(Lond) 62:413–424. https://doi.org/10.
1017/S0022172400040158
28. Grose C, Perrotta DM, Brunell PA, Con Smith
G (1979) Cell-free varicella-zoster virus in
cultured human melanoma cells. J Gen Virol
43(1):15–27.
https://doi.org/10.1099/
0022-1317-43-1-15
29. Rubinson DA, Dillon CP, Kwiatkowski AV,
Sievers C, Yang L, Kopinja J, Zhang M, McManus MT, Gertler FB, Scott ML, Van Parijs L
(2003) A lentivirus-based system to functionally silence genes in primary mammalian cells,
stem cells and transgenic mice by RNA interference. Nat Genet 33(3):401–406. https://
doi.org/10.1038/ng1117
30. Manicassamy B, Manicassamy S, BelichaVillanueva A, Pisanelli G, Pulendran B, Garcı ´a-Sastre A (2010) Analysis of in vivo dynamics
of influenza virus infection in mice using a GFP
reporter virus. Proc Natl Acad Sci U S A 107
(25):11531–11536.
https://doi.org/10.
1073/pnas.0914994107
31. Hoffmann E, Neumann G, Kawaoka Y,
Hobom G, Webster RG (2000) A DNA transfection system for generation of influenza A
virus from eight plasmids. Proc Natl Acad Sci
U S A 97(11):6108–6113. https://doi.org/
10.1073/pnas.100133697
32. Li J, Are ´valo MT, Chen Y, Posadas O, Smith
JA, Zeng M (2014) Intranasal immunization
with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum
immunity against influenza viruses. Hum Vaccines Immunother 10(5):1211–1220. https://
doi.org/10.4161/hv.28407
33. Priore SF, Moss WN, Turner DH (2012) Influenza A virus coding regions exhibit hostspecific global ordered RNA structure. PLoS
One 7:e35989. https://doi.org/10.1371/
journal.pone.0035989
34. Xu L, Bao L, Li F, Lv Q, Ma Y, Zhou J, Xu Y,
Deng W, Zhan L, Zhu H, Ma C, Shu Y, Qin C
(2011) Adaption of seasonal H1N1 influenza
virus in mice. PLoS One 6(12):e28901.
https://doi.org/10.1371/journal.pone.
0028901
35. Brown EG, Liu H, Chang Kit L, Baird S, Nesrallah M (2001) Pattern of mutation in the
genome of influenza A virus on adaptation to
increased virulence in the mouse lung: Identification of functional themes. Proc Natl Acad
Sci U S A 98(12):6883–6888. https://doi.
org/10.1073/pnas.111165798
36. Poon LLM, Leung YHC, Nicholls JM, Perera
P-Y, Lichy JH, Yamamoto M, Waldmann TA,
Peiris JSM, Perera LP (2009) Vaccinia virusbased multivalent H5N1 avian influenza vaccines adjuvanted with IL-15 confer sterile
cross-clade protection in mice. J Immunol
182(5):3063–3071.
https://doi.org/10.
4049/jimmunol.0803467
37. Valkenburg SA, Li OTW, Mak PWY, Mok
CKP, Nicholls JM, Guan Y, Waldmann TA,
Peiris JSM, Perera LP, Poon LLM (2014)
IL-15 adjuvanted multivalent vaccinia-based
universal influenza vaccine requires CD4+ T
cells for heterosubtypic protection. Proc Natl
Acad Sci U S A 111:5676–5681. https://doi.
org/10.1073/pnas.1403684111
38. Wen X, Huang X, Mok BW-Y, Chen Y,
Zheng M, Lau S-Y, Wang P, Song W, Jin D-Y,
Yuen K-Y, Chen H (2014) NF90 exerts antiviral activity through regulation of PKR phosphorylation and stress granules in infected cells.
J Immunol 192(8):3753–3764. https://doi.
org/10.4049/jimmunol.1302813
39. Blaschke V, Reich K, Blaschke S, Zipprich S,
Neumann C (2000) Rapid quantitation of
proinflammatory and chemoattractant cytokine expression in small tissue samples and
monocyte-derived dendritic cells: Validation
of a new real-time RT-PCR technology. J
Immunol Methods 246(1-2):79–90. https://
doi.org/10.1016/S0022-1759(00)00304-5
40. Nicholls JM, Wong LPW, Chan RWY, Poon
LLM, So LKY, Yen HL, Fung K, Van
Poucke S, Peiris JSM (2012) Detection of
highly pathogenic influenza and pandemic
influenza virus in formalin fixed tissues by
immunohistochemical methods. J Virol Methods 179(2):409–413. https://doi.org/10.
1016/j.jviromet.2011.11.006
41. Buchy P, Mardy S, Vong S, Toyoda T, Aubin
JT, Miller M, Touch S, Sovann L, Dufourcq JB,
Richner B, Van Tu P, Tien NTK, Lim W, Peiris
JSM, Van der Werf S (2007) Influenza
A/H5N1 virus infection in humans in Cambodia. J Clin Virol 39(3):164–168. https://doi.
org/10.1016/j.jcv.2007.04.010
42. Ding H, Tsai C, Gutie ´rrez RA, Zhou F,
Buchy P, Deubel V, Zhou P (2011) Superior
neutralizing antibody response and protection
in mice vaccinated with heterologous DNA
prime and virus like particle boost against
HPAI H5N1 virus. PLoS One 6(1):e16563.
https://doi.org/10.1371/journal.pone.
0016563
43. Shahangian A, Chow EK, Tian X, Kang JR,
Ghaffari A, Liu SY, Belperio JA, Cheng G,
Deng JC (2009) Type I IFNs mediate development of postinfluenza bacterial pneumonia in
mice. J Clin Invest 119(7):1910–1920.
https://doi.org/10.1172/JCI35412
44. Van Spaendonk RML, Ramesar J, Van
Wigcheren A, Eling W, Beetsma AL, Van
Gemert GJ, Hooghof J, Janse CJ, Waters AP
Live Vaccine Attenuation
355
1017/S0022172400040158
28. Grose C, Perrotta DM, Brunell PA, Con Smith
G (1979) Cell-free varicella-zoster virus in
cultured human melanoma cells. J Gen Virol
43(1):15–27.
https://doi.org/10.1099/
0022-1317-43-1-15
29. Rubinson DA, Dillon CP, Kwiatkowski AV,
Sievers C, Yang L, Kopinja J, Zhang M, McManus MT, Gertler FB, Scott ML, Van Parijs L
(2003) A lentivirus-based system to functionally silence genes in primary mammalian cells,
stem cells and transgenic mice by RNA interference. Nat Genet 33(3):401–406. https://
doi.org/10.1038/ng1117
30. Manicassamy B, Manicassamy S, BelichaVillanueva A, Pisanelli G, Pulendran B, Garcı ´a-Sastre A (2010) Analysis of in vivo dynamics
of influenza virus infection in mice using a GFP
reporter virus. Proc Natl Acad Sci U S A 107
(25):11531–11536.
https://doi.org/10.
1073/pnas.0914994107
31. Hoffmann E, Neumann G, Kawaoka Y,
Hobom G, Webster RG (2000) A DNA transfection system for generation of influenza A
virus from eight plasmids. Proc Natl Acad Sci
U S A 97(11):6108–6113. https://doi.org/
10.1073/pnas.100133697
32. Li J, Are ´valo MT, Chen Y, Posadas O, Smith
JA, Zeng M (2014) Intranasal immunization
with influenza antigens conjugated with cholera toxin subunit B stimulates broad spectrum
immunity against influenza viruses. Hum Vaccines Immunother 10(5):1211–1220. https://
doi.org/10.4161/hv.28407
33. Priore SF, Moss WN, Turner DH (2012) Influenza A virus coding regions exhibit hostspecific global ordered RNA structure. PLoS
One 7:e35989. https://doi.org/10.1371/
journal.pone.0035989
34. Xu L, Bao L, Li F, Lv Q, Ma Y, Zhou J, Xu Y,
Deng W, Zhan L, Zhu H, Ma C, Shu Y, Qin C
(2011) Adaption of seasonal H1N1 influenza
virus in mice. PLoS One 6(12):e28901.
https://doi.org/10.1371/journal.pone.
0028901
35. Brown EG, Liu H, Chang Kit L, Baird S, Nesrallah M (2001) Pattern of mutation in the
genome of influenza A virus on adaptation to
increased virulence in the mouse lung: Identification of functional themes. Proc Natl Acad
Sci U S A 98(12):6883–6888. https://doi.
org/10.1073/pnas.111165798
36. Poon LLM, Leung YHC, Nicholls JM, Perera
P-Y, Lichy JH, Yamamoto M, Waldmann TA,
Peiris JSM, Perera LP (2009) Vaccinia virusbased multivalent H5N1 avian influenza vaccines adjuvanted with IL-15 confer sterile
cross-clade protection in mice. J Immunol
182(5):3063–3071.
https://doi.org/10.
4049/jimmunol.0803467
37. Valkenburg SA, Li OTW, Mak PWY, Mok
CKP, Nicholls JM, Guan Y, Waldmann TA,
Peiris JSM, Perera LP, Poon LLM (2014)
IL-15 adjuvanted multivalent vaccinia-based
universal influenza vaccine requires CD4+ T
cells for heterosubtypic protection. Proc Natl
Acad Sci U S A 111:5676–5681. https://doi.
org/10.1073/pnas.1403684111
38. Wen X, Huang X, Mok BW-Y, Chen Y,
Zheng M, Lau S-Y, Wang P, Song W, Jin D-Y,
Yuen K-Y, Chen H (2014) NF90 exerts antiviral activity through regulation of PKR phosphorylation and stress granules in infected cells.
J Immunol 192(8):3753–3764. https://doi.
org/10.4049/jimmunol.1302813
39. Blaschke V, Reich K, Blaschke S, Zipprich S,
Neumann C (2000) Rapid quantitation of
proinflammatory and chemoattractant cytokine expression in small tissue samples and
monocyte-derived dendritic cells: Validation
of a new real-time RT-PCR technology. J
Immunol Methods 246(1-2):79–90. https://
doi.org/10.1016/S0022-1759(00)00304-5
40. Nicholls JM, Wong LPW, Chan RWY, Poon
LLM, So LKY, Yen HL, Fung K, Van
Poucke S, Peiris JSM (2012) Detection of
highly pathogenic influenza and pandemic
influenza virus in formalin fixed tissues by
immunohistochemical methods. J Virol Methods 179(2):409–413. https://doi.org/10.
1016/j.jviromet.2011.11.006
41. Buchy P, Mardy S, Vong S, Toyoda T, Aubin
JT, Miller M, Touch S, Sovann L, Dufourcq JB,
Richner B, Van Tu P, Tien NTK, Lim W, Peiris
JSM, Van der Werf S (2007) Influenza
A/H5N1 virus infection in humans in Cambodia. J Clin Virol 39(3):164–168. https://doi.
org/10.1016/j.jcv.2007.04.010
42. Ding H, Tsai C, Gutie ´rrez RA, Zhou F,
Buchy P, Deubel V, Zhou P (2011) Superior
neutralizing antibody response and protection
in mice vaccinated with heterologous DNA
prime and virus like particle boost against
HPAI H5N1 virus. PLoS One 6(1):e16563.
https://doi.org/10.1371/journal.pone.
0016563
43. Shahangian A, Chow EK, Tian X, Kang JR,
Ghaffari A, Liu SY, Belperio JA, Cheng G,
Deng JC (2009) Type I IFNs mediate development of postinfluenza bacterial pneumonia in
mice. J Clin Invest 119(7):1910–1920.
https://doi.org/10.1172/JCI35412
44. Van Spaendonk RML, Ramesar J, Van
Wigcheren A, Eling W, Beetsma AL, Van
Gemert GJ, Hooghof J, Janse CJ, Waters AP
Live Vaccine Attenuation
355