Preliminary Review

Indian Journal of Medical Microbiology, (2004) 22 (3):143-146

Review Article

AVIAN INFLUENZA A (H5N1): A PRELIMINARY REVIEW *S Padhi, PK Panigrahi, A Mahapatra, S Mahapatra

Abstract Humanity has been at the receiving end of many viral diseases since ages. Sudden emergence and re-emergence of new viral diseases in human beings has surprised the medical scientists from time to time. “Avian influenza” or “Bird flu” by H5N1 epidemics is one such surprise. Although many aspects about this disease are clear, there are some dark areas regarding vaccine development that need to be further explored and understood, so as to effectively contain the spread of this disease. The present article details out almost everything known about this interesting disease along with the review of the recent literature. Key words: Avian influenza, influenza A (H5N1), epidemic

“Avian influenza” or “Bird flu” is an infectious disease of birds, ranging from a mild to a severe form of illness. It is caused by 15 subtypes of the influenza A virus, subtype of avian influenza. Viruses of low pathogenicity can, after circulation for some time in a poultry population, mutate into highly pathogenic viruses. To date, all outbreaks of the highly pathogenic form have been caused by influenza A/H5N1 virus, the only subtype that causes outbreaks of severe disease in humans.1 The ability of the strain H5N1 to evade the body’s defence mechanism by evading cytokines (the first line of defence against ‘flu’) may be responsible for the high pathogenicity of this particular strain.2 The ‘bird flu’, now sweeping many countries of Asia, is caused by the H5N1 strain of the influenza A virus. Like Severe Acute Respiratory Syndrome (SARS), bird flu is suspected to have originated in China, probably in the first half of 2003.3 Avian flu first ‘jumped’ the “species barrier” from birds to humans in 1997 and caused an outbreak in Hongkong.2 Following the death of six people by H5N1 bird flu, Hongkong conducted a mass slaughter of chickens. To protect its poultry, Chinese poultry producers use an inactivated H5N1 virus for vaccination of birds. However, the vaccination does not confer complete immunity, which is evident from the fact that the vaccinated birds may develop the disease possibly due to infection by new strains resulting out of genetic reassortments, as is the case with H5N1 strain now sweeping Asia. According to *Corresponding author Department of Microbiology (SP, AM), Department of General Surgery (PKP), Department of Pathology (SM), MKCG Medical College, Berhampur, Orissa - 760 004, India. Received : 14-02-2004 Accepted : 14-04-2004

some health experts, the vaccination of birds in China could have contributed to the current problem.3 The present outbreak was first reported in poultry of Thailand as chicken cholera in November 2003. On 15th December 2003, South Korea confirmed that the outbreak was, infact ‘avian flu’. On 23rd January 2004, Thailand confirmed the first human case of ‘avian-flu’.4 At present, it has spread to countries such as Vietnam, Cambodia, Taiwan, Japan, South Korea, China, Hongkong, Indonesia and Laos. It is believed that migratory wildfowl, which can carry numerous viruses without being infected, are most likely to blame for the initial spread of the disease. Other factors – the transport of infected chickens across borders, both legally and illegally, as well as months of government inactivity despite mounting evidence of avian flu outbreaks- came into play to produce the current problem.5 Human cases have been reported only in Thailand and Vietnam, whereas in other countries only infection in poultry has been reported.6 Till 10th February 2004, the death toll in humans from the disease had been 18 and tens of thousands of chickens had been killed to keep the disease under control.7 Morphology and genetic structure of the virus The morphology of influenza A (H5N1) is basically that of an orthomyxovirus as it is a subtype of the type A influenza virus. The typical virion is enveloped, spherical (100 nm), with a nucleocapsid of helical symmetry surrounding a minus sense single stranded 8 segmented RNA (Figure). The envelope is internally lined by a matrix protein (M) and externally with glycoprotein peplomers-rod shaped haemagglutinin (HA) which are homotrimers of class I membrane glycoproteins and mushroom shaped neuraminidase (NA) molecules which are tetramers of a class II membrane protein.

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through infected aerosols, discharges and surfaces, as large amounts of the virus are excreted in bird droppings and can survive for some time in the environment. There is no evidence of human to human transmission till date.3 Fortunately, these viruses lack the ability to ‘hop’ easily between people, which has probably helped to contain the problem. However, in the future, a strain might acquire this ability, either by mutation or by recombination of genetic material with a human influenza virus and the ensuing virus would then be highly pathogenic and transmissible causing an “influenza pandemic”.8 Signs and symptoms

Figure: Schematic diagram of influenza A virus HA - Haemagglutinin; NA - Neuraminidase; M(M1 & M2) Matrix protein; NS - Non-structural protein; NP Nucleoprotein; PA, PB1, PB2 - Polymerase proteins

Based on the variation on HA and NA molecules there exists 15 HA and 9 NA subtypes of influenza A virus. The avian strains differ from human strains in that they have all the 15 subtypes of HA in contrast to only three in case of humans. The virulent avian influenza H5N1 strains differ from other avian strains in that, there lies a link between HA cleavage and degree of virulence. In virulent strains the HAs contain multiple basic aminoacids at the cleavage site, which are cleaved intracellularly by endogenous proteases. In contrast, in case of avirulent avian strains as well as non-avian influenza A viruses, the HAs lack the basic aminoacid residues, hence not subjected to cleavage by such proteases. Moreover, all types of influenza A viruses are antigenically labile, well adapted to evade host defences and lack mechanisms for “proof reading”; hence constant, permanent and small changes in antigenic composition are very common, which is known as antigenic drift. Another important characteristic of great public health concern is antigenic shift which results from reassortment of genetic material from different species resulting in variability of HA spikes, keeping the basic structure of the virus constant. Modes of transmission to humans The disease is transmitted to humans by direct or indirect contact with infected wild ducks and chickens

They are very similar to that of the disease caused by other influenza viruses. Fever, malaise, myalgia, sore throat and cough are found in most of the patients while conjunctivitis is seen in some. Persistent high fever is an useful sign. Life threatening complications like viral pneumonia, respiratory distress syndrome and multi organ failure may result in the death of the patient. Diagnosis A patient is suspected to be suffering from “avian influenza” (‘bird flu’) if he/she has any respiratory illness and has had recent direct or indirect contact by handling, or by having taken care, or by exposure to sick chickens or other birds. Besides the classical clinical presentations as described above, X-ray of the chest is useful in detecting early viral pneumonia. Specimens like nasopharyngeal aspirate, endotracheal aspirate, sputum and serum from clinically suspected cases are subjected for laboratory investigations for further confirmation. Laboratory Diagnosis Rapid antigen detection by immunofluorescence assay and enzyme immuno assay, virus isolation by culture in HeP-2, RD cells or MDCK cell lines and identification by immunofluorescence assay using specific monoclonal antibody and haemagglutination inhibition assay have been used for diagnosis. 9 Detection of influenza- specific RNA by reverse transcriptase-polymerase chain reaction, by using primer sets specific for HA sequence of influenza A/H5 and of N1 are some of the other tests that have been developed. Serological identification by measuring the specific antibodies by haemagglutination inhibition test, enzyme immuno assay and the virus neutralisation test, more specifically the micro neutralisation test, have also been developed. Following kits are presently available:

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1.

2.

Indian Journal of Medical Microbiology

Immunoflourescence assay- WHO influenza reagent kit for the identification of Influenza A/H5 virus (1997-1998, 2003 or 2004 version) which includes influenza type A/H5- specific monoclonal antibody pool along with influenza B, A/H1 and A/H3 subtype specific monoclonal antibodies.

shown resistance to amantadine and rimantadine, two of the antiviral drugs commonly used for influenza. However, other antivirals (oseltamavir and zanamavir) are still effective against this strain of H5N1.6

Virus culture - Madin-Darby Canine Kidney cells (MDCK). ATCC CCL34.

At present there is no available human vaccine for avian flu and production of a new vaccine would not begin until the disease shows “significant human-tohuman transmission”.5

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3.

Vol.22, No.3

Inactivated virus, goat serum to A/Term/South Africa/61/H5, chicken pooled serum to A/ Goose/Hong Kong/437-4/99.

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WHO influenza reagent kit: reference antigens and reference antisera.

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Receptor destroying enzyme (RDE).

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Red blood cells (chicken, turkey, human type O, or guinea-pig red blood cells) in Alsever’s solution.

Polymerase chain reaction - Gene primers from Hong Kong, Government Virus Unit.

All laboratory results for influenza A/H5N1 should be confirmed by a WHO collaborating center for influenza or by another WHO- recommended reference laboratory. The WHO reference laboratories are as below10: Queen Mary Hospital, University of Hong Kong. National Influenza Center, Kowloon, Hong Kong. National Institute of Infectious disease, Tokyo, Japan. National Institute of Medical Research, UK. Department of Infectious disease, Memphis, USA. Centers for Disease Control and Prevention, Atlanta, USA. Treatment If ‘bird flu’ is suspected in a person, treatment should be started immediately without waiting for laboratory confirmation. Treatment for infection by the H5N1 strain is essentially similar to that employed for infections due to the other influenza viruses. Unfortunately, the current strain of H5N1 has already

Vaccine

Containment The culling of sick and exposed birds is the key to containing the outbreak. For the current outbreak in Asia, governments are culling poultry to try to contain the virus. Patients are being treated and isolated and investigations are under way to determine the source of infection. Prevention The ban on importing of live chickens and other poultry products from countries affected with ‘bird flu’ is a critical step to prevent the entry of ‘bird flu’ into India. Other important steps to be followed are : • • • • •

Wearing of masks and gloves by persons handling poultry. Cleaning kitchen surfaces and utensils before and after use Cooking chicken till boiling temperature is reached Controlling human traffic into poultries. Reporting to authorities any unusual death or illness of chickens or other birds as well as illness of workers in poultry farms.

Conclusion Even after tremendous development in molecular biology, the mysteries surrounding the complex viral genetic reassortments giving rise to new pathogenic viral mutants, remain unclear to the scientists till date. But we can hope that, in future the virologists and biotechnologists will be able to unveil these secrets and save a number of precious human lives.

References 1.

Available at : http://www.doh.gov.ph/bird_flu.htm. Accessed February 9, 2004.

2.

Whitfield J. Deadly flu evades body’s defences. Nature news Service/Macmillan Magazines Ltd.

2003. Available at: http://www.nature.com/nsu/ 020819/020819_14.html. Accessed February 4, 2004. 3.

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China. Likely source of bird flu: New Scientist. Available at: http://www.economictimes.indiatimes.

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com/articleshow/449636.cms. Accessed February 9, 2004. 4.

Sixth bird flu death in Vietnam. Available at :http:// www.allzenews.com/click/22339-36k. Accessed February 9, 2004.

5.

Elegant S. Is a human pandemic next? Time 2004; 163(5):14-20.

6.

CDC-Avian Influenza (Bird Flu) Outbreak. Available at : http://www.cdc.gov/flu/avian. Accessed February 10, 2004.

7.

WHO- Confirmed Human Cases of Avian Influenza A (H5N1). Available at : http://www.who.int/csr/ disease/avian_influenza/country/

cases_table_2004_02_09/en/. Accessed February 9, 2004. 8.

Pearson H, Cyranoski D. Bird Flu attacks in Hanoi. Natural News Service/Macmillan Magazines Ltd. 2004. Available at :http://www.nature.com/nsu/ 040112/040112-3.html.

9.

Recommended laboratory tests to identify influenza A/H5 virus in specimens from patients with an influenza like illness. Available at : http://www.int/ en/. Accessed March 18, 2004.

10. WHO reference laboratories for diagnosis of influenza A/H5 infection. Available at: http:// www.who.int/csr/disease/avain_influenza/ guidelines/referelabs/en/. Accessed March 18, 2004.

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