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Avian Influenza Surveillance in Domestic Pigeons Columba livia domestica. Submitted to Avian Influenza Department, National Institute of Virology, Pune. Under Kishor Vaigyanik Protsahan Yojana (KVPY), Indian Institute of Science, Bangalore, 2008.

By, PRANAV SUDHIR PANDIT Fourth Year B.V.S.c & A.H. K.N.P. College of Veterinary Science, Shirawal, Satara.

Under the Guidance of Dr. SHAILESH PAWAR Scientist-B, Avian Influenza, National Institute of Virology (NIV)Microbial Containment Complex (MCC), 130/1, Sus Road, Pashan, Pune-411021, India.

1

Acknowledgements Dr. A.C. Mishra, Director National Institute of Virology, for granting me permission to work in the prestigious institute and encouraging me to do my small project. Prof. Dipankar Chattarji for giving me this wonderful opportunity to work in NIV and help in official work regarding it. Dr. Satish Pande for his constant support and encouragement. Dr. Alok Chakrabarti for his time-to-time guidance in laboratory especially guiding me about PCR Amit Pawashe and Aditya Ponkshe for their help in fieldwork during sample collection. Khude Sir and Vishal Thite for their constant help in lab.

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Contents:

Page Number

a. Acknowledgement

1

b. Contents

2

1. Introduction to Avian Influenza.

3

2. Project Outline

6

i. Sample Collection ii. Transport of Samples and Storage iii. Aliquot Preparation iv. Egg Inoculation and Harvesting v. Haemagglutination Test 3. Results

10

4. Sample Collection from White-backed Vulture Gyps bengalensis

12

5. Training on maintainance of Madin Darby canine kidney (MDCK) cell line.

13

6. Introduction to biosafety & biosecurity, as a part of training

14

7. Appendix

18

8. References

19

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1.0 Avian Influenza: Introduction Avian Influenza is acute disease of birds caused by Orthomyxovirus. Influenza A virus subtypes are distributed worldwide and are frequently recovered from clinically normal birds. Outbreaks of severe clinical disease usually caused by subtypes expressing H5 and H7 determinants. The acute infection is often referred to as fowl plague or highly pathogenic avian Influenza and is categorized as List A disease by the OIE. 1.1 Etiological Agent The virus particles are spherical or pleomorphic, its enveloped virus, 80 to 120 nm in diameter. The envelope, which is derived from host cell membrane lipids, contains glycosylated and nonglycosylated viral proteins. Surface projections of glycoprotein form spikes or peplomers which, in influenza A and B viruses, are of two types: a heamagglutinin (HA) responsible for virus attachment and envelope fusion, and neuraminidase (NA) capable of cleaving viral receptors and promoting both entry of virus into cells and release of viron from infected cells.

Fig.1 Diagrammatic representation of Influenza Virus

Influenza virus haemagglutinate erythrocytes from a wide range of species. Antibodies to the HA glycoprotein are responsible for virus neutralization. The nucleocapsid has helical symmetry. The genome, which is composed of six to eight segments, consists of linear, negative sense, single stranded RNA. Replication occurs in cell nuclei with release of virons by budding from plasma membranes. Virons are labile in environment and are sensitive to heat, lipid solvents, detergents, irradiation and oxidizing agents.

4

The family Orthomyxoviridae contains four genera namely Influenzavirus A, Influenzavirus B, Influenzavirus C, and Thogotovirus. Influenza B and C viruses are pathogens of humans; Thogotovirus and Dhori virus are tick borne arboviruses isolated from camels and humans in parts of Africa, Europe and Asia. Influenza A virus the most important member of family is significant Pathogen of animals and humans. Isolates of influenza A virus are grouped into subtypes on the basis of their HA and NA antigens. Currently, 16 HA and 9 NA antigens are recognized. New subtypes of influenza emerge periodically. Two mechanisms, point mutation and genetic reassortment, is responsible for emergence of new subtypes. Point mutation gives rise to antigenetic drift in which variation occurs within a subtype. Genetic reassortment, a more complex process producing antigenetic shift, results in development of new subtypes. To assess the risk posed by the emergence of new variant viruses, World Health Organization has adopted a precise classification of isolates. This system is based on the influenza virus type, host, geographical origin, stain number, year of isolate and subtype. An example of this classification system, Influenza virus A/equine/Prague/1/56(H7N7), indicates that this virus is isolated from horse in Prague during 1956.

1.2 Epidemiology Infection is maintained in wild Bird populations. Migrating waterfowls are considered to be responsible for spreading the virus to domestic birds. Naturally occurring infections of AIV are reported from free-living birds representing more than 90 species in 13 avian orders. Most of the species are associated with aquatic habitats and two avian orders, the Anseriformes (ducks, geese, and swans) and the Charadriiformes (gulls, terns, and shorebirds). Species within the family Anatidae of the order Anseriformes (specifically ducks, subfamily Anatinae) have accounted for most of the reported AIV isolations. Although ducks become infected with influenza A virus, they rarely show any signs of illness. The virus is shed in feces after replication in intestine. Live bird markets may also contribute to spread of infection. The critical community size (CCS) for Low Pathogenic Avian Influenza (LPAI) virus in wintering dabbling ducks indicates to population of 1200-1500. The critical community size, defined as number of individuals able to maintain infection in an environment for a certain period of time. Considering the mortality associated with HPAI H5N1 in some wild species, the CCS for the virus must be higher than that required for LPAI viruses.

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1.3 Pathogenesis Spread of influenza virus in tissues is dependent in the type of proteases present in a given tissue and structure of viral haemagglutinin molecule. The production of infectious virions requires cleavage of viral haemagglunin. In the majority if influenza A virus subtypes, haemagglutinin cleavage takes place in the epithelial cells of respiratory tract and digestive tracts. Because of amino acid composition at their cleavage sites the haemagglutinins of virulent subtypes are susceptible to cleavage in many tissues, facilitating the development of generalized infection.

1.4 Clinical Signs The incubation period, which is variable, is up to seven days. Clinically, the disease may be in apparent, mild or, in some instances, severe with high mortality. Factors, such as overcrowding, poor ventilation and concurrent infections, may predispose to the development of sever disease. Highly virulent subtypes cause explosive outbreaks of disease with high mortality. Clinically signs are more apparent in birds, which survive for few days. Respiratory distress, diarrhoea, oedema in cranial region, cyanosis, sinusitis and lacrimation are features of clinical presentation. The infection of laying birds results in dramatic drop in egg production.

Fig 2: Clinical Signs Showing Neurological signs, Cyanotic and swollen combs, conjunctivitis and respiratory signs.

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2.0 Project Outline. An AI Survellience virus was conducted in Domesticated Pigeons Columba livia domestica, in Saswad town. Fifty cloacal swabs were collected from 3 different aviaries and all samples were processed for virus Isolation. The flowchart of work was as follows

Outline of Project Work Preparation of Sampling Kit ↓ Collection of Samples ↓ Transport & Storage ↓ Aliquot Preparation ↓ Egg Inoculation and Harvesting ↓ Haemagglutination test for detection of AI virus

2.1 Preparation of Kit for sample collection. (Field Work) Following consumables and non-consumables were included in the Kit prepared for sample collection. • • • • • • •

Ice Box, Ice Packs Virus Transport Medium (VTM), with antibiotics 5 ml screw capped vials (Autoclaved) N95 masks Gloves Sterile Cotton Swabs. Adhesive tape and labels

• • • • • • •

Sealable plastic bags Autoclavable disposable bags Lead Pencil Cello tape Spoon and stick Scissors & Blade Wet Ice.

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2.2 Preparation Virus Transport Medium (VTM) VTM was prepared as given in standard operating procedures (SOP) and prescribed antibiotics were added. (Refer Appendix 6.2). The cold chain was maintained during its preparation and it was transferred to sample collection vials. 3ml of this VTM was added in screw-capped vials, which were sealed using Parafilm. 2.3 Collection of Samples Samples collected were cloacal swabs. From live Pigeons were taken by inserting a swab deeply into vent and vigorously swabbing the wall. The swab should be deeply stained with fecal material. The swab was then directly placed into VTM. 2.4 Transportation of Samples. Screw capped vials were sealed with parafilm to avoid contamination, leakage. All samples were transported to laboratory at +4°C on an ice-box. The cold chain was strictly maintained everywhere henceforth in processing the samples. 2.5 Aliquot Preparation Each sample was first centrifuged to at 5000 RPM for 5 mins at +4°C. And then was divided into two aliquots. One aliquot was used for virus isolation and other was kept at -70°C till further use. 2.6 Egg Inoculations and Harvesting The samples were inoculated by allantoic route for amplification of Influenza virus.

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Fig. 3. Allantoic Route of Inoculation

0.2 Ml of each sample was inoculated in two ten-day-old embryonated chicken SPF (specific pathogen free) eggs with two eggs as controls. After inoculation the eggs were examined daily for any mortality. Dead eggs were stored at 4°C. After 72 hrs all eggs were transferred to 4°C for chilling for overnight. Allantoic fluid from inoculated eggs was harvested using syringe, and then suspected samples were processed for second blind passage. 2.7 Heamaggltination Test. Haemagglutination (HA) Test was carried out using chicken erythrocytes (0.5%) and horse erythrocytes (1%) using the harvested allontoic fluid, immediately after harvesting. Procedure of HA test REQUIREMENTS:

Equipment

Consumables

Biosafety cabinet,

U/V bottom 96-well microtiter plates, RBCs, ice,

micropipette, multichannel

syringes, needles, alsever’s solution, graduated

micropipette, centrifuge etc.

tubes, PBS (pH 7.2), filter tips etc.

Procedure: 1. 50 µl. of diluent (PBS) was added to each well of the 96-well-plate. 2. 50 µl. of antigen (Tissue Culture/Allantoic fluid) added to the first well of each row. 3. Two-fold dilution was carried out by transferring 50 µl from the first well of each column using multichannel micropipette. Proceed till the 8th column. Discard remaining 50 µl from the 8th column. 4. 50 µl of or 0.50% Fowl RBCs or 1% Horse RBCs was added to all wells using multichannel pipette. 5. Mixing was done by manually agitating the plate thoroughly.

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6. The plates were incubated at room temperature for 60 min. Cell control was checked for complete settling of RBC. INTERPRETATION:


Fowl/Horse RBCs will form a button or a ring at the bottom of the wells and recorded as ‘O’. If haemagglutination occurs, i.e. RBCs remain in suspension, it is recorded using a “+” symbol.




The highest dilution of virus that causes complete haemagglutination, is considered the end point in HA titration. The HA titer is the reciprocal of dilution of virus in the last well with complete Haemagglutinatiion.




If partial agglutination is seen, the titer of the last well showing complete agglutination must be added with the titer of the well showing partial agglutination and divided by two to obtain the haemagglutination titer.

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3.0 Results All samples were negative for any Haemagglutinating Virus for both Chicken as well as Horse RBCs. Samples from individuals with clinical history of respiratory disease were given second passage. The further passaging also could not detect any HA virus in those samples. Table 1. Haemagglutination Test Results of Samples from Columba livia domestica. Sr. No.

AI No.

NIV No.

Date of Collection

Sample Type

Location

1

2800

88663

22/7/08

Cloacal Swab

Saswad

2

2801

88664

22/7/08

Cloacal Swab

Saswad

3

2802

88665

22/7/08

Cloacal Swab

Saswad

4

2803

88666

22/7/08

Cloacal Swab

Saswad

5

2804

88667

22/7/08

Cloacal Swab

Saswad

6

2805

88668

22/7/08

Cloacal Swab

Saswad

7

2806

88669

22/7/08

Cloacal Swab

Saswad

8

2807

88670

22/7/08

Cloacal Swab

Saswad

9

2808

88671

22/7/08

Cloacal Swab

Saswad

10

2809

88672

22/7/08

Cloacal Swab

Saswad

11

2810

88673

22/7/08

Cloacal Swab

Saswad

12

2811

88674

22/7/08

Cloacal Swab

Saswad

13

2812

88675

22/7/08

Cloacal Swab

Saswad

14

2813

88676

22/7/08

Cloacal Swab

Saswad

15

2814

88677

22/7/08

Cloacal Swab

Saswad

16

2815

88678

22/7/08

Cloacal Swab

Saswad

17

2816

88679

22/7/08

Cloacal Swab

Saswad

18

2817

88680

22/7/08

Cloacal Swab

Saswad

19

2818

88681

22/7/08

Cloacal Swab

Saswad

20

2819

88682

22/7/08

Cloacal Swab

Saswad

21

2820

88683

22/7/08

Cloacal Swab

Saswad

22

2821

88684

22/7/08

Cloacal Swab

Saswad

Species Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica

Health Status

HA Results

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy Skin Lesions

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Negative

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23

2822

88685

22/7/08

Cloacal Swab

Saswad

24

2823

88686

22/7/08

Cloacal Swab

Saswad

25

2824

88687

22/7/08

Cloacal Swab

Saswad

26

2825

88688

22/7/08

Cloacal Swab

Saswad

27

2826

88689

22/7/08

Cloacal Swab

Saswad

28

2827

88690

22/7/08

Cloacal Swab

Saswad

29

2828

88691

22/7/08

Cloacal Swab

Saswad

30

2829

88692

22/7/08

Cloacal Swab

Saswad

31

2830

88693

22/7/08

Cloacal Swab

Saswad

32

2831

88694

22/7/08

Cloacal Swab

Saswad

33

2832

88695

22/7/08

Cloacal Swab

Saswad

34

2833

88696

22/7/08

Cloacal Swab

Saswad

35

2834

88697

22/7/08

Cloacal Swab

Saswad

36

2835

88698

22/7/08

Cloacal Swab

Saswad

37

2836

88699

22/7/08

Cloacal Swab

Saswad

38

2837

88700

22/7/08

Cloacal Swab

Saswad

39

2838

88701

22/7/08

Cloacal Swab

Saswad

40

2839

88702

22/7/08

Cloacal Swab

Saswad

41

2840

88703

22/7/08

Cloacal Swab

Saswad

42

2841

88704

22/7/08

Cloacal Swab

Saswad

43

2842

88705

22/7/08

Cloacal Swab

Saswad

44

2843

88706

22/7/08

Cloacal Swab

Saswad

45

2844

88707

22/7/08

Cloacal Swab

Saswad

46

2845

88708

22/7/08

Cloacal Swab

Saswad

47

2846

88709

22/7/08

Cloacal Swab

Saswad

48

2847

88710

22/7/08

Cloacal Swab

Saswad

49

2848

88711

22/7/08

Cloacal Swab

Saswad

50

2849

88712

22/7/08

Cloacal Swab

Saswad

Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica Columba livia domestica

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

Healthy

Negative

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4.0 Sample Collection from White-backed Vulture Gyps bengalensis A highly endangered Species of Vulture White-backed Vulture Gyps bengalensis was rescued at 13 KM from Shrigonda, Dist. Ahmednagar by Local Forest Officers. The bird was apparently normal with no external injuries and was feeding well. On 05/08/08 the biometry of the bird was carried out and different samples for virus Isolation were collected. Samples included Blood, serum, Cloacal swab, tracheal swab and fecal material. The samples were processed and then inoculated in embyonated eggs using allantoic route. The harvested allantoic fluid was tested in haemagglutination test. 3.2.1 Observations and results Table 2. Biometry observations of Gyps bengalensis. Biometry Tail: Tarsus: Middle Toe: Talon: Wing cord: Beak Depth of beak:

mm 290 120 94 24 610 68 35

3.2.2 HA Test Results All the samples from this vulture were found to be negative for haemagglutinating viruses in HA test with Chicken (0.5%) and Horse RBCs (1%). Table 3. Haemagglutination Test Results of Samples from Gyps bengalensis. Sample Name CC CD T F B S

Sample Cloacal Swab (Conc.) Cloacal Swab (Dil.) Tracheal Swab Fecal Material Blood Serum

Species

DOC

Health Status

HA Results

Gyps bengalensis Gyps bengalensis Gyps bengalensis Gyps bengalensis Gyps bengalensis Gyps bengalensis

5/8/2008 5/8/2008 5/8/2008 5/8/2008 5/8/2008 5/8/2008

Appearently Healthy Appearently Healthy Appearently Healthy Appearently Healthy Appearently Healthy Appearently Healthy

Negative Negative Negative Negative Negative Negative

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4.0 Training on maintainance of Madin Darby canine kidney (MDCK) cell line Madin Darby canine kidney MDCK is canine cell line prepared from Cocker Spaniel Dog Kidney. This cell line is fairly used for in vitro cultivation of Influenza virus. The cell line was passaged using following procedure. 4.1 Procedure •

Full grown T-25 MDCK bottle was taken

•

The growth medium was decanted.

•

Washing of monolayer with plain DMEM (without FCS)

•

Repeated this procedure with plain DMEM

•

Added 2 ml of TPVG for 1 minute

•

Decanted

•

Repeated twice

•

Kept the bottle at 37°C for 3-5 minutes

•

16 ml DMEM was added with 10% FCS

•

4ml from suspension of cells was distributed to four new T-25 Bottles

•

Incubated at 37°C in CO2 (5%) incubator for next 3-5 days. The monolayers were observed under inverted microscope every 24 hours and they showed full growth on 3rd day. Fig: MDCK Cell line Growth.

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5.0 Introduction to biosafety & biosecurity, as a part of training: Before initiating work in Avian Influenza (AI) laboratory, introduction to biosafety levels and standard operating practices in AI laboratory was given as a part of training. 5.1 Biosafety and Biosecurity. Bio-safety refers to biocontainment and related precautions taken to prevent and avoid accidental spillage of organism to environment, safety of lab workers and safety of public and other animals from that organism. While bio-security consists policies and measures taken to protect economy, environment and public health from biological harm of disease pests and bioterrorism. Biocontainment can be classified by the relative danger to the surrounding environment as biological safety levels (BSL). These are called BSL1 through BSL4. At the lowest level of biocontainment, the containment zone may only be a bio-safety cabin that utilizes HEPA filters. At the highest level the containment involves isolation of the organism by means of building systems, sealed rooms, and sealed containers, personal isolation equipment commonly referred to as personal protective equipments and elaborate procedures for entering room, and decontamination procedures for leaving the room. In most cases this also includes high levels of security for access to the facility, ensuring that only authorized personnel may be admitted to any area that may have some effect on the quality of the containment zone.

5.1.1 Biosafety Level 1 This level is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. It includes several kinds of bacteria and viruses including canine hepatitis, Escherichia coli, varicella (chicken pox), as well as some cell cultures and non-infectious bacteria. At this level precautions against the biohazardous materials in question are minimal, most likely involving gloves and some sort of facial protection. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Usually, contaminated materials are left in open (but separately indicated) trash receptacles. Decontamination procedures for this level are similar in most respects to modern precautions against everyday microorganisms (i.e.: washing one's hands with anti-bacterial soap, washing all exposed surfaces of the lab with disinfectants, etc). In a lab environment, all materials used for cell and/or bacteria cultures are

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decontaminated via autoclave. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science. 5.1.2 Biosafety Level 2 This level is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. Includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting, such as C. diff, hepatitis A, B, and C, influenza A, Lyme disease, dengue fever, Salmonella, mumps, Bacillus subtilis, measles, HIV, scrapie. BSL-2 differs from BSL-1 in that: 1. Laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; 2. Access to the laboratory is limited when work is being conducted; 3. Extreme precautions are taken with contaminated sharp items; and 4. Certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment. 5.1.3 Biosafety Level 3 This level is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents that may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Includes various bacteria and viruses that can cause severe to fatal disease in humans, but for which vaccines or other treatment exist, such as anthrax, West Nile virus, Venezuelan equine encephalitis, Eastern equine encephalitis, SARS, tuberculosis, typhus, Rift Valley fever, Rocky Mountain spotted fever, yellow fever. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents. This is considered a neutral or warm zone. All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features. It is recognized, however, that some existing facilities may not have all the facility features recommended for Biosafety Level 3 (i.e., double-door access zone and sealed penetrations). In this

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circumstance, an acceptable level of safety for the conduct of routine procedures, (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, susceptibility testing, etc.), may be achieved in a Biosafety Level 2 facility, providing 1. The exhaust air from the laboratory room is discharged to the outdoors, 2. The ventilation to the laboratory is balanced to provide directional airflow into the room, 3. Access to the laboratory is restricted when work is in progress, and 4. The recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations is made only by the laboratory director. 5.1.4 Biosafety Level 4 This level is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, agents which cause severe to fatal disease in humans for which vaccines or other treatments are not available, such as Bolivian and Argentine hemorrhagic fevers, smallpox (there is a vaccine), Marburg virus, Ebola virus, Lassa fever, Crimean-Congo hemorrhagic fever, and other various hemorrhagic diseases. When dealing with biological hazards at this level the use of a Hazmat suit and a self-contained oxygen supply is mandatory. The entrance and exit of a Level Four biolab will contain multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designed to destroy all traces of the biohazard. Multiple airlocks are employed and are electronically secured to prevent both doors opening at the same time. All air and water service going to and coming from a Biosafety Level 4 lab will undergo similar decontamination procedures to eliminate the possibility of an accidental release. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents are handled at this level until sufficient data is obtained either to confirm continued work at this level, or to work with them at a lower level. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics. Qualified scientists who are trained and experienced in working with these agents supervise them. The laboratory director strictly controls access to the laboratory.

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The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. A specific facility operations manual is prepared or adopted. Building protocols for preventing contamination often uses negatively pressurized facilities, which, if compromised, would severely inhibit an outbreak of aerosol pathogens. Within work areas of the facility, all activities are confined to Class III biological safety cabinets, or Class II biological safety cabinets used with one-piece positive pressure personnel suits ventilated by a life support system. The Biosafety Level 4 laboratory has special engineering and design features to prevent microorganisms from being disseminated into the environment. The laboratory is kept at negative air pressure, so that air flows into the room if the barrier is penetrated or breached. Furthermore, an airlock is used during personnel entry and exit.

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6.0 Appendix 6.1 Preparation of Virus Transport Medium. Hanks A (20X): 25ml. Hanks B (20X): 25ml. Phenol Red (0.5%): 1ml. DDW: 394 ml. Autoclave in 10 lbs for 10 mins. At 110° C Add Bovine Serum albumin (10%): 50ml. NaHCO3 (3.5%): 5ml. Adjust pH to 7.2% with 1N NaOH and store at 4°C. 6.2 Preparation of VTM with antibiotics For 100 ml of VTM Add 4.8 ml. of Gentamicin and 2 ml. of Streptomycine. 6.3 DMEM- Dulbeccos Modified Eagles Medium For one Liter DDW:

950 ml

NaHCO3:

3.7 gm

Adjust volume to one liter Adjust pH to 7.2 by Passing CO2 and filter through 0.22 µ Millipore. 6.4 FCS- Fetus Calf Serum Thaw 50 ml in 100 ml Inactivate at 56°C for 20 mins. Check for sterility and store at 4°C. 6.5 TPVG-Trypsin inPBS with Versene and Glucose PBS (10X): 50 ml DDW: 371.5ml Phenol Red (0.5%): 1 ml Autoclave and sterile Trypsin (2%): 25ml EDTA (0.2%): 50ml Glucose (10%): 2.5ml, Adjust pH 7.5 with 0.1 N NaOH and store at 4°C

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References: 1. http://en.wikipedia.org/wiki/Biosafety_level: Biosafety level. 2. Veterinary Microbiology and Microbial Disease; Quinn P.J., B.K. Markey, M.E. Carter, W.J. Donnelly and F.C. Leonard. Blackwell Science, 2002. 3. Biosafety in Microbiological and Biomedical Laboratories, U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention and National Institutes of Health, Fifth Edition, 2007, U. S. Government Printing Office Washington: 2007. 4. Guidelines on Wild Bird Surveillance for Highly Pathogenic Avian Influenza H5N1 Virus1, Vittorio Guberti2 and Scott H. Newman, Journal of Wildlife Diseases, 43(3) Supplement 2007, pp. 29–34 Wildlife Disease Association 2007. 5. Training Manual on Avian Influenza Collection of Samples, (Western Zone) Regional Disease Diagnostic Laboratory Disease Investigation Section and Dept of Animal Husbandry, Maharashtra, Aundh, Pune 411007. 26th April, 2006. 6. Standard Operating Procedures, Department of Avian Influenza, National Institute of Virology, Pune.