1

- -

,

- (RSV),

(MPV)

2004-2009 .

„ ”

01.06.13

:

. - , . .

2015

2

167 , 23 , 31

338 (12 326 ).

„ ”, ,

( )

-

, 06/11/2014 .

.....................2015 .

............ . ,

№26, ,

.

, .

„

”, , - .

3

I. ............................................................................................................................... 1

II. ......................................................................................................................... 4

III. ............................................................................................................... 5

1. RSV..................................................................................................... 5

2. , ................................... 5

3. , RSV……………………….. 6

4. .................................................................................................................................... 6

4.1. RSV .. ............................................. 6

4.1.1. ............................................................. 6

4.2. ( ) RSV..

7

4.3. RSV ....... 7

4.4. .................................................................................. 8

4.4.1. Trizol LS (Invitrogen, USA)............................................ 8

4.4.2. RIBOSORB (SACACE, Italy)........................................... 8

4.4.3. QIAAMP® VIRAL RNA MINI KIT (QIAGEN)............. 8

4.4.4. IPREP PURELINK TM

VIRUS KIT ............................... 9

4.5. RT-PCR RSV………………………… 10

4.5.1. RT-PCR RSV.............. 10

4.6. RT-PCR MPV……………….……….. 11

4.6.1. RT-PCR MPV................... 12

4.7. PCR ………………………………................................................ 13

4.8. Real Time RT- PCR ............................................................. 13

4.9 ................................................................. 13

IV. ……………………………………………………………………... 15

1. ...................................... 15

1.1. RSV MPV ...........................................................................................

16

1.2. ( 1N1)2009 ........................................................................

17

2 . RSV ............................................................. 18

3. RSV -

–Directigen RSV......................................................

23

4. RSV MPV............................................................... 26

4.1. RT-PCR 2006/ 2007................................................................ 27

4.2. RT-PCR 2007/ 2008............................................................... 30

4.3. RT-PCR 2008/ 2009............................................................... 34

4.4. ................................................................................................................................ 38

5. ( 1N1)2009....................................................................... 44

V. ......................................................................................................................... 58

VI. . ........................................................................... 65

VII. . ............................................................................... 67

VIII. ……………………………………... 68

4

:

- OP - RSV- - MPV-

PCR-

SARS-CoV, NL63, HKU1- . Coronaviridae

hBoV- KI, WU- . Polyomaviridae

ECDC-

CDC –Centers for Diseases Control and Prevention

AMPV- -

-

-

m -

ORF-

-2-

MRC-5-

BHK-

-

LLC-MK2-Rhesus Monkey Kidney Epithelial Cells -

ELISA ( - ) EIA-

- Nested PCR- -

Real Time PCR - -

PBS -Phosphate buffer solution / / RT-PCR- -

DEPC -diethylpyrocarbonate

FITC -fluorescin isothiocyanate

c.DNA - dNTP s –

-

5

I.

( )

.

,

. ,

,

.

World Lung Fondation

4,25

.

, - (RSV) -

3

. , 50%

15-35%

.

,

( ,

.).

(

)

. , ,

, ,

- (RSV), , ,

, .

,

. ,

.

, .

2001 . van den Hoogen

,

(MPV). ,

7-10% .

6

, MPV ,

RSV, .

- , , ,

. ,

RSV MPV ,

.

,

RSV,

, ,

RSV MPV . in

vitro,

, RSV,

,

.

.

,

- -

(PCR).

,

-2001 ., SARS-CoV -2003 , NL63 -2003 . HKU1 (2005 .),

(С V) -2005 . KI 2007 . АU-2007 .

( 5N1/) (1997 .)

, MERS-CoV2012 .

,

RT-PCR MPV .

RSV

(ECDC),

RSV

.

7

( 5N1/) (1997 .)

.

20- , 1999 .

,

.

17 2009 . CDC

A(H1N1)2009,

, .

24 2009 . ,

, 10 2009 .,

,

6- . 10

2010 .

„ ”, 2002 .

( 1N1)2009, ,

RSV

,

, ,

.

.

8

II.

,

- ,

,

( 1N1)2009 .

:

1.

.

2. RSV

- .

3. RT-PCR

RSV MPV Real Time RT-PCR

.

4. RSV MPV

5

.

5. Real Time RT-PCR

2009 .

.

6.

(H1N1)2009.

9

III.

1. RSV

RSV- Randall

-2, .

2. ,

RSV MPV

5 . 2004-2009 . RSV

846 , 452

MPV, 371 .

04.05.2009 . - 08.01.2010

( ), ,

. 2429

( 9 ).

23.09.2009 .-12.10.2009 . 152

.

RSV

, RSV.

-

. ,

+4 .

.

. RSV MPV,

Directigen RSV,

RT–PCR. BSL3

( )

,

rRT PCR.

10

9 ( ).

,

. ,

2ml ,

,

.

3. ,

RSV

RSV -2,

. -

MRC-5,

.

4.

4.1. RSV

-2 MRC-5 24

37 5% 2

. 0,2ml/ ( -

).

10 . 50%

, - ,

.

+4 .

4.1.1.

RSV

,

.

,

, .

11

- 24-36

. -

. ,

.

.

4.2. ( )

RSV

(chamber-slides)

,

, 16 , 0,25ml/ .

IMAGENTM

RSV Dako Cytomation.

, . ,

, 25 μl

.

16 0,4.

, .

-

, 3

.

4.3.

DIRECTIGEN RSV (BECTON DICKINSON, USA)

RSV

-

,

, 48 10 . -

RSV -

, ,

.

12

4.4.

- ,

.

4.4.1. TRIzol LS (Invitrogen, USA)

TRIzol LS e

. .

,

,

.

, .

. ,

.

.

. 250µl

, 25µl dd 20

(RNAse free), –70º

RT-PCR.

4.4.2. RIBOSORB (SACACE, ITALY)

Ribo-Sorb , -

.

;

100µl, -50µl.

4.4.3. QIAamp® Viral RNA Mini Kit

(QIAGEN)

- ( , )

.

„ ” ,

Carrier RNA, AVL

( ). ,

13

. ,

- , -

. ,

.

, .

140µl,

60/80l .

4.4.4. IPREP PURELINK TM

VIRUS

KIT

,

iPrep Purification

instrument, 40 .

Magtration® ( ) PSS (Precis ion System

Science),

, .

,

. ,

.

ChargeSwitch®

Technology (CST),

, .

. . CST®

.

.

8,5, ,

50 100µl. 200µl

400 µl.

14

4.5. RT-PCR

-

RSV in-h use

RT-PCR. RSV

N ( 858

1135– ).

:

N1 5’GGAACAAGTTGTTGAGGTTTATGAATATGC3'

N2 5'CTTCTGCTGTCAAGTCTAGTACACTGTAGT3'

RT-PCR Mastercycler

(Eppendorf) :

READY-TO- GO RT-PCR BEADS (AMERSHAM BIOSCIENCES, UK)

ABGENE REVERSE IT-ONE STEP RT-PCR (INVITROGEN, USA)

4.5.3. ABGENE ABSOLUTE MAX QRT PCR

e - ,

.

-Randall, - DEPC-treated H2O.

4.5.1. я RT-PCR RSV

RSV-Randall, -2,

RNA Real Time PCR (Respiratory Syncytial Virus

Real Time RT-PCR Kit, Liferiver, Shanghai Bio-Tech)-

RSV Chromo 4 (BioRad).

FA BHQ1

HEX/VIC/JOE .

1 107cop/ml RNA, .

,

15

RSV

30 380cop/ml . 3 104cop/ml.

( 10-1

10-6) 3 104

cop/ml

( 3 000 0,03 cop/ml).

RT-PCR. RT-

PCR

(H3N2) (H1N1). . 1

2%

RT-PCR. -

, RSV , 10-3

( 1:1 000 30 cop/ml).

.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

. 1 RT-PCR

RSV-Randall

- ; 1- ; 2 3 RSV – ; 4 5

; RSV 10-1 (6) 10-6

(11)

4.6. RT-PCR

MPV

M ,

16

.

:

M1 5`CCC TTT GTT TCA GGC CAA C 3`

M2 5`GCA GCT TCA ACA GTA GCT G 3`

RT-PCR Mastercycler

(Eppendorf) : Qiagen® One Step RT-PCR Kit (Qiagen) kit

e - ,

.

MPV Catacusino Institiut, Romania,

- DEPC-treated H2O.

4.6.1. я RT-PCR MPV

Cantacuzino Institute, Romania (Tecu C, et al. 2007)

MPV, LLC-MK2 1/10 1/10000

( 2) - , , 10-4

. (Tecu

C, et al. 2007)

MPV,

MPV Qiagen,

,

.

.2 RT-PCR

MPV. (Tecu C, et al. 2007)

17

4.7. PCR

PCR ( ),

з280bp RSV ~416bp MPV,

2% 0,5g/ ml.

–φ -174 (4l) DNA Ladder 100-1500bp

(Invitrtogen)

4.8. REAL TIME RT- PCR

Real Time RT-PCR (rRT-PCR) Super Script One-Step

RT-PCR with Platium Taq (Invitrogen, USA) iScript One Step RT-PCR Kit for

Probes (BioRad, USA) Real Time PCR Chromo 4 (BioRad),

(Taqman®)

( /H1, A/H3 B), CDC Atlanta, .

FAM/BHQ

( /H1, A/H3 B)

.

: InfA -

; swInfA

; sw 1

; InfB ; Inf 1 Inf 3

.

,

.

rRT-PCR:

A/Brisbane/59/2007(H1N1),

A/Brisbane/10/2007(H3N2) /Brisbane/60/2008,

(WHO-CCs)- ,

.

A/California/04/2009

CDC Atlanta, USA ,

0.01M PBS.

.

18

я :

CDC,

,

, “ ” (swInfA / swH1),

(threshold line)

40- ,

.

,

. “Case definition” ,

, ( - )

PCR A(H1N1)2009

.

.

.

4.9.

:

4.9.1.

4.9.1.1.

4.9.1.2. : ,

4.9.2. -

. α=0,05

.

4.9.2.1. -

(PОКrsШЧ χ2 tОst, CСТ sqЮКrО tОst).

4.9.3. (MS E cel

2007)

19

IV.

1.

2004/2005 .,

RSV 2 ,

.

.

,

.

5 ,

.

-

- , .

RSV

CDC

, - .

RSV

. -

.

MPV

5 , ,

.

17 2009 ., CDC

,

.

A(H1N1)2009

. .

„ ” ,

20

.

.

,

,

, .

1.1. RSV

MPV

№1 №2

846 RSV, 452

MPV 371 ,

.

MPV 2008 .

.

. №1 RSV

MPV 0 5 . /

2004/2005 . 2 .

** MPV 81 .

/

2004/2005* 2005/2006 2006/2007 2007/2008** 2008/2009

- - 33 - - -

II - - 141 101 130 241

13 - - - -

- - 18 - 32

- - 29 - -

- 10 - - - -

- - - - 98

23 174 148 130 371

21

. №2

1.2. ( 1N1)2009

№ 3 4

2429

( 1N1) , 04.05.2009 .-08.01.2010 .

. №3

/

. №4

(H1N1)2009

2004/2005 2005/2006 2006/2007 2007/2008 2008/2009

13 36 54 51 137

- 65 66 72 98

10 73 28 7 136

23 174 148 130 371

1174

596

659

2429

396

446

1 518

# 31

* 38

** 2 429

22

* – , , , , ,

, , , , ,

, , , , , ,

# - , , , .

**

.

2 . RSV

RSV ,

,

.

- , -

, . . « » .

, -

. ,

- MPV

2001 . RT-PCR

RSV,

.

,

.

2004-2009 . RSV

-2, „ ”,

. -

, -2

, ,

95%,

23

-2 24-29% .

2006/2007 2007/2008 .

MRC-5 ( ), „

”, . ,

. № 5

№5 RSV

-2

MRC-5

MRC-5

-2

2004/2005 23 2 (8,7%) - -

2005/2006 174 0 - -

2006/2007 148 2 (1,4%) 4 (2,7%) 3 (2%)

2007/2008 130 2 (1,5%) 0 0

2008/2009 371 2 (0,5%) - -

. 24 .

, -

.

( 48 10

).

, –

, , ,

.

, 48 .

-

.

, . ( .3 , 3 3 , .4 , 4 , .5)

24

3 . -2 , - ,

10 0,25

3 . -2 RSV-

, - , 10 0,25

25

3 -2 RSV-

, - , 10 0,25

4 . MRC-5 , - ,

10 0,25

26

4 . MRC-5 RSV –

, - , 10 0,25

5. -2 RSV -

, , 20 0,5,

IMAGEN RSV (Dako Cytomation)

27

№5 ,

( 846) RSV

15 .

-2 , MRC-5 -

- .

2004/2005 2006/2007

, (2007/2008

2008/2009) .

, . -

(9 ) 2006/2007,

RT-PCR.

. ,

.

, ,

.

.

- (4 ) MRC-

5 -2 2006/2007 ., ,

RSV

,

.

.

3.

RSV -

–DIRECTIGEN

RSV

( )

, PCR .

( 85%)

28

RSV, .

, .

, RSV (

15-20 .)

.

2003/2004 ,

48 ,

.

RSV,

– ,

. RT-PCR

( 2006/2007),

RSV

. №6

.

. №6 Directigen RSV

-

2004/2005 2005/2006 2006/2007

13 16 24

Directigen RSV 11* 4** 16

IF RSV 12* 10*** -

* .

** X, XI, XII 2005 .

*** I, II, III, IV 2006 .

29

2004/2005

RSV 23 . 11

Directigen RSV ( 12 )

.

10 , , 2005 .

RSV RT-

PCR RSV .

4 , ,

.

-2005/2006

,

( №6). 16 ( -2) 14

(87,5%) RSV,

.

,

,

( 177 ). ,

.

,

,

(RSV

). , Directigen RSV

RSV , ,

.

2006/2007,

RT-PCR. 7

24

.

30

. №7 24

, 2006/2007

Directigen

RSV

Directigen

RSV

RT-PCR

24

9

(37,5 %) 0

16

(66,6 %)

24

(100 %)

24 RT-PCR

, 9 , Directigen RSV

, 16

48 .

4. RSV MPV

RSV MPV RT-

PCR ( )

.

RT-PCR “ ”,

2005 ., 2006/2007 .

RSV 5 .

RT-PCR , RSV,

MPV.

5 ,

- .

MPV RT-PCR 2008 .

5 .

31

4.1. RT-PCR 2006/ 2007 .

2006/2007 . RT-PCR

RSV 148 ,

,

. RSV

.

RSV , 2 2007 . (

6.) RSV

,

2007 ., RSV

.

RSV 148

5 . , -

(631 ), ,

. ,

– RSV

( 3N2) .

RSV,

. 2006 . . .

.

№ 8 , 148

RT-PCR, 56 (37,8%) RSV,

.№9 , ,

RSV RT-PCR 84%. ,

,

. ( №10) -

(37 ) - ,

- . RSV

RT-PCR 24-48 .,

- .

32

RSV ( ) 27,7% 39,2%

, - ,

.

.7 RT-PCR

2% .

~280b ,

.

. №8 RSV RT-PCR, 2006/2007

(p<0.001, Pearson chi square=17.47).

. №9 RSV RT-

PCR

2006/2007

49 50-51 52-1 2 3

4 5 6

12 12 5 38 26 28 10 17

( )

2

(16,6%)

3

(25%)

4

(80%)

32

(84,2%)

7

(26,9%)

5

(17,9%)

1

(10,0%)

2

(11,8%)

RT-PCR

II - 101 49 (48,5%)

“ ” 29 2 (6,9%)

18 5 (27,7%)

148 56 (37,8%)

33

6. R -PCR RSV , 2006/2007 .

. №10. 56 RSV RT-PCR

, 2006/2007,

0- 11 12-35 36-59

RSV

/

RSV

/

RSV

/

54

20 /36

55,5%

7/18

38,8% 0/0

66 2/20

10%

3/22

13,6%

3/24

12,5%

28 15/17

88,2%

6/8

75% 0/3

37/73

50,6%

16/48

33,3%

3/27

11,1%

34

RSV 0-11

(p<0.001, Pearson chi square=15,37), 12-35

(p<0.001, Pearson chi square=10,35), 36 .

(p<0.001, Pearson chi square=13,69)

.7 RT-PCR 2% -

з280 . φX 174

4.2. RT-PCR RSV MPV 2007/ 2008

,

.

2007 . EISS (European Influenza Surveillance Scheme),

35

ECDC (European Centre for Disease

Prevention and Control),

, .

EISS ECDC,

RSV,

RSV. 2008 .

5 RSV MPV.

-

- 5 .

( 2007 .- 2008 .) RSV

130 5 ( .№1,

11, . 8), 2008 . 81

. RT-PCR RSV 11

(8,46%), 13 ( 81 ) (16.05%) MPV. (

15) . №12 24

.

MPV -

0 11 . (10 ), .

RSV - ,

( №12).

RSV,

,

2006 .

2007 ., RSV

.

RSV -2.

.8

,

, .

RSV MPV,

, ,

. MPV

36

(81 ),

.

. №11. RT-PCR RSV

MPV 2007/2008

. №12. RSV MPV 24

, 2007/2008

2007

2007

2007

2008

2008

2008

RSV

18 13 18

42 32 7

MPV

0 0 0

0 0 0

3 RSV

7%

2 MPV

4,7%

8 RSV

25%

8 MPV

25%

3 MPV

42%

0- 11 12-35 36-59

RSV MPV RSV MPV RSV MPV

/

/

/

51

7/33

21,2%

9/33

27,2%

2/17

11,7%

2/17

11,7% 0/1 0/1

72 2/39

5,1%

1/39

2,5% 0/27 0/27 0/6

1/6

16,6%

7 0/0 0/0 0/6 0/6 0/1 0/1

9/72

12,5%

10/72

13,8%

2/50

4%

2/50

4% 0/8

1/8

12,5%

37

RSV MPV 0-11 ,

( p<0.05, Pearson chi square=4,24 p<0.05,

Pearson chi square=9,16).

, - .

.

. 8 RT-PCR RSV, MPV

, 2007/2008

.9 RT-PCR MPV 2%

– з 416bp . φX 17

38

4.3. RT-PCR RSV MPV 2008/ 2009

2008/2009 . 371

5 , Real

Time -PCR RT-PCR RSV MPV,

.

« », . ( . №13)

. №13 5 ,

2008/2009

/

-

Real Time RT-PCR :

RSV MPV

241

16

6,6%

47

19,5%

18

7,5%

2 RSV (0,8%)

3 AdV (1,2%)

5 / 3N2/ (2%)

98 2

2%

4

4%

1

1% 0

32 0 7

21,8%

6

18,75%

10 AdV

31,3%

371 18

4,8%

58

15,6%

25

6,7%

RSV

,

, . RSV

- (15,6% 4,8%).

-2 RSV.

, (58

) .

,

, RSV, MPV AdV.

39

№13 ,

5 , -

,

RSV -58 (15,6%), MPV 25

(6,7%) -18 (4,8%). . №14

83 RSV MPV

18 ( )

. RSV

- , MPV e

.

.№14. RSV, MPV

, 2008/2009

0-11 ,

, (p<0.05, Pearson chi square=7,06) 36-

59 , (p<0.05, Pearson chi square=7,02).

,

,

0- 11 12-35 36-59

RSV MPV

RSV MPV

RSV MPV

/

/

/

137

28/100

28%

5/100

5%

3/100

3%

2/31

6,5%

3/31

9,6%

1/31

3,2% 0/6

1/6

16,6% 0/6

98 9/39

23%

2/39

5,1%

5/39

12%

6/41

14,6%

4/41

9,7%

1/41

2,4% 0/18

3/18

16,6%

3/18

16,6%

136 7/31

22% 0/31 0/31

3/46

6,5%

5/46

10,8%

4/46

8,7%

3/59

5%

2/59

3,4%

1/59

1,7%

44/170

26%

7/170

4,1%

8/170

4,7%

11/118

9,3%

12/118

10,2%

6/118

5%

3/83

3,6%

5/83

6%

4/83

4,8%

40

RSV (p<0.001, Pearson

chi square=22,69).

. 10 , MPV 2008/2009

,

.

10 , RSV, MPV Ad, 2008/2009

-

, ,

( ).

,

, (

)

, , -

.

, 3

, 7

.

,

41

.

. ( .№15) AdV

,

-2 RSV MPV.

AdV , -

,

.

, -

RSV MPV.

– ,

.

RSV. ( . 10).

, , 22

. ,

, ,

- AdV MPV.

.

. №15

5 . .

PCR

„

”

,

38

20

7 RSV

2 MPV 0

„

”

,

15

7 7 AdV

3 MPV 10 AdV

5 3 AdV

1 MPV

42

4.4.

RSV MPV RT-PCR

( 24 48 .),

,

,

.

,

.

. . „ ”

,

.

2006/2007 RSV ,

RT-PCR 37,8% 6,1%

8,46% 1,5% 15,6% 0,54% . ( .№16)

-

RT-PCR .

RT-PCR ( 19,2% 2%

, )

,

, RSV,

,

.

.

43

. №16 RSV

RT-PCR

RSV

:

2006/2007

2007/2008

2008/2009

9

6,1%

2

1,5%

2

0,54%

13

2%

RT-PCR 56

37,8%

11

8,46%

58

15,6%

125

19,2%

148 130 371 649

%

(p<0.001, Pearson chi square=16,89),

% RT-PCR (p<0.001, Pearson chi square=45,71).

,

.

RSV

.11.

, RSV

2006/2007 , - (

№ 17),

.

9 RSV

2006/2007, .

RSV

RT-PCR - 84%,

.

, RSV

– ,

,

-

. 2008/2009

( № 17), MPV .

44

. №17 RSV MPV

* -

RSV

(p<0.001, Pearson Chi square=33.95).

%

2006/2007 .,

, .

2008/2009 . ((p<0.05, Pearson Chi square=4,42).

% MPV

2007/2008 2008/2009 . (p<0.05, Pearson Chi square=7,74).

% 2008/2009

.

%

2008/2009 .

(p<0.05, Pearson Chi square=4,79).

2006/2007 2007/2008 2008/2009

RSV RSV MPV RSV MPV

/

/

/

51/130

39,2 %

11/130

8,5%

13/130

13%

47/241

19,5%

18/241

7,5%

16/241

6,6%

*

5/18

27,7% - -

11/130

8,4%

7/130

5,3%

2/130

1,5%

45

. 11 RT-PCR

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47

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%

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596 186 31

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.

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,

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/

(

1-1/2) /California/4/2009, A/California/7/2009

A/England/195/2009/H1N1/ ( .№20 №21)

55

. №20

( 1N1)2009, ,

.

Antigenic analyses of pandemic influenza A(H1N1) viruses (25/08/2009)

Haemagglutination inhibition titre1

Post infection ferret sera

Viruses Collection Passage A/NJ2 A/NJ A/Cal A/Cal A/Eng

date History 8/76 8/76 4/09 7/09 195/09

R15/79 F34/82 C4/F14/09 C4/16/09 NIBSC

F17/09

REFERENCE VIRUSES

A/New Jersey/8/76 Ex >5120 640 320 320 320

A/California/4/2009 C1,M1,E1 2560 160 2560 1280 2560

A/California/7/2009 E1+4 2560 160 2560 1280 1280

A/England/195/2009 MDCK3 2560 160 2560 1280 2560

TEST VIRUSES

A/Bulgaria/179/2009 26.6.2009 MDCK2 1280 80 1280 1280 1280

A/Bulgaria/340/2009 24.7.2009 MDCK2 1280 80 1280 1280 1280

A/Bulgaria/293/2009 15.7.2009 MDCK2 2560 160 2560 2560 1280

A/Bulgaria/330/2009 22.7.2009 MDCK2 2560 160 2560 1280 1280

A/Bulgaria/311/2009 20.7.2009 MDCK2 2560 160 2560 2560 1280

A/Bulgaria/324/2009 22.7.2009 MDCK2 2560 320 2560 2560 2560

A/Bulgaria/197/2009 08.6.2009 MDCK4 2560 160 1280 1280 1280

A/Bulgaria/325/2009 22.7.2009 MDCK3 2560 160 1280 1280 1280

A/Bulgaria/335/2009 23.7.2009 MDCK3 5120 160 1280 2560 2560

A/Bulgaria/363/2009 28.7.2009 MDCK3 5120 160 1280 2560 2560

A/Bulgaria/342/2009 24.7.2009 MDCK3 5120 160 1280 2560 1280

A/Bulgaria/354/2009 24.7.2009 MDCK3 5120 160 1280 2560 1280

1. < = 40; 2. hyperimmune

rabbit serum

56

. №21

( 1N1)2009, ,

.

Antigenic analyses of pandemic influenza A(H1N1) viruses (23/03/10)

Haemagglutination inhibition titre1

Post infection ferret sera

Viruses Collection Passage A/Cal A/Cal A/Eng A/Auck A/Bayern A/Lviv

date History 4/09 7/09 195/09 3/09 69/09 N6/2009

C4/F14/09 C4/31/09 NIBSC F17/09

C4/17/09 C4/33/09 C4/34/09

REFERENCE

VIRUSES

A/California/4/2009 C1,E2 2560 2560 1280 2560 1280 2560

A/California/7/2009 E6 2560 2560 1280 2560 1280 2560

A/England/195/2009 MDCK3/SIAT1 1280 2560 1280 2560 1280 2560

A/Auckland/3/2009 Ex+3 2560 2560 1280 5120 1280 2560

A/Bayern/69/2009 MDCK4/SIAT1 80 320 40 80 320 320

A/Lviv/N6/2009 MDCK4/SIAT1 640 1280 160 320 2560 2560

TEST VIRUSES

A/Bulgaria/1325/2009 03.11.2009 SIAT2 2560 5120 1280 5120 2560 2560

A/Bulgaria1369/2009 03.11.2009 SIAT3 2560 2560 1280 2560 1280 2560 1. < = 40

WHO CC

,

. ( NA),

- V106I N248D,

,

H275Y. ( .17, 18) 03.02.2010 . 225

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(H1N1)2009.

.

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. ( 19)

19. A(H1N1)2009

rRTPCR, 40-53 2009 .

61

20 ,

,

.

- ,

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, 99%

, .

20. -

2009 .

152 28 , 2009 .,

,

. rRT-PCR

5 (0,9%), - 7 (1,3%).

,

.

,

WHO/Europe.

62

V.

RSV, MPV

2004-2009 .

,

.

RSV .

RT-PCR 2005 .

2006/2007. 2008 .

RT-PCR

2001 . ,

2008/2009 5

RSV, MPV

. 2009 .

rRT-PCR ( 1N1)2009.

RSV 846

, 452 MPV, 371

. ,

04.05.2009 .- 08.01.2010 . 2429

157 ,

,

.

, RSV MPV

- , 0 5 .

0 11 ., 12 35 .

36 59 .

.

RSV

- , MPV

63

.

5 , ,

.

,

-

, , .

„ ”

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-

RT-PCR, . ,

RSV ,

, ,

,

- .

15 RSV,

- -2 MRC-5.

RSV MPV

, MPV

, ,

.

2009 . ,

- BSL3

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.

Directigen RSV

RSV, ,

.

64

,

48 .,

.

,

,

,

.

RSV

,

, (2005 .)

,

.

RSV,

,

RT-PCR , .

MPV - ,

. ,

, RSV MPV.

RT-PCR RSV

.

„ ” 2006 . ,

INSTAND, RSV .

RT-PCR .

, -

RSV ,

.

RT-PCR

, - ,

, ,

65

. 2006 . RT-

PCR RSV .

2006/2007 . RSV -

,

,

.

MPV.

5 -

.

-

, , RSV MPV

.

RSV MPV

.

RSV

2006/2007, ,

RSV MPV,

.

,

2008/2009.

, RSV

(ECDC). 2007 .

ECDC

RSV.

2008 .

.

H5N1, 1997 .

.

66

, ,

2007 . ,

- External Quality Assessment (EQA).

„ ”

H1N1 2009 .

, 2009 .

. ,

,

.

. 2006 .

.

,

, ,

.

2429

15 . 29 .,

,

.

1918 . ,

, . . „ ”,

. ,

65 . . «

», .

. 34% 1950 .

.

, -

„ ” ,

67

.

.

, ,

90% 18% .

, A(H1N1) 2009

,

.

-

.

0.3-

1.5%. ,

. ,

. . „ „

.

.

ECDC,

.

„ ” Global

Influenza Surveillance Network (GISN)

WHO CCs

. ,

, ,

,

.

68

, RSV MPV,

.

( ) 80- 20–

RSV

(

.

.

,

.

( 1N1)2009

2009 .

e

Real Time RT-PCR.

69

VI. .

1.1.

RSV MPV,

-

.

1.2.

15 29 .,

.

2. (

) RSV,

- ,

,

.

3.1. in-house RT-PCR

RSV MPV

.

3.2. RT-PCR RSV

, .

3.3.

, 2009 .

Real Time RT-PCR,

.

4.1. RSV, MPV

5

.

4.2. RSV

( , ) -

(0-11 )

70

4.3. RT PCR

MPV 5

.

5.1.

( 1N1)2009 , ,

.

5.2.

.

6.

(

) ,

.

A/California/7/2009.

71

VII. .

1.

( )

RSV,

- ,

,

.

2. in-house RT-PCR

RSV MPV

.

3. RSV MPV

.

4. CDC Real Time RT-PCR,

.

5.

- Real Time RT-

PCR.

6.

,

.

72

VIII. :

:

1. ., ., ., ., ..

. , 3, 2004, . 28-32

2. Pavlova S., Hadziolova T, Kotzeva R. Diagnostic studies on the etiological role

of respiratory syncytial virus and influenza viruses in hospitalized children.

Problems of Infectious and Parasitic Diseases. 2006, vol 34/2, pp 29-31

3. ., ., . RT-PCR ,

. , 2007, LIV, Supplement I ,119-122.

4. ., ., . “ ”

, . , 2008, LV, ,

. 30-34

5. Pavlova S., Hadzjiolova T., Arabadjieva P., Kotseva R.. Application of PCR for

the Respiratory Viruses Diagnosis. Second Congress of Virology (Days of

Virology in Bulgaria), S 2008,151-159.

6. . , . , . . 2008/2009 .

, IV, . 1/2012, 17-22

7. . , . . (H1N1)2009 . 48/2012, №2, 17-

26

1. Hadzhiolova T., Pavlova S., Kotseva R. Possibilities for laboratory diagnosis of

respiratory syncytial virus. Biotechnology 2/ 2005/19:67-71.

2. Pavlova S.,T.Hadzhiolova,P.Arabadjieva, R.Kotseva. Application of RT-PCR for

diagnosis of respiratory syncytial virus and human metapneumovirus infections

in Bulgaria,2006-7 and 2008-9. Eurosurveillance, vol 14, issue 23, 1-8.

3. Hadzhiolova T., Pavlova S., Kotseva R, Laboratory investigation of the first

suspected human cases of infection with avian influenza A(H5N1) virus in

Bulgaria, 2008, Eurosurveillance, vol 13, issue 30, 1-6.

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:

1. ., ., , . -

. . “ ”, Vol XL., 3, 2004, . 10-14

2. ., ., . -

. . . 2006; VIII; 2: 6-9

3. ., ., . . . 2007, 6, 5-8

4. ., ., . – . . 2008, 3, 16-18

, , :

1. ., ., . DТrОМtТРОЧ FХЮ – A+ DТrОМtТРОЧ RSV

RSV. II , , 30 -1 2003.II - , 30

-1 2003.

2. ., ., ., ., ..

14-th Annual Assembly of IMAB-26-30 May

2004, Varna.

3. Pavlova S., Hadzhiolova T., Kotseva R., Mladenova Z., Dundarova D

Kalvatchev Zl. Possibilities for laboratory diagnosis of respiratory syncytial

virus, Days of Virology, 23-25.09.2004, Sofia.

4. ., ., ., ., .

- . XI

, 2004 .

5. ., ., . . .

; . 2006 . .

6. ., ., . -

. XI . . 05-07. 10.2006 .

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7. ., ., ., ., T.

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- , 19-22 2007 .

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the Respiratory Viruses Diagnosis. Second Cogress of Virology (Days of

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9. ., ., . “ ”

/ 5N1/.. IV

, BIO-RAD, , 12-14.11.2008 .

10. . , . . (H1N1)2009 . 8- , , 2010 .

:

1. . : . , . , .

, . , . . 2003 .

2. RT-PCR - . : . , . , . , . .

2004 .

3. PCR / .

: . , . , . , . , .

4. Real Time RT-PCR ( 1N1). . , . .