutions, a protein stabilizer, bovine albumin, and a spectrum of antibiotics to minimize bacterial and fungal growth. Non-phosphate based VTM is selected if the specimen is only suggested for the PCR test. The collected specimen is placed in VTM and refrigerated at 4°C or with refrigerated packs for transportation to the designated laboratories [100]. The clinical specimens should be well labeled and sent to the laboratory as soon as possible. If specimens cannot be processed, then it should be either stored at 4°C or frozen at ≤-70°C. Samples should not be repeatedly freezed0 and thawed [102]. In the case of viral antigen detection by immunofluorescence staining, specimen processing should be done as soon as possible not more than 1-2 hours after collection [83]. A guide for field operations is recommended regarding the collection, storage, processing, and even transportation of samples for H5N1 detection [100]. Laboratory diagnosis Since most of the respiratory infections show similar manifestations, H5N1 can only be diagnosed in account with endemic areas and contact with dead or infected poultry or with confirmed cases. Direct detection of virus or viral antigen Virus isolation or reverse transcriptase PCR (RT-PCR) Virus isolation is considered to be the gold standard method and can be carried out in the laboratory either by inoculation of embryonated eggs or by using cell lines like Mardin-Darby Canine Kidney (MDCK). The obtained viral isolate can be later used for the study of pathogenicity, antiviral resistance, and DNA sequencing and analysis. But culture needs a biosafety level 3 (BSL- 3) which is not available at ease. So, RT-PCR is used as the first diagnostic tool in contrast to culture [85, 86]. Real-time PCR or quantitative PCR (qPCR) qPCR methods are preferred to conventional RT-PCR. There is a panel of qPCR assays covering even the specific detection of different NA genes and HA subtypes like H5, H3, and H1. H5 and N1 specific primers are used as they exclude false-negative results due to mutation in genes [72, 103]. Loop-mediated isothermal amplification (LAMP) tests are no longer in use [104, 105]. Serology The Antigen detection For the detection of viral antigens by using specific monoclonal antibodies against H5 and N1 direct immunofluorescence and enzyme immunoassay (EIA) are commonly used. The EIA method is considered simple, convenient, sensitive, and even applicable as Point of Care testing (POCT). The sensitivity of EIA is 1,000-folds low compared to viral isolation. Thus, subtype-specific diagnostic methods like RT-PCR should be carried out simultaneously [106]. Antibody detection Since seroconversion is delayed and needs paired sera, antibody detection provides a retrospective scenario of infection. It can be done by detecting a four-fold rise Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 30 while comparing acute and convalescent sera. Hemagglutination Inhibition (HI) assay is conventional yet the preferred method. But HI has low sensitivity for the detection of subtype-specific antibodies [107-109]. The use of horse erythrocytes while detecting antibodies against H5N1 shows effective results [110]. One of the studies done in 1997 during the Hong Kong outbreak reported similar results and reported that the micro- neutralization test is more effective, reliable, and sensitive as compared to HI [111]. Western blot analysis with recombinant H5 is also done for the conformation [112-114]. Hematological Profile It includes leukopenia, relative lymphopenia, and even thrombocytopenia. Disseminated intravascular coagulation occurs but is rare [33, 84]. Biochemical parameters Levels of liver enzymes are elevated, along with lactate dehydrogenase and creatinine kinase, serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) [85, 86]. Others Blood culture, sputum culture, and CSF analysis are carried out in case of serious complications. Chest X-ray reveals effusions, multifocal consolidation, lymphadenopathy, and even shows diffuse ground-glass appearance as in the case of ARDS [71, 78]. Differential diagnosis Diagnosis of AIV should be differentiated with atypical pneumonia, community-acquired pneumonia (CAP), corona-virus disease 2019 (COVID-19), endemic respiratory infections, hantavirus pulmonary syndrome, middle east respiratory syndrome (MERS), pediatric pneumococcal infections, pneumococcal infections (Streptococcus pneumoniae), respiratory syncytial virus infection, seasonal influenza, and severe acute respiratory syndrome (SARS) [83, 94, 115]. Definition of exposures to poultry and humans Human exposure definition differs from the environments and the situation (Table 1). Table 1. Definition of exposures to poultry and humans [116]. Exposure to live poultry in occupations Poultry-related exposure at workplace (e.g., persons engaged with poultry raising, trafficking, selling, and quarantine) within 2 weeks of the start of symptoms Exposure to poultry at live bird markets Visiting a live poultry or bird wholesale or retail market within 2 weeks before the start of symptoms Exposure to sick or dead poultry Close or direct physical contact with infected or dead poultry or poultry products (e.g., meat) or feces within 2 weeks before the start of symptoms Exposure to backyard poultry Close or direct contact with poultry raised in the backyard within 2 weeks before the start of symptoms Any exposure to poultry Close or direct or indirect contact to healthy, or sick, or dead poultry (including birds-e.g., chickens, ducks, geese, pet birds, pigeons) in live bird markets, or backyards, or farms, or neighborhoods, or consumption of improperly processed poultry products Exposure through patient contact A patient with a history of close contact within 2 weeks before the start of symptoms with a person with a confirmed or suspected influenza H5N1 virus infection (at any time from the day before the onset of symptom to death, or during the period during which the patient was hospitalized) Case definitions [117, 118] For prompt diagnosis and treatment, WHO has defined H5N1 infection as: Person under investigation A person who is decided to be investigated for possible H5N1 infection by assigned public health authorities Suspected H5N1 case A person with fever (>38ºC) exhibiting unexplained acute lower respiratory distress along with cough, shortness of breath, or dyspnoea. AND In the 7 days prior to symptom onset, one or more of the following exposures: a) Contact with a person within 1-meter distance during caring, speaking, or touching whether s/he is a suspected, probable, or confirmed H5N1 case. b) Exposure either to poultry in an area suspected or confirmed with H5N1 infections not more than a month or wild animals during handling, slaughtering, de-feathering, etc. and even contact with the contaminated feces. c) Intake of poultry products raw or undercooked in an area where animal or human H5N1 infections is reported or confirmed in the past month. d) Close contact with an animal infected with confirmed H5N1 other than poultry or wild birds (e.g. cat or pig) e) Handling animal or human samples in a laboratory or other facility suspected of having the H5N1 virus. Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 31 Probable H5N1 case (notify WHO) Probable definition 1: A person who satisfies the conditions for a suspected case AND Additional one of the following criteria: a) Infiltrates or proof of acute chest radiograph pneumonia, with proof of respiratory failure (hypoxemia, severe tachypnea) OR b) Confirmed laboratory influenza A infection but inadequate evidences in laboratory. Probable definition 2: A person dying from an unidentified acute respiratory illness who is known to be epidemiologically linked to a probable or confirmed case of H5N1 by time, place, and exposure. Confirmed H5N1 case (notify WHO) A person who satisfies the condition of a suspected or probable case AND Either of the following findings in a national, regional, or international laboratory of influenza whose H5N1 test results are recognized as confirmatory by WHO: a) H5N1 virus isolation; b) H5 PCR positive by tests using two separate targets of PCR, e.g. influenza A and H5 HA specific primers; c) On test of an acute serum sample (collected 7 days or less after the start of symptom) and a convalescent serum sample, the H5N1 neutralization titer must be at least fourfold rise. The convalescent neutralizing antibody titer must also be at least 1:80; d) In a single serum sample collected at day 14 or later after the start of symptom a microneutralization H5N1 antibody titer must be at least 1:80 and a positive in a different serological assay, for e.g. titer of at least 1:160 in a horse red blood cell HI or western blot specific to H5. Management/Treatment Hospitalization H5N1 can be a serious disease in humans that needs hospitalization, isolation, and intensive care [119]. Antiviral agents The primary therapy is the use of antiviral medication. The WHO and CDC guidelines (2015) recommend use of a neuraminidase inhibitor [92]. Amantadine Amantadine interferes with M2 protein and affects the release of infectious viral nucleic acid [120]. It is found to be effective for both prophylaxis and short-term treatment [121]. Since most of the H5N1 virus shows resistant patterns to amantadine or rimantadine, combination therapy with oseltamivir is recommended [92]. Rimantadine (brand name: Flumadine) It inhibits uncoating affecting viral replication. But H5N1 develops resistance to it [92]. Oseltamivir (brand name: Tamiflu) It affects the receptor of host cells towards viral HA [92]. But the spread of oseltamivir-resistant H5N1 from 2007 to 2009 surprised the influenza community [122]. Failures of treatment have been reported in single-drug oseltamivir regimens due to resistance. There are current researches on the relative success of high-dose and/or extended oseltamivir treatment courses [83]. If the use of high-dose tends to be more effective, it will affect the supply of antiviral drugs in the case of a large epidemic, in addition to medical considerations for patients who are moderately or seriously ill [92]. Zanamivir (brand name: Relenza) In an animal model, it shows better results but has not been tested in humans. But in severe cases, inhaled zanamivir may not be able to reach the distal airways [83, 92]. Uricosuric Agents Probenecid can be used as adjunctive therapy [92]. But no study has been carried out to confirm the suitable dose. Studies are still going on other drugs like arbidol and peramivir [123]. Immunomodulators Low dose corticosteroids are used to treat acute lung injury (ALI)/ARDS caused due to H5N1 [83, 124]. But the long term and high dose use may result in serious complications increasing the probability of opportunistic infections [92]. Other immunomodulators used are non- steroid anti-inflammatory drugs (NSAIDs), antipyretics, growth hormones, etc. But the use of such intervention modalities has not been proven any significant benefits [125, 126]. Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 32 Miscellaneous supportive therapy in a critical case These include oxygen therapy, ventilatory support [127], and non-ventilatory treatments for ALI/ARDS [128]. Prevention and control Immunization Based on different studies, three vaccines of H5N1 were licensed as Sanofi Pateur's vaccine, (monovalent killed vaccine approved by the US Food and Drug Administration (FDA), United States), Glaxo Smith Kline's vaccine-Prepandrix (approved by the European Union), and CSL Limited's vaccine-Panvax (approved by Australia) [129-131]. Even though none of the vaccines are available for the civilians yet [132]. Since the egg inoculation method is not applicable, tissue or cell culture and recombinant viruses are used for the production of vaccine and the adjuvants like aluminum hydroxide can be used to increase immunogenicity [133]. The inactivated vaccines produced by using the H5N1 was found to be immunogenic with high doses of HA [134]. But due to rapid mutation, immunization is not quite effective and practicable [135]. For travelers, prophylaxis by antiviral is not recommended, instead avoiding contact with dead birds and poultry, use of properly cooked food, and if needed use of N95 respirator mask, goggles while traveling in outbreak areas is recommended [136]. Disease management for preventing outbreaks The most effective way to prevent an outbreak is to avoid the source of exposure. People should avoid contact with the wild as well as suspected domestic birds and even should avoid contact with contaminated feces or surface. Active surveillance and strengthening biosecurity done in poultry workers, animals, and the environment in suspected sites contribute to a great extent. [136]. One health concept or collaboration of different sectors play a vital role in early detection and prevention. Proper supervision of animal transportation, inspection on the border while transporting, and provision of isolation stations on different sites play a vital role [137]. Skilled manpower in the field of veterinary to help with identifying the diversity and emerging disease will help in early warning [136]. Stamping-Out, proper carcass disposal, and surveillance and monitoring of animal health are needed [138]. Minimizing the occupational risk of exposure and vaccination of exposed poultry workers and animals should be prioritized [136]. Pandemic preparedness Pandemic preparedness can be implemented effectively only if it is planned by a collaboration of global, federal, and state or local levels. It requires the involvement of public health personnel, different health care professionals, researchers, and even private sectors to prepare effective measures for pandemic situations. Thus, the instant response becomes possible that will help to tackle the pandemic scenario in a relevant way [139]. Pandemic preparedness includes mass surveillance, continuous monitoring, early diagnosis, formulation of triage policies [140], development and distribution of different medical interventions like therapeutics, personal protective equipment (PPE), the rapid response of health care system, and proper interaction [139]. Precautionary measures for general publics As a general precaution, avoiding contact with suspected wild birds, contaminated poultry products, contaminated water, and proper hand hygiene is recommended for the public. Besides, awareness programs on the public level regarding the transmission and different preventive measures should be made available by the government [136, 138]. Hospital infection control Specific measures taken in a hospital setting not only protect the health care workers but also guide them to control the infection that in turn prevents the spread of disease in the community. Health workers must ensure the environment cleaning, proper disinfection and must be trained about occupational hazards and various modes of virus transmission [141]. N95 masks are more effective than multiple surgical masks. Pre-exposure prophylaxis should be considered if there is evidence of transmission of the virus or likely to be at risk of exposure [33, 142]. Chemoprophylaxis by oseltamivir is recommended for a person at high risk. The appropriate dose is 75mg of oseltamivir, a single dose for 7-10 days [143, 144]. Household and close contacts There are many routes of transmission for the H5N1 virus as such there are different protective measures to avoid the infection. Proper handwashing plays a significant role in avoiding the self-inoculation of the virus, thus preventing the disease [145]. Besides, households with illness should follow extra specific measures of post- exposure chemoprophylaxis that include oseltamivir [146, 147]. Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 33 Table 3. Major events related to avian influenza in Nepal [16, 42, 150-154]. Time Major events 2004 Surveillance of influenza started from Jhapa, eastern Nepal. Oct 2005 First report of serologic evidence of AIV infection (H9N2) in poultry in Nepal. 2009 Domestic ducks seropositive for antibodies against H5 and H9, but not H7. 2009 With the deployment of Real-Time PCR (qPCR) at the National Public Health Laboratory (NPHL), a molecular diagnostic assay-based influenza surveillance was initiated. Jan 2009 The first case of H5N1 detected in the Jhapa district, eastern Nepal Jun 2009 During the 2009 pandemic influenza virus outbreak, detection and molecular characterization of pandemic influenza virus A H1N1 in a human specimen obtained at Tribhuvan International Airport. Oct 2009 In Kathmandu valley, the community spread of pandemic H1N1 2009 was identified. Apr 2010 Highly equipped National Influenza Centre was established at NPHL. Apr 2011 For the isolation and characterization of influenza and parainfluenza viruses, the Madin Darby Canine Kidney (MDCK) cell line was successively cultured and propagated at NPHL. Jun 2011 From a clinical specimen obtained and stored at NPHL, the influenza virus was isolated and characterized successively. Nov 2011 The NPHL isolated and characterized a total of 28 influenza viruses and sent them to the WHO Collaborating Centre (WHOCC), National Institute of Infectious Diseases, Japan. Mar 2019 The first human death by H5N1 in Nepal. Avian influenza in the context of Nepal Epidemiology and outbreaks history Nepal had not observed HPAI H5N1 until 2009, although adjoining India and China reported several episodes of outbreaks. In Jhapa, a district bordering India and close to Bangladesh, the first HPAI outbreak was identified in January 2009. Although Nepal has undergone sporadic HPAI outbreaks since January 2009, until January 2012, no clinical outbreaks were identified in the capital city of Kathmandu. As per the National Agriculture census 2011, Nepal has a population of domestic poultry of around 53.6 million birds (Table 2). The 45% of this poultry are commercial birds and 55% are backyard birds. In Chitwan and Kathmandu Valley, the majority of the layers and broilers are raised. There were an estimated 58 hatcheries and 800 poultry producers in Nepal [148]. Most of the parent stocks are imported to Nepal from foreign countries. Occasional HPAI outbreaks has occurred in some of these stocks. In most of these countries, parent flocks are, however, subjected to strict biosecurity measures and national HPAI surveillance. It is extremely unlikely that parent stocks are contaminated with the HPAI virus. The import from Tibet is negligible. However, smuggling from India poses a greater risk of HPAI infected parent stock across the border. Also, there are minimal biosecurity practices across the industry, and migratory wild birds also use the same bodies of water used by domestically raised ducks. With arrays of wildlife reserves and national parks, migratory birds and wild birds often visit the water bodies in Nepal [149]. TFigure 2: Map of Nepal showing risk districts and wild water bird zones. Level of risk as represented by a different color: Black color represents the 27 high-risk districts, blue color represents the 18 medium-risk districts, white color represents the 32 low-risk districts, 6 green bubbles represent the wild water bird zones [Figure adapted from Reference 149]. Based on the national HPAI surveillance plan, Kathmandu and Chitwan are identified as high-risk disease areas in Nepal (Figure 2). The high-risk designation is based on the higher commercial poultry density, the higher poultry influx from other districts, Table 2. Poultry population and products in Nepal [148]. Poultry Population Fowl 45,171,185 Ducks 376,916 Laying hens 7,907,468 Laying ducks 174,978 Meat Weight in metric tonnes Chickens 40,346 Ducks 217 Eggs Numbers in 1000 Hens 788,310 Ducks 13,060 Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 34 the larger free-ranging ducks, the larger natural and man- made ponds and lakes visited by migratory birds annually, the existence of live bird markets, and the lower bio-security of commercial poultry farms [16, 153]. Since the first outbreak of 2009, 256 outbreaks have been reported to OIE by Nepal. Outbreaks peaked during 2012 followed by silent 4 years. Outbreaks have been resurgent in recent years. Most of the infected population includes commercial broilers and commercial layers (Table 4) (Figure 3) [16]. Recently Epidemiology and Disease Control Division, Ministry of Health and Population confirmed the first human death by H5N1 on 2nd May 2019. The 21 years old male was admitted on 24th March 2019 and died on 29th March 2019. It has been the first and the only case of identified human death by H5N1 infection in Nepal [42, 154]. Isolation and study of influenza in human/birds/poultry National Influenza Centre at National Public Health Laboratory (NPHL) is currently recognized by WHO and so is a member of the WHO Global Influenza Surveillance Network. Initially, influenza viruses were detected in suspected cases by Rapid Diagnostic Test (RDT), but now qPCR is employed. The center is primarily focused on collecting appropriate clinical specimens from patients, storing, transporting, and processing. Initial identification of virus type and subtype is done and isolates are forwarded to the WHO Collaborating Centre for Reference and Research on Influenza and alert the WHO Global Influenza Programme [152]. Figure 3. Map showing avian influenza outbreak clusters since 2009 in Nepal and its periphery [Figure adapted from Reference 155]. To date, 256 outbreaks have been reported by Nepal to OIE. Besides these notifications, several surveillance studies have been done in a bird population in Nepal. The first report of serologic evidence of AIV infection in poultry in Nepal was reported in October 2005 which was later determined to be the H9N2 subtype. This was clear evidence of the introduction of the virus in Nepal before 2005. The antibodies to influenza A were detected in the Table 4: Avian influenza outbreak history in Nepal [16]. Years Total outbreaks New outbreaks District Species Affected population Cases Killed and disposed 2019 15 14-Feb Makwanpur Birds Commercial layers 9,769 41,125 28-Feb Kathmandu Birds Layers 2,600 2,600 06-Nov Bhaktapur Birds Commercial broilers 1,535 3,961 17-Mar Kathmandu Birds House crow 200 0 2018 3 03-May Chitwan Birds Commercial layers 1,500 12,091 20-May Kathmandu Birds Ducks 270 240 2017 4 17-Feb Kaski Birds Chicken and ducks 98 297 01-Mar Sunsari Birds Commercial layers 3,650 2,550 02-Mar Sunsari Birds Swans and storks 15 0 2016 0 - - - - 0 0 2015 0 - - - - 0 0 2014 1 13-Feb Sunsari Birds Commercial layers 570 1,430 2013 0 - - - - 0 0 2012 210 27-Aug Lalitpur Birds Commercial layers 2,500 0 2011 13 10-Nov Bhaktapur Birds Chicken and ducks 88 308 2010 8 26-Jan Kaski Birds Chicken and ducks 153 11,128 25-Oct Chitwan Birds Commercial poultry 66 11,437 2009 2 08-Jan Jhapa Birds Poultry 14 24,689 Total 256 23,028 111,856 Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 35 chicken while ducks and pigeons were still serologically negative for the virus. This also outlines the absence of HPAI and notifiable H5 and H7 subtypes until that time. This also proved the brewing adaptation and introduction of another influenza A viruses in Nepal [150]. H5 subtype was not detected in 2007 by the RT- PCR test in suspected dead birds [156]. Similarly, domestic ducks were found seropositive for antibodies against H5 and H9 but not against H7 in 2009. Though none of the seropositive ducks were symptomatic for HPAI or LPAI virus infection [151]. H9N2 was reported in a fecal sample of migratory birds in southern Nepal. However, H7N9 and other HPAI viruses were not detected [157]. Different studies reported pandemic H1N1 in Nepal [158, 159]. Pandemic H1N1 detected in Nepal was the lineage of the novel influenza H1N1 virus (A/California/07/2009) [159]. HPAI virus has been a major threat to the Nepalese poultry industry since its outbreak in India in 2003. A substantial number of customers temporarily stopped buying chicken meat and switched to other sources of meat. The loss to entire value chain actors was estimated at NRs 1,154 million per year ($1=NRs. 73.06, the average exchange rate from 2001 to 2010) [160] from 2001 to 2010 [161]. Since the 2009 outbreak, the impacts have spilled across the country. The economic loss is even devastating. Loss of more than NRs 4.5 million has been attributed to the Pokhara bird flu outbreak alone, the third outbreak in Nepal. The commercial and backyard farmers including butchers on a small scale are primarily affected by the outbreak. Nepal government had tried to compensate for the loss during the outbreak, but the compensation offered is lower as compared to the gate price of the poultry and products [161, 162]. Outbreak Preparedness in Nepal A pandemic imposes an increased pressure on infrastructures to contain the disease, be it a developed nation or a developing nation. Developing nations face much more stress in resource mobilization which in the case is already scarce. But in global context, pandemic help to better understand the disease and effects of preventive measures. Pandemic preparedness includes the holistic approach from virologists, epidemiologists, animal and human health professionals, military and paramilitary forces, press, media, and administrations. Continuous coordination among all parties is warranted for the execution of swift countermeasures in case of a new pandemic outbreak. But as is the case in most developing countries, this co-ordination is still lacking in Nepal [163]. Besides this, AIV is dynamically evolving and adapting to newer hosts so there is still the need to establish tests that are quick and easy to use to characterize new influenza strains [164]. Cross country movements of birds and products should strictly follow OIE recommendations. Farms should be designed to minimize the interaction with wild bird populations and there should be maximum biosecurity practices [165- 167]. Public awareness level about the disease in Nepal Different studies reported that most of the Nepalese population were aware of AIV and that poultry workers were at risk of infection. Televisions, radios, newspapers, and social media had been conveying the message to the general population about AIV. However, on preventive measures, only hand washing was widely accepted by most folks even though the majority of the population was familiar with most of the preventive behaviors. Thus, there is a strong degree of acceptance of many specific government regulation policies in public. But the implemented control measures were not considered sufficient even though preventive measures are frequently conveyed by the government (Table 4) [168, 169]. Policy in controlling outbreaks in Nepal Between 2007 and 2011, the World Bank-funded the AIV surveillance and awareness campaign as the Avian Influenza Control Project (AICP) in Nepal. Since 2011, Nepal has continued the AICP with its internal resources. The management program focuses largely on outbreak- associated culling of poultry in tandem with washing and disinfection. control policy is mainly focused on the Table 4: Excerpt of the preventive measures released by the Ministry of Health and Population, Nepal in the public interest [154]. Frequent hand washing using soap. Personal hygiene and cleanliness. Environmental cleanliness. Avoid close contact with sick poultry and wild birds. Immediately visit nearby health institutions on suspicion of ill birds or persons in the vicinity. Keep children far away from dead and sick birds. Safe handling and personal safety should be considered while preparing poultry meat. The handler should clean hands and tools after preparing meat. Consume only well-prepared poultry meat (cooked to at least 70ºC). Don’t do drugs without consultation with physicians. Avoid close contact with dead birds or their droppings. Bury the dead birds carefully if found. Nepal J Biotechnol. 2 0 2 1 J u l ; 9 (1): 2 4 - 4 1 Shrestha et al. ©NJB, BSN 36 outbreak-related culling of poultry in conjunction with cleaning and disinfection. However, despite of current AICP, the continuing incidence of outbreaks has raised concerns about the effectiveness of the money expended. Poultry and product export from Nepal is negligible. But domestic demand is nearly self-sufficient, though parent stocks and vaccines are imported. Thus, it is crucial to preserve the poultry industries as a pandemic can wipe out the industry owing to the small geography of the country. Thus, AICP alone cannot be fully relied upon, instead, the vaccination control program can be implemented along with bio-security, as proven effective in Pakistan [170]. Nevertheless, there is always a possibility of the circulation of the asymptomatic virus and the future spread of infection [171]. Control and management strategies in Nepal The control and management of AIV are primarily focused on outbreak zone/s in Nepal. Once the outbreak is identified, the area is sealed and poultry production is banned for 45 days. Surveillance is intensified in the proximity of epidemic regions and other regions at higher risks. The suspected poultry are quarantined and culled. Since the first outbreak, 111,856 poultry have been killed to control the further spread. Thus, occasional outbreaks in different areas have seriously hampered the poultry industry (Table 2). The control and management strategies are thus targeted to outbreak areas only after the outbreak has been identified, rather than focusing on surveillance and adopting preventive measures in the outbreak prone and high-risk areas. The control measures were adopted by Nepal to curb AIV outbreaks, as reported to OIE (Table 5). Table 5. Measures taken by Nepal to curb AIV outbreaks (as reported to OIE). Control of movement in the country Surveillance outside containment and/or protection zone Surveillance within containment and/or protection zone Screening Traceability Quarantine Official destruction of animal products Official disposal of carcasses, by-products, and wastes Stamping out Disinfection Vaccination prohibited No treatment of affected animals Conclusion More than 20 years ago, H5N1 in humans was first identified. The reservoirs and key sources of human H5N1 infections are infected birds. Human-to-human transmission is very low and initial manifestations of illness are non-specific so detailed histories along with possible travel in endemic areas should be considered for case detection. Outbreaks have been recurrent in recent years in Nepal. Commercial rapid antigen tests are insensitive and only suitable for screening. Confirmatory diagnosis can only be done by molecular techniques. Oseltamivir has been warranted for the treatment of severe cases. The understanding of epidemiology, natural history, and human H5N1 disease control is still inadequate; thus, warranting the need for co-ordination in clinical and epidemiological research among institutions globally. Author’s Contribution BB and HP compiled the literature review. DS and SS prepared the initial draft. DS finalized the draft. All authors read and approved the final draft. Competing Interest The authors declare no conflict of interest or competing interest. Acknowledgements None References 1. Dictionary M-W: Influenza. In: Merriam-Webster Dictionary. Merriam-Webster Corporation; 2019. 2. Smith W, Andrewes CH, Laidlaw PP: A virus obtained from influenza patients. The Lancet 1933, 222(5732):66-68. https://doi.org/10.1016/S0140-6736(00)78541-2 3. 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