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.

www.ijmm.org
July, 2004 Padhi et al – Avian Influenza
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
www.ijmm.org
144
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
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:
145 Indian Journal of Medical Microbiology
1. 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.
2. Virus culture - Madin-Darby Canine Kidney cells
(MDCK). ATCC CCL34.
• Inactivated virus, goat serum to A/Term/South
Africa/61/H5, chicken pooled serum to A/
Goose/Hong Kong/437-4/99.
• WHO influenza reagent kit: reference antigens
and reference antisera.
• Receptor destroying enzyme (RDE).
• Red blood cells (chicken, turkey, human type O,
or guinea-pig red blood cells) in Alsever’s
solution.
3. 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
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.
www.ijmm.org
Vol.22, No.3
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
Vaccine
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-to-
human transmission”.5
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.
2003. Available at: http://www.nature.com/nsu/
020819/020819_14.html. Accessed February 4, 2004.
3. China. Likely source of bird flu: New Scientist.
Available at: http://www.economictimes.indiatimes.
July, 2004 Padhi et al – Avian Influenza 146

com/articleshow/449636.cms. Accessed February 9, cases_table_2004_02_09/en/. Accessed February 9,

2004. 2004.
4. Sixth bird flu death in Vietnam. Available at :http:// 8. Pearson H, Cyranoski D. Bird Flu attacks in Hanoi.
www.allzenews.com/click/22339-36k. Accessed Natural News Service/Macmillan Magazines Ltd.
February 9, 2004. 2004. Available at :http://www.nature.com/nsu/
040112/040112-3.html.
5. Elegant S. Is a human pandemic next? Time 2004;
163(5):14-20. 9. Recommended laboratory tests to identify influenza
A/H5 virus in specimens from patients with an
6. CDC-Avian Influenza (Bird Flu) Outbreak. Available
influenza like illness. Available at : http://www.int/
at : http://www.cdc.gov/flu/avian. Accessed
en/. Accessed March 18, 2004.
February 10, 2004.
10. WHO reference laboratories for diagnosis of
7. WHO- Confirmed Human Cases of Avian Influenza
influenza A/H5 infection. Available at: http://
A (H5N1). Available at : http://www.who.int/csr/
www.who.int/csr/disease/avain_influenza/
disease/avian_influenza/country/
guidelines/referelabs/en/. Accessed March 18, 2004.

L V Prasad Eye Institute

HYDERABAD, INDIA

Announcement

Applications are invited from candidates interested in making a career in Ocular Microbiology
at LV Prasad Eye Institute. The job encompasses all aspects of diagnostic work related to
eye infections/inflammations, in addition to ample scope for research and teaching. The
position is open to individuals trained in clinical (bacterial/fungal/viral/parasitic) microbiology,
molecular microbiology, microbial genetics etc.

Essential Qualifications : MBBS and MD (Medical Microbiology) or

PhD (Medical Microbiology)
Preferred added Qualifications : Experience in molecular techniques and virology
Desired Qualifications : Experience in ocular microbiology
Application Deadline : August 31, 2004
Age Limit : 35 years
Emoluments : To be fixed according to the salary structure
comparable to national centres, based on experience

Mail your CV with a letter of reference to the following address:

Prof. D. Balasubramanian
Director of Research
L V Prasad Eye Institute
L V Prasad Marg, Banjara Hills, Hyderabad – 500 034
Email : dbala@lvpei.org

www.ijmm.org