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Annual reports of OIE Reference Laboratories and Collaborating Centres, 20
11

1

OIE Reference Laboratory Reports

Activities in 20
1
1

Name of disease (or topic)
for which you are a
designated OIE Reference
Laboratory:

Highly pathogenic avian influenza
and low
pathogenic avian influenza (poultry)

Address of laboratory
:

Avian Virology

Animal Health and Veterinary Laboratories Agency

AHVLA Weybridge

Woodham Lane

New Haw

Addlestone

Surrey KT15 3NB

UNITED KINGDOM

Tel.:

+44 (0)1932 357
.339

Fax:

+44 (0)1932 357239

e
-
mail address:

Ian.brown
@ahvla.gsi.gov.uk

website:

http://vla.defra.gov.uk/science/sci_ai_reflab.htm

Name
(including Title and
Position)
of
Head of
Laboratory (Responsible
Official):

Catherine Brown


Chief Executive

Name(including Title and
Position) of OIE Reference
Expert:

Prof. Ian Brown

Name (including Title and
Position) of writer of this
report

(if different from above):



Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

2

Annual reports
of OIE Reference Laboratories

and Collaborating Centres, 2011

Part I:

Summary of general activities related to the disease

1.

Test(s) in use/or available for the specified disease
/topic

at your laboratory

Test

For

Specificity

Total

M gene real
-
time RT
-
PCR

Viral RNA

Group

687

H5

real
-
time RT
-
PCR

Viral RNA

H5

83

H5 genetic analyses

Viral RNA

H5 gene lineage and pathotype

23

N1

real
-
time RT
-
PCR

Viral RNA

N1

35

H7

real
-
time RT
-
PCR

Viral RNA

H7

39

H7 genetic analyses

Viral
RNA

H7 gene lineage and pathotype

4

HI

Antibody

Subtype

18383

AGP

Antibody

Group type

4535

ELISA

Antibody

Group type

688

Egg Inoculation/HA

Virus isolation

Virus

1662

IVPI

Virus

Pathogenicity

1

2.

Production and distribution of diagnostic reagents

Type of reagent

Amount supplied
nationally

(
including for own use
)

Amount supplied to other
countries

Control positive/negative
serum for AGDPT

20 ml

64/46 ml

Influenza antisera

(all subtypes)

100 ml

952 ml

H1
×

20

ml, H2
×

16

ml,

H3

×

13

ml, H4
×

16

ml, H5

×

453

ml,
H6
×

17

ml, H7
×

239

ml, H8
×

11

ml,
H9
×

87

ml, H10
×

13

ml, H11
×

9

ml,
H12
×

12

ml, H13
×

12

ml,

H14
×

11

ml, H15
×

12

ml, H16
×

9

ml

Antigens for HI test

150 ampoules of 1ml for
covering all virus subtypes.

1760 ampoules of 1

ml for H1
×

9,


H2
×

2, H3
×

2, H4

×
2, H5
×

1351,

H6
×

4, H7
×

358, H8
×

2, H9
×

16,
H10
×

2, H11
×

2, H12
×

2, H13
×

2,
H14
×

2, H15
×

3, H16
×

1

Antigens/control sera for
mandatory EU annual survey

H7N7: 35

ml,

serum 2

ml;

H7N1: antigen 3

ml,

serum
1

ml


H5N3: antigen 40

ml,

serum 3

ml;

H5N1: antigen 4

ml,

serum 1

ml

H7N7: 416

ml,

serum 17

ml;

H7N1: antigen 61

ml,

serum 3

ml

H5N3: antigen 519

ml,

serum 21

ml;

H5N1: antigen 61

ml,

serum 3

ml

Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

Annual reports of OIE Reference Laboratories and Collaborating Centres, 20
11

3

Part II:

Activities specifically related to the
mandate

of OIE Reference Laboratories

3.

International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines

a)

Establishment and maintenance of a network with other OIE Reference Laboratories
designated for the same pathogen or disease and organisation of regular inter
-
laboratory
proficiency testing to ensure comparability of results.

b)

Organisation of inter
-
laboratory proficiency testing with laboratories other than OIE
Reference Laboratories

for the same pathogens and diseases to ensure equivalence of
results

a) & b):

This year’s panel to assess conventional methods for virus typing and serology was dist
ributed to
40

countries
(including some laboratories designated as OIE reference laborator
ies for AI)
including the
two OIE twinning project countries of Botswana and South Africa, 38 of the 40 countries (from three
continents) returned results for analysis.
Several countries mistyped non
-
notifiable viruses as H5 or H7
and this was due to a com
mon neuraminidase subtype recognized between reference serum and antigen.
Results were posted on FluLabNet (an interactive laboratory network web based forum) and also
presented to EU members at the EU annual national reference laboratory meeting in Brusse
ls.

Further
input by supplying reagents and assessment assistance was given to the organisers to implement the
second proficiency panel for the Southern African DC region.

In addition the annual panel to test proficiency and standardisation of PCR methodology was also issued.
This ring test was distributed to AI National Reference Laboratories (including some laboratories
designated as OIE reference laboratories for AI) in t
he European Union (EU) (26) and other third
countries (from three continents) or EU non
-
NRLs (22) for the application of molecular diagnostics to AI
where
real
-
time

PCR has a key role.

The 22 latter institutions include AI laboratories beyond the
immediate

European geographic region, with more laboratories having participated than in 2010.

The
AHVLA analysed results of such
real
-
time

PCR tests, collated data, informed laboratories of their results
and provided support and training if further action was requ
ired.

‘Mini panels’ continued to be made
available and distributed to international laboratories as a first step towards participating in the full
exercise.

This was the sixth successive year (since 2006) in which the molecular EU AI Panel was
distributed,

and a general plateauing of successful AI generic detection by
real
-
time
PCR is being
observed (
c.

91
-
96% success rate), with similar observations for specific
real
-
time
PCR’s for the
detection of notifiable AI (NAI) due to H5 and H7 viruses.

Institutions

that have access to sequencing
facilities were asked to identify the molecular pathotype for NAI, where an onus is placed on the
participating laboratory to not only provide the correct sequence, but also to declare the NAI specimen as
being eith
er LPAI o
r HPAI.

As with the 2010 panel, the 2011 version provided an opportunity for
laboratories to evaluate their methods for detecting pandemic (H1N1) 2009 influenza. A reduction in the
use of non
-
recommended / non
-
validated tests that have previously caused pr
oblems due to poor
performance was again noted in 2011.

Individual requests for advice and assurance concerning various aspects of molecular approaches to AI
detection and pathotyping continued to be received during 2011.

Following the molecular ring trial
,
twenty laboratories engaged in constructive dialogue to resolve testing anomalies. Most of these were
successfully resolved in year.

AHVLA participated in an OFFLU initiative working towards harmonised approaches for proficiency
testing at an internatio
nal level.

4.

Preparation and supply of international reference standards for diagnostic tests or vaccines

AHVLA coordinated the OFFLU technical group to produce an H5 international reference serum. A cross
group analyses was completed to identify two refe
rence sera that produced robust and accurate typing of H5
viruses, done using a panel of global strains. Production of standard sera is currently in progress
.

Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

4

Annual reports
of OIE Reference Laboratories

and Collaborating Centres, 2011

Continuing supply of ‘Eurasian’ lineage H5 and other influenza A virus inactivated preparations a
s reference
standards or controls for AI PCR methodologies
.

5.

Research and development of

new procedures for diagnosis and control

Overarching principles laid down in the OIE validation template are applied to all assessments described
below.

Ongoing

validation of AI RRT PCR

Throughout 2011, all new H5 and H7 submissions to AHVLA were carefully tested by the corresponding type
-
specific RRT PCRs to monitor continuing fitness for purpose of these assays.

Relevant sequence data was also
collected and ins
pected for any evolving trends.


It has been previously noted that H5N1 HPAI isolates of newly
-
emerging clade 2.3.2 and 2.3.4 viruses are not
sensitively detected by the validated Spackman
et al
.

(2002) M gene RRT PCR which is in use by many AI
Reference L
aboratories globally.

Clinical specimens obtained from chickens and ducks infected with viruses
from these clades in Vietnam during 2009 have been tested by a range of AI RRT PCRs.

Bayesian statistical
analysis of these swab specimen results has shown that

this M gene RRT PCR (hereafter referred to as the
“wet” M gene RRT PCR in this section) is insensitive in comparison to the validated H5 HA2 RRT PCR.

However, sensitivity for testing swabs from these clade 2.3.2 and 2.3.4 infected birds was also largely r
estored
by using the “bead” M gene RRT PCR that utilises freeze
-
dried reagents that were described originally by
Petrauskene
et al
.

(
Avian Diseases
[
2010
]
,
54
[
s1
]
: 686
-
689
).

The statistical analysis also demonstrated that
inclusion of feathers for AI RRT P
CR testing resulted in improved sensitivity for the identification of infected
birds, reflecting the high viral titres that appear to occur in H5N1 HPAI diseased chickens and ducks.

Therefore inclusion of feathers in the sampling regime resulted in more se
nsitive identification of infected
birds by the “wet” M gene RRT PCR. However, the non
-
optimal sensitivity of the “wet” M gene RRT PCR
remains a concern in view of the continuing spread of clade 2.3.2 viruses in several Asian countries.

A clade
2.3.2 submission received from Nepal during 2011 included four tracheal samples that were positive by the H5
and N1 RRT PCRs, but two were negative by the “wet” M gene RRT PCR.

Therefore vigilance and ongoing
investigations of molecular test perfor
mance remain important in view of continuing H5N1 HPAI clade 2.3.2
outbreaks
.

Statistical assessment of the validity of testing LPAI swabs either singly or as pools of five

Validations of AI RRT
-
PCRs have focused on testing individual swabs, but pooling h
as the attraction of
facilitating a higher test throughput at lower cost
, particularly

during AIoutbreak
-
related surveillance.
However, concerns exist that e.g. pooling a weak positive swab with four negative swabs may result in a

detrimental effect upon A
I RRT
-
PCR sensitivity.

To address this question, swabs (tracheal and cloacal) were
collected daily from turkeys that had been successfully infected with a H2N2 AIV which was shed consistently
but at low titre for over a week.

These swabs were tested either

(i) singly or (ii) pooled with four AI negative
swabs from uninfected turkeys.

Bayesian statistics was used in

the data analysis and the results indicated that
there was no significant deleterious effect of diluting positive with negative swabs on the Ct
value of either of
the AI RRT
-
PCRs. Flock level modelling showed that pooled sampling

was able to widen the detection
window compared to individual sampling, for the same number of RRT
-
PCR tests. It was also found that the
‘bead’ M
-
gene AI RRT
-
PCR was more

sensitive than the ‘wet’ as
say.

H9 RRT PCR and molecular epidemiology

Although not a notifiable disease, AI poultry infections caused by H9 viruses are an endemic problem in many
Asian and Middle
-
Eastern countries and differential diagnosis from NAI is re
quired.
Continuing evaluation of
H9 RRT PCR tests have been extended to include 132 clinical specimens obtained from five outbreaks, and
these include two 2011 H9 submissions received from Saudi Arabia (28 tissues) and the United Arab Emirates
(seven tissu
e pools).

Analysis of these AI RRT PCR results by Bayesian statistics has shown the H9 RRT PCR
(originally described by Monne
et al

(J Clin Microbiol
[
2008
], 46: 1769
-
1773
) to be highly effective in H9
AIV detection, with a sensitivity that is very similar

to that of the M gene RRT PCR.

The H9 RRT PCR was
also used to successfully identify H9 infection in a 2011 submission from Iraq that included 33 samples, while
this method also identified H9 infection by testing clinical specimens from a UK chicken farm.

Because no
virus isolates were successfully obtained from thisoutbreak, the H9 RRT PCR demonstrated its value in
successfully identifying the infecting H9 subtype.


Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

Annual reports of OIE Reference Laboratories and Collaborating Centres, 20
11

5

The investigation of H9 molecular epidemiology has continued, where t
he HA gene of 33 H9 i
solates grown in
EFEs were amplified by conventional RT
-
PCR and sequenced.

Phylogenetic analysis has shown the 11 South
Asian (
ie

from Bangladesh, India, Nepal and Pakistan) and 12 Middle
-
Eastern H9s (from Iran, Iraq, Israel,
Jordan, Kuwait, Lebanon, Libya
, Saudi Arabia and UAE) to belong to the established G1 lineage of H9 AIVs
which has been previously been observed to be widespread in many parts of Asia.

Interestingly, the seven
European H9 isolates appear to be more closely related to the previously obs
erved “Korean
-
like” (or Y439
-
like) lineage of H9 AIVs.


In addition preliminary antigenic characterisation of H9 viruses from poultry using newly raised panels of
ferret antiserum revealed considerable antigenic diversity with important implications for bo
th diagnostics and
control including selection of appropriate candidate vaccine strains.

N1 RRT PCR

Description of the successful performance of this validated assay with clinical specimens has been included in
the study of the Vietnamese 2009 H5N1 HPAI cl
ade 2.3.2 and 2.3.4 outbreaks (Slomka
et al

2012a).


Validation of AI recombinase polymerase amplification (RPA)

In partnership with a biotechnology company that has developed its own isothermal amplification technology,
namely recombinase

polymerase amplification (RPA) we assessed utility for AI detection.

Appropriate RPA
primers and probes were designed for the generic detection of AI viruses, and va
lidation included testing of
69

diverse AIV isolates, including 47 H5 and H7 AIVs, plus 48

H5 and H7 clinical specimens from poultry
(3

swabs, nine feathers and six tissues) which were tested in parallel by AI RPA and M gene RRT PCR.

All
117 samples were M gene RRT PCR positive, and 105 were detected by the AI RPA.

The 12 detection failures
all

occurred among low
-
titre samples which registered ct values of >30 by the M gene RRT PCR, these being
observed in five AIV isolates and seven AIV positive clinical specimens.

However, 32 samples with
Ct>3

(2

isolates and eight clinical specimens) were suc
cessfully detected by the AI RPA. The AI RPA has
shown promise as a potentially simple and rapid (20 minutes to complete the assay) molecular test which
utilises a simple platform that may be suitable for regional laboratories in developing world countries

which
are unable to invest in RRT PCR technology.

Following this proof of principle, it is speculated that the AI RPA
may also be developed into a penside molecular test for AI.

Validation of a novel AI molecular pathotyping assay

The EU FLUTEST project h
as included adaptation of the plexor
-
probe technology by SVA (Uppsala, Sweden)
for the purpose of discriminating LP and HP pathotypes among H5 and H7 AIV isolates.

The key collaborative
input from AHVLA was to provide a substantial proportion of the 65 Eas
tern Hemisphere H5 and H7 AIV
isolates for validation of the molecular pathotyping assay:

All H5 and H7 isolates gave correct pathotyping
results by comparison to the respective cleavage site sequences.

In addition, AHVLA provided 39 H5 and
21

H7 clinical
specimens from LP and HP virus
-
infected birds. The samples originated both from field
specimens and experimentally infected birds, and included 26 specimens from H5N1 HPAIV Asian
-
lineage
outbreaks. Swabs, tissue samples, and feathers from infected birds we
re included.

M gene RRT PCR results for
these clinical specimens displayed a wide range of Ct values from 19.7 to 35.5, but all 60 were correctly
pathotyped.


Investigation of commercial lateral flow devices (LFDs) for AI detection

Testing of swabs (trache
al and cloacal) and feathers from 46 chickens and 48 ducks obtained from Vietnam
during 2009 was by the Anigen and Quickvue lateral flow devices (LFDs) as well as AI RRT PCR which
served as the sensitive gold
-
standard for the identification of H5N1 infecte
d birds.

AI LFDs continue to have
the attraction of being simple penside tests, and our activities have continued to include objective and rigorous
assessments of this methodology. This study has supported our previous findings which showed that for
diseas
ed H5N1 HPAI infected galliformes, a proportion of swabs in infected birds harbour virus at sufficiently
high titres to register positive results by these commercial LFDs (Slomka, 2011 see section 12).

The
Vietnamese study showed that the higher viral titr
e observed in chicken feathers resulted in a high proportion
of LFD positive results.

Testing of both swabs plus feathers from the diseased chickens gave sensitivities of
84.6 and 65.4% relative to M gene RRT PCR for the Anigen and Quickvue LFDs respective
ly.

In the case of
H5N1 HPAI infected and diseased ducks, however, tracheal and cloacal shedding is at a lower titre where these
LFDs failed to register any positive results, but duck feathers contained higher titres which did result in a
number of success
ful positive LFD results.

Sensitivity for duck feather testing was 53.3 and 33.3% relative to
M gene RRT PCR for the Anigen and Quickvue LFDs respectively.


Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

6

Annual reports
of OIE Reference Laboratories

and Collaborating Centres, 2011

The above investigations of LFDs in H5N1 HPAI infected poultry continue to show that while LFDs are not as
sensitive as validated and optimised AI RRT PCRs and indeed virus isolation (VI), they can be useful in testing
diseased galliformes when viral shedd
ing (tracheal and cloacal) when HPAI virus titres are sufficiently high.

The systemic nature of HPAI infections would also appear to be responsible for the feather tropism and the
accompanying high viral titres observed in both galliforme and duck feathers

which in turn has resulted in a
proportion of infected birds being successfully identified as positive through LFD testing (Slomka
et al

2012b).

Such LFD evaluations can ultimately influence the correct use of LFDs during H5N1 HPAI outbreaks.
However, the
re continues to be limited information concerning the value of LFDs in testing LPAI outbreaks,
and to this effect an investigation was organised to assess the value of this technique using swabs collected
from ducks and turkeys that had been experimentally

infected with a H5N2 LPAI virus.

Briefly, 72 turkey and
192 duck swabs (tracheal and cloacal) were tested by the Anigen and Synbiotics LFDs and M gene RRT PCR.

Results revealed sensitivities in turkeys for the two LFDs of 0.05 and 0% respectively for the
Anigen and
Synbiotics LFDs, while in ducks a sensitivity of 7.9% was observed for both LFDs.

This study showed that
both LFDs are highly insensitive for successfully diagnosing LPAI infection, and was conducted as part of the
EU Epizone project short
-
term
mission programme.

Use of RNA controls and standards for AI RRT PCR

The EU Epizone and
FLULABNET

projects have continued to support assessment at AHVLA of AI RRT PCR
standards.

These are “Tuff RNA” preparations, where the target region is cloned into a
cowpea mosaic virus
vector, and this can be used as a positive control for the corresponding AI RRT PCR.

In addition to the generic
M gene RRT PCR target, Tuff RNA controls containing H5, H7 and N1 RRT PCR targets have been assessed
successfully.

In additi
on, a Tuff RNA control for the M gene “perfect match” RRT PCR target for detection of
European swine influenza viruses (SIVs) and A(H1N1)pdm09 has also been assessed.

6.

Collection, analysis and dissemination of epizootiological data relevant to internatio
nal
disease control

AHVLA continues to monitor the epidemiology of AI (relating to all virus subtypes but especially NAI) at a
global level in a timely manner. This

information has been widely disseminated at international conferences,
meetings and through

publication in the scientific literature and on the web (see 12/13 below). All laboratory
reports relating to submissions to AHVLA from OIE member countries are sent immediately to the OIE.
During the year there were eleven individual submissions with pos
itive results for AI (
Nepal x3, United Arab
Emirates x2, Finland, South Africa x2, Germany, Netherlands and Bulgaria)

and two submissions of mixed
positives with AI and NDV (
Ireland and Saudi Arabia

).
In total a further four submissions produced negative
results.

Twenty
-
nine full
-
length HA sequences form the Vietnamese H5N1 HPAI isolates obtained in 2009 were
uploaded to the public databases.

These included 18 clade 2.3.2 viruses (eight from chickens, seven from Pekin
ducks and three from Muscovy ducks) a
nd 11 clade 2.3.4 viruses (all from chickens).

This has contributed to
the ongoing molecular epidemiology of these H5N1 HPAI clades which are currently a significant problem in
a number of Asian countries.

Six of these viruses were also sequenced in the M gene segment, and this
revealed presence of the S31N adamantane
-
resistance mutation in the M2 protein in four of the six viruses.

This mutation occurred in both clade 2.3.2 and 2.3.4 isolates (Slomka
et al
2012a). In addition 23 full length
HA sequences of NAI were submitted. All sequences were submitted to GISAID/Genbank.

Collation of data from annual surveys for avian influenza in poultry and wild birds implemented by EU
Member States: to undertake consist
ency checks and raise queries with each Member State as required;
undertake epidemiological analyses and compile reports on a quarterly (wild bird surveillance only) and annual
basis; recommend improved data collection methods and implementation of agreed
changes amongst EU
Member States. Annual review to ensure better targeting of programme and revision of surveillance guidelines
was done See item 12/13.

7.

Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen

an
d the disease concerned

A quality assurance system is in place at AHVLA with most tests being used accredited to the ISO17025

standard.

All SOP’s are updated and revised on a regular basis where necessary. The establishment is

regularly
inspected regularly

by the UKAS accreditation authorities.

Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

Annual reports of OIE Reference Laboratories and Collaborating Centres, 20
11

7

AHVLA has a specified bio
-
safety officer for the site and regional laboratories with whom the Virology
department work closely. There are formal co
rporate structures in place for

management of Health and Safety.
All
staff working with avian influenza viruses are bioterrorism cleared

and formally supported through a
programme of occupational health. In year a new algorithm for handling influenza viruses according to risk
was developed covering both ACDP level 2 and 3 p
athogens. All facilities used for handling infectious
material are licensed in accord with national

legislation (Specified Animal Pathogens Order) operating at
BSL3+ and are subject to regulatory

inspection by the UK Health and Safety Executive. . Liaison
with other
OIE reference laboratories is

carried out when necessary, discussing new viral agents and related issues
.

8
.

Provision of consultant expertise to OIE or to OIE Member Countries

1.

Continuation of role as parent laboratory in two funded twinning
projects between AHVLA
-
Weybridge
and the ARC
-
Onderstepoort Veterinary Institute (ARC
-
OVI), South Africa and with Botswana National
Veterinary Laboratory (BNVL). The objective of the project with ARC
-
OVI is to increase the capacity
and training, with the ov
erall aim of the candidate laboratory becoming a regional OIE reference
laboratory for the diagnosis of Newcastle disease and avian influenza that will serve the SADC region. In
the case of BNVL the overall goal is to establish the candidate laboratory as
a regional centre for ND/AI
within the SADC region. Both twinning projects will conclude in 2012.

2.

A

contribution was made to the AI section of the FAO initiated “
Detection of Selected Diseases”
document which was commenced during 2011.

This summarises
a range of diagnostic and investigative
tests that are directly relevant to AI investigations, and included AI RRT PCRs, conventional AI RT
PCRs, isothermal amplification methods, LFDs, ELISAs and other serological approaches.


3.

Continual and proactive s
upport to OFFLU initiatives including provision of expertise, leading

technical
groups, contributing and writing texts. In addition, Ian Brown represented OFFLU at the WHO

vaccine
composition meetings in February and September.

4.

Ian Brown was part of a t
wo person international mission to provide disease and technical consultancy to
the South African Veterinary Authorities in relation to the control of NAI in the Ostrich sector.

5.

Ad
-
hoc technical support via email to numerous OIE member countries on AI d
iagnosis and surveillance.


6.

Defra UK AI Expert Committee (Ian Brown, Dennis Alexander and Richard Irvine)
.

9
.

Provision of scientific and technical training to personnel from other OIE Member Countries


Personnel were trained at AHVLA
-
Weybridge from the following countries: Hungary, Germany, Republic of
Ireland, Romania, Denmark, Slovakia, Poland, Philippines and Spain. Two training programmes held in
February and July for five participants consisted of t
wo weeks training in diagnostic techniques in two
modules; one week focused on ‘conventional’ virological methods and one week on molecular
-
based methods
for diagnosis of AI. The programme was a balance of key lectures to supplement practical application w
ith
strong influence on the latter and group discussion, and was concluded by an interactive lecture on AIV
nucleotide sequencing and phylogenetic analysis.

10
.

Provision of
diagnostic testing facilities to
other OIE Members

Thirty three submissions of sam
ples were received from the following countries OIE: Bulgaria, Canada,
Ethiopia, Finland, Germany, Israel, Malta, Nepal, Republic of Ireland, Saudi Arabia, South Africa,
Switzerland, The Netherlands, United Arab Emirates and USA. ,

Samples received consis
ted of the following categories; 170
×

allantoic fluid samples, 64 swabs, 58
×

tissue/organ suspensions and 299
×

serum samples.

As a result of virus isolation carried out on tissue and swab samples and the characterisation of the allantoic
fluids submitt
ed the following breakdown of isolates has been recorded. All activity should be considered as
confirmatory

testing.

All laboratory reports relating to submissions to AHVLA from OIE member countries are
sent immediately to the OIE
.

Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

8

Annual reports
of OIE Reference Laboratories

and Collaborating Centres, 2011

Virus identification

Number

Paramyxoviruses

93

Influenza A viruses

84

H1N1

1

H3N8

10

H4N6

4

H5N1

7

H5N2

7

H6N1

2

H6N2

3

H6N8

1

H6N9

1

H7N1

1

H7N4

1

H7N7

2

H8N4

3

H9

20

H9N2

11

H10N7

3

H10N8

1

H11N9

1

H12N5

2

H13N2

1

H13N6

1

H16N3

1

To be typed

0

Not
viable

107

UK Virus identification

Number

Paramyxoviruses

32

Influenza A viruses

0

11
.

Organisation of international scientific meetings on behalf of OIE or other international
bodies


17
th

Annual Meeting of the national avian influenza laboratories of countries of the European Union and
EFTA, Brussels, April. 70 participants. Proceedings published

http://ec.europa.eu/food/animal/diseases/controlmeasures/avian/docs
.



Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

Annual reports of OIE Reference Laboratories and Collaborating Centres, 20
11

9

1
2
.

Participation in international scientific collaborative studies

EU FLUTEST project

In January 2011 AHVLA hosted a lead scientist from SVA (Uppsala, Sweden
), responsible for the
development of the plexor
-
probe based molecular pathotyping assay.

This working visit was done in order to
thoroughly validate this assay with a substantial number of AIV laboratory isolates and clinical specimens
(section 5) and res
ulted in a published output later in 2011 (Leijon
et al
, 2011, please refer to section 13).

EU Epizone short
-
term mission

Assessment of commercially available AI LFDs for testing clinical specimens from LPAI infected birds
(section 5) was organised within
an Epizone short
-
term mission in September 2012, where AHVLA hosted a
visit from a colleague Friedrich Loeffler Institute, Germany) for three weeks.


Presentation and statement at “Upcoming technologies for the early and rapid diagnosis of infectious disea
ses”

Marek Slomka represented AHVLA and gave a presentation on AI penside testing at an internationalmeeting
organised by the FAO and IAEA in Vienna, May 2011.

This involved making a presentation (Slomka 2011b,
please see section 12) on A summary statement

was produced by the FAO / IAEA Joint Division in
consultation with experts after the conclusion of the meeting.

AHVLA continued to host and provide site moderation for an international interactive web based
forum(FLULABNET) for the scientific community. S
ubject matter included exchange of information on AI
training, meetings and various AI technical matters.


AHVLA participated in numerous collaborative research projects funded by the European Union (coordinator of
FLU
-
LAB
-
NET, FLUTEST; with work

package l
eaders in FLUTEST, EPIZONE, FLUPATH; participant in
FLURESIST, FLUTRAIN) with increasing emphasis on partnership with institutes in countries suffering from
infection with H5N1 HPNAI ‘Eurasian lineage’ viruses. These projects provided strategic research on

a wide range
of topics addressing emerging issues for control, diagnosis and international networking plus include studies with
additional non notifiable avian influenza viruses. These projects involve collaborations with partner institutes
ranging betwee
n 6 and 37. Space does not permit extensive detail on each (available on request).

1
3
.

Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at int
ernational
conferences)



Scientific publications in peer
-
reviewed journals

1.

Lyall, J., Irvine, R. M., Sherman, A., McKinley, T. J., Nunez, A., Williams, A., Outtrim, L., Brown, I. H.,
Sang, H. and Tiley, L. (2011). Suppression of avian influenza
transmission by genetic modification of
chickens.
Science 14 January 2011
Vol. 331
no. 6014
pp 223
-
226.DOI:10.1126/science.1198020
.

2.

Reid, S., Shell, W., Barboi, G., Onita, I., Turcitu, M., Cioranu, R., Marinova
-
Petkova, A., Goujgoulova,
R., Webby, R., W
ebster, R., Russell, C., Slomka, M., Hanna, A., Banks, J., Alton, B., Barrass, L., Irvine
R. and Brown, I. (2011) First reported incursion of highly pathogenic notifiable avian influenza A H5N1
viruses from clade 2.3.2 into European poultry. Transboundary
and Emerging Diseases,Vol. 58 Issue 1
pages 76
-
78

3.

Mastin, Alexander; Alarcon, Pablo; Pfeiffer, Dirk; Wood, James; Williamson, Susanna; Brown, Ian;
Wieland, Barbara (2011) Prevalence and risk factors for swine influenza virus infection in the English pi
g
population [Internet]. Version 17. PLoS Currents: Influenza. 2011 Jan 15 [revised 2011 Feb
1]:PMC3032880.

4.

Lloyd, L. E., Jonczyk, M., Jervis, C. M., Flack, D. J., Lyall
, J., Foote, A., Mumford, J. A., Brown, I. H.,
Wood, J. L. and Elton, D. M. (2011), Experimental transmission of avian
-
like swine H1N1 influenza
virus between immunologically naïve and vaccinated pigs. Influenza and Other Respiratory Viruses,
Volume 5 Issu
e 5 pages 357
-
364 September (2011):

no. doi:

10.1111/j.1750
-
2659.2011.00233.x

Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

10

Annual reports
of OIE Reference Laboratories

and Collaborating Centres, 2011

5.

Howard, W.A., Essen, S.C., Strugnell, B.W., Russell, C., Barrass, L., Reid, S.M. and Brown, I.H. (2011).
Reassortant pandemic (H1N1) 2009 virus in pigs, United Kingdom. Emerg
ing Infectious Diseases
Volume 17 Number (6) 1049
-
1052.

6.

WHO; OIE; FAO H5N1 Evolution Working Group

2011.
31 Names including Brown, I.H., (2011).

Continued evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature.
Influenza
and Othe
r Respiratory Viruses 6(1) 1
-
5.

7.

Leijon, M., Ullman, K., Thyselius, S., Zohari, S., Pedersen, J.C.,
Hanna, A., Mahmood, S., Banks, J.,
Slomka, M.J.,

and Belák S (2011).

Rapid PCR
-
based molecular pathotyping

of H5 and H7 avian
influenza viruses Journal of Clinical Microbiology, Vol 49
: 3860
-
3873.

8.

Dent, J.E., Kiss, I.Z., Kao, R.R., and Arnold, M. (2011The potential spread of highly pathogenic avian
influenza virus via dynamic contacts between poultry premis
es in Great Britain. BMC Veterinary
Research, 7:59

http://www.biomedcentral.com/1746
-
6148/7/59


9.

Li, Y., Reddy, K., Reid, S. M., Cox, W. J., Brown, I. H., Britton, P., Nair, V., and Iqbal, M. (2
011).
Recombinant herpesvirus of turkeys as a vector
-
based vaccine against highly pathogenic H7N1 avian
influenza and Marek’s Disease
. Vaccine 29, 8257
-
8266.

10.

Kuchipudi SV,

Dunham SP, Nelli R, White GA Coward VJ, Slomka MJ,

Brown IH and

Chang
KC.(2011)

Rapid death of duck cells infected with influenza: a potential mechanism for host resistance to
H5N1.

Immunology and Cell Biology, advance online publication, 22 March, 2011, pages 1
-
8;
doi:10.1038/icb.2011.

11.

Slomka MJ, To TL, Tong HH, Coward VJ,
Mawhi
nney IC, Banks J and Brown IH (2011).

Evaluation of
lateral flow devices for identification of infected poultry by testing swab and feather specimens during
H5N1 highly pathogenic avian influenza. outbreaks in Vietnam.

Influenza and Other Respiratory
Viruses,
in press Published online on 12 December 2011: DOI: 10.1111/j.1750
-
2659.2011.00317.x

12.

Brookes, S., Brown, I.H., (2009) A/H1N1/pdm09 virus: dynamics of infection in pigs and people.
Research in Veterinary Record 169 (6) 151
-

152 August 2011. Do
i:10.1136/vr.d4945

13.

Dundon, W.G., Heidari, A., Fusaro, A., Monne, I., Beato, M.S., Catt
oli, G., Koch, G. Starick, E.,
Brown,
I.H., Aldous, E.W., Briand, F.X., Le Gall
-
Recule, G., Jestin, V., Jørgensen, P.H., Berg, M.,Zohari, S.,
Metreveli, G., Munir, M.
, Stah, I.K., Albina, E., Hammoumi, S., Gil, P., de Almeida, R.S., Smietanka, K.,
Domanska
-
Blicharz, K., Minta, Z., Van Borm, S., van den Berg T, Martin, A. M, Barbieri, I, Capua I

(2011).
Genetic data from avian influenza and avian paramyxoviruses generat
ed by the European network
of excellence (EPIZONE) between 2006 and 2011
--
review and recommendations for surveillance.
Veterinary Microbiology 27;154(3
-
4):209
-
21

14.

Kuchipudi, S.,
Dunham, S., Nelli, R., White, G., Coward, V., Slomka, M., Brown, I., Chan
g, K.C, 2011.
Host pro
-
inflammat
ory cytokine response as a fundamental ke
y to host survival or death

in ducks and
chickens infected with highly pathogenic avian influenza virus.
British Poultry Abstracts

7 (1) 43
-
44
.



Book chapters

1.

Brown, I.H., Alexander
, D.J., Phin, N. & Zuckerman, M. (2011) Influenza

Chapter 30.
In: Oxford
textbook of Zoonoses: biology, clinical practice, and public

health control,

2
nd

ed Palmer, S.R., Lord
Soulsby (ed), Torgensen, P.R. (ed)& Brown D.W.G. (ed) Oxford University Press pp

332
-
352

2.

Brown, I.H.

(2011) History and Epidemiology of Swine Influenza in Europe.
Current Topics In
Microbiology and Immunology: Swine influenza. Editors J.

Stech & R.

Webby. Springer.
www.
springer
link.com/index/v165143460m7u34q.pdf
.



Highly pathogenic avian influenza
and low pathogenic avian influenza (poultry)

Annual reports of OIE Reference Laboratories and Collaborating Centres, 20
11

11



Other
communications

I.H.Brown
, Presentations at the 17
th

Annual Laboratory meeting on AI and ND April
2011.
http://ec.europa.eu/food/animal/diseases/controlmeasur
es/avian/crls_proceedings_en.htm
VLA
website with AI laboratory protocols

:
http://www.vla.gov.uk/science/science
-
viral
-
ai
-
reflab
-
prot.htm

Public
website for influenza laboratories
www.flu
-
lab
-
net.eu




Presentations at international conferences and meetings

1.

Brookes, S., Garcon, F., Germundsson, A., Gardner, R., Nash, B., Donnelly, C., COSI and Brow
n, I.
Pandemic H1N1/09 in pigs: a reservoir host and model for human disease. ESWI conference, Malta,
September 2011

2.

Hanna, A., Govil, J., Krill, D., Mahmood, S., Shell, W., Manvell, R., Banks, J., Brown, I. and Slomka, M.
Validation of H9 real time RT
PCRs and HA phylogenetic studies of diverse H9 avian influenza viruses.
5
th

Annual EPIZONE Conference, Arnhem, Netherlands, June 2011

3.

Williams C.

Brown, I.H., Swift, A., Ellis, J., Kearney, Wreghitt, T., Joe., Hoschler, K., Reacher, M.,

Ellis., J.,
Kearney, J., Phin, N., Gent, N., Nair, P., Rolfe, K., : Outbreak of Avian Influenza H9 in a
poultry farm, East of England. Health Protection Agency, Warwick, 14 September 2010

4.

Williams, C.J., Swift, A., Gent, N., Reacher, M., Wreghitt, T., Rolfe, K., El
lis, J., Hoschler, K. , Brown, I.,
Phin, n., Nair, P., Kearney, J.,: Outbreak of Avian influenza H9 in a poultry farm, East of England, 2010.
I.H. Brown, Presentation at
2011 European Scientific Conference on Applied Infectious

Disease
Epidemiology (ESCAIDE)


6
-
8th November 2011, Stockholm (Stockholm Waterfront Congress Centre)
http://ecdc.europa.eu/en/escaide/Pages/ESCAIDE2011_Home.aspx

5.

Slomka, M
.J., (2011b).

Considerations for penside testing for avian influenza in poultry: The present, the
future and

what to avoid.

Early and rapid diagnostic tool consultancy. Presentation at “Upcoming
technologies for the early and rapid diagnosis of infectious
diseases”.

IAEA

Headquarters, Vienna,

Austria, 18
-

20 May 2011.

6.

Slomka, M.J., Banks, J., Mahmood, S. Collins, C. Russell & I.H. Brown (2011).

In vivo

transmission of a
waterfowl isolate of H5N2 low pathogenicity avian influenza virus from ducks to turk
eys

Influenza 2011
Conference, 7
-
9 September 2011, St Hilda’s College, Oxford
to novel H2N3 swine influenza virus.

Submitted to the Journal of Veterinary Diagnostic Investigation.

_______________