Gao YW. Evolution of H5N1 Influenza Virus from Tigers and Lions and Construction of Recombinant Canine Adenovirus Type2 Expressing Hemagglutinin Gene. Ph.D Thesis, Jilin University
Evolution of H5N1 Influenza Virus from Tigers and Lions
and Construction of Recombinant Canine Adenovirus Type
2 Expressing Hemagglutinin Gene
Ph. D. Candidate: Gao Yu-wei
Advisor：Xia Xian-zhu，Membership of Chinese Academy of Engineering
Since late 2003, H5N1 virus has reached epizootic levels in domestic fowl in a
number of Asian countries. More recently, the H5N1 virus spread to poultry in 45
countries in Asia, Europe, and Africa. Moreover, H5N1 virus was reported to be able
to transmit to humans with a high mortality rate. Of 192 laboratory-confirmed cases,
109 patients died of H5N1 virus infection as of 6 April 2006. These events have led to
significant global concern about the potential for this virus to evolve to pandemic
proportions with the capacity to cause millions of deaths. The first outbreak of H5N1
influenza virus infected tigers was reported in China in 2002. This report is the first of
H5N1 virus infection causing death in nonhuman mammals.
Twenty-one specimens of lung of tigers and lions, died of high fever, anorexia and
twitch, were collected from Beijing, Harbin, Taiyuan, Yichang, Guilin, Datong,
Zhengzhou, Shanghai, Nanjing, and Tangshan from 2002~2005. All of specimens
detected by RT-PCR with H5N1 virus special primers, 18 specimens were positive. 15
isolates of influenza-like virus were obtained from the positive specimens. The isolates
were confirmed to be H5N1 subtype influenza A virus by characterization of their
virology. Two hundreds and seventy-five sera from 1998~2005 were tested for
evidence of exposure to H5N1 virus by hemagglutination inhibition test. Among those
from 2002~2005, 98 were HI antibody positive. These results indicated that H5N1
influenza rapidly spread to capture tigers and lions from south to north in China in a
short time.
To test the pathogenicity of the Tiger H5N1 influenza viruses(TIV), the cell and
animal infection experiments were carried out. TIV infected PK-15, Vero, F81 and
BHK-21 cell with obvious cytopathic effect(CPE). The amount of IVPI in chickens is
to 2.23, hence the TIVs could be determined to be strains of HPAI subtype. The
average half mouse lethal dose (MLD50) of TIVs was from10-6.8 to 10-7.1/50μl. Three
cats infected with TIVs showed clinical signs, including anorexia, depression and
raised body temperature. One survived in the infection and the other two died. On
necropsy, dull red focus of infection was found on the lung of dead cats. Histologic
lesions observed in the infected cats showed diffusing alveolar damage and a number
of macrophage infiltrating and a few of protein serosity effusions in alveolus.
Influenza A virus antigen was demonstrated in endochylema of bronchia epithelial
cells and sporadic macrophage by the method of immunohistochemistry. H5N1 TIVs
were recovered from the lung, brain, heart and kidney tissues. The virus titers in the
lung were the highest, ranged from10-5.9 to 10-6.8 /g.
To shed light on the evolution and genetic backgrounds of these TIVs, 9 of 15
isolates were selected for whole genome analysis. Homology analysis showed that 8
gene segments of TIVs had high homology with the gene segments of H5N1 influenza
A virus (96%~100%). Phylogenetic analysis showed that the TIVs from
2002~2004 isolates clustered within the lineage of the 1996~2004 H5N1 avian
and human isolates from china, the TIVs from 2005 isolates clustered within the
lineage of the 2003-2005 H5N1 avian and human isolates from China, Japan,
South Korea, and Russia. Genetic analysis revealed that the HA proteolytic
cleavage site of TIVs harbor multiple basic amino acids(RRRKKR) , indicatives of
high virulence. All 9 isolates of TIVs had a glutamine at position 222 and a glycine at
position 224 of HA protein,which are related to receptor binding sites specific for
avian species. All 9 isolates of TIVs contained a 20 amino acid deletion in the NA
stalk (49~68), and contained a 5 amino acid deletion at positions 80~84 of NS1
protein. There is no mutations related to amantadine resistance properties in M2
protein of TIVs and therefore, the TIVs were presumably sensitive to amantadines.
To find out the molecular determination of the H5N1 virus infected tigers, the
receptor binding specificity of all the TIVs and the type of influenza virus receptor in
tiger trachea were studied. All of the sialic acid was removed from chicken red blood
cells (CRBCs) using Vibrio cholerae neuraminidase (VCNA). Subsequently,resialylation
was performed using α2,3-(N)-sialyltransferase orα2,6-(N)-sialyltransferase and 1.5 mM CMP-sialic acid. To assay the receptor binding specificity of tiger isolates, the Hemagglutination assay was used with treated CRBCs.
All TIVs binds the α2,3 sialic acid(Avian) cellular receptor. To identify the type of
receptor in tiger trachea, tigertrachea sections were incubated with
digoxigenin-labeled SNA and MAA lectin, respectively. Subsequently the sections
were incubated with FITC-conjugated anti-DIG antibody. The results showed that
there are abundant α2,3 sialic acid linked in tiger trachea.
To develop a new type of vaccine for Felidae influenza prevention, recombinant
replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of
H5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003（H5N1）
HA gene was cloned into PVAX1. The HA expression cassette which included CMV
and HA and PolyA was ligated into the E3 deletion region of pVAXE. The
recombinant plasmid was named pEHA. The pEHA and the pPoly2-CAV2 were
digested with Nru I/Sal I, respectively. The purified Nru I/Sal I DNA fragment
containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the
recombinant plasmid pCAV-2/HA. The recombinant genome was released from
pCAV-2-HA, and was transfected into MDCK cells by Lipofectamine. The
recombinant virus named CAV-2-HA was obtained. Anti-H5N1influenza virus HI
antibody (1:8~1:16) could be detected in cats immunized with CAV-2-HA.
Keywords: tiger; H5N1 influenza virus; evolution; interspecies transmission;
recombinant; canine adenovirus type 2; hemagglutinin
and Construction of Recombinant Canine Adenovirus Type
2 Expressing Hemagglutinin Gene
Ph. D. Candidate: Gao Yu-wei
Advisor：Xia Xian-zhu，Membership of Chinese Academy of Engineering
Since late 2003, H5N1 virus has reached epizootic levels in domestic fowl in a
number of Asian countries. More recently, the H5N1 virus spread to poultry in 45
countries in Asia, Europe, and Africa. Moreover, H5N1 virus was reported to be able
to transmit to humans with a high mortality rate. Of 192 laboratory-confirmed cases,
109 patients died of H5N1 virus infection as of 6 April 2006. These events have led to
significant global concern about the potential for this virus to evolve to pandemic
proportions with the capacity to cause millions of deaths. The first outbreak of H5N1
influenza virus infected tigers was reported in China in 2002. This report is the first of
H5N1 virus infection causing death in nonhuman mammals.
Twenty-one specimens of lung of tigers and lions, died of high fever, anorexia and
twitch, were collected from Beijing, Harbin, Taiyuan, Yichang, Guilin, Datong,
Zhengzhou, Shanghai, Nanjing, and Tangshan from 2002~2005. All of specimens
detected by RT-PCR with H5N1 virus special primers, 18 specimens were positive. 15
isolates of influenza-like virus were obtained from the positive specimens. The isolates
were confirmed to be H5N1 subtype influenza A virus by characterization of their
virology. Two hundreds and seventy-five sera from 1998~2005 were tested for
evidence of exposure to H5N1 virus by hemagglutination inhibition test. Among those
from 2002~2005, 98 were HI antibody positive. These results indicated that H5N1
influenza rapidly spread to capture tigers and lions from south to north in China in a
short time.
To test the pathogenicity of the Tiger H5N1 influenza viruses(TIV), the cell and
animal infection experiments were carried out. TIV infected PK-15, Vero, F81 and
BHK-21 cell with obvious cytopathic effect(CPE). The amount of IVPI in chickens is
to 2.23, hence the TIVs could be determined to be strains of HPAI subtype. The
average half mouse lethal dose (MLD50) of TIVs was from10-6.8 to 10-7.1/50μl. Three
cats infected with TIVs showed clinical signs, including anorexia, depression and
raised body temperature. One survived in the infection and the other two died. On
necropsy, dull red focus of infection was found on the lung of dead cats. Histologic
lesions observed in the infected cats showed diffusing alveolar damage and a number
of macrophage infiltrating and a few of protein serosity effusions in alveolus.
Influenza A virus antigen was demonstrated in endochylema of bronchia epithelial
cells and sporadic macrophage by the method of immunohistochemistry. H5N1 TIVs
were recovered from the lung, brain, heart and kidney tissues. The virus titers in the
lung were the highest, ranged from10-5.9 to 10-6.8 /g.
To shed light on the evolution and genetic backgrounds of these TIVs, 9 of 15
isolates were selected for whole genome analysis. Homology analysis showed that 8
gene segments of TIVs had high homology with the gene segments of H5N1 influenza
A virus (96%~100%). Phylogenetic analysis showed that the TIVs from
2002~2004 isolates clustered within the lineage of the 1996~2004 H5N1 avian
and human isolates from china, the TIVs from 2005 isolates clustered within the
lineage of the 2003-2005 H5N1 avian and human isolates from China, Japan,
South Korea, and Russia. Genetic analysis revealed that the HA proteolytic
cleavage site of TIVs harbor multiple basic amino acids(RRRKKR) , indicatives of
high virulence. All 9 isolates of TIVs had a glutamine at position 222 and a glycine at
position 224 of HA protein,which are related to receptor binding sites specific for
avian species. All 9 isolates of TIVs contained a 20 amino acid deletion in the NA
stalk (49~68), and contained a 5 amino acid deletion at positions 80~84 of NS1
protein. There is no mutations related to amantadine resistance properties in M2
protein of TIVs and therefore, the TIVs were presumably sensitive to amantadines.
To find out the molecular determination of the H5N1 virus infected tigers, the
receptor binding specificity of all the TIVs and the type of influenza virus receptor in
tiger trachea were studied. All of the sialic acid was removed from chicken red blood
cells (CRBCs) using Vibrio cholerae neuraminidase (VCNA). Subsequently,resialylation
was performed using α2,3-(N)-sialyltransferase orα2,6-(N)-sialyltransferase and 1.5 mM CMP-sialic acid. To assay the receptor binding specificity of tiger isolates, the Hemagglutination assay was used with treated CRBCs.
All TIVs binds the α2,3 sialic acid(Avian) cellular receptor. To identify the type of
receptor in tiger trachea, tigertrachea sections were incubated with
digoxigenin-labeled SNA and MAA lectin, respectively. Subsequently the sections
were incubated with FITC-conjugated anti-DIG antibody. The results showed that
there are abundant α2,3 sialic acid linked in tiger trachea.
To develop a new type of vaccine for Felidae influenza prevention, recombinant
replication-competent canine adenovirus Type 2 expressing hemagglutinin gene of
H5N1 subtype tiger influenza virus was constructed. A/tiger/Harbin/01/2003（H5N1）
HA gene was cloned into PVAX1. The HA expression cassette which included CMV
and HA and PolyA was ligated into the E3 deletion region of pVAXE. The
recombinant plasmid was named pEHA. The pEHA and the pPoly2-CAV2 were
digested with Nru I/Sal I, respectively. The purified Nru I/Sal I DNA fragment
containing the HA expression cassette was cloned into pPoly2-CAV2 to generate the
recombinant plasmid pCAV-2/HA. The recombinant genome was released from
pCAV-2-HA, and was transfected into MDCK cells by Lipofectamine. The
recombinant virus named CAV-2-HA was obtained. Anti-H5N1influenza virus HI
antibody (1:8~1:16) could be detected in cats immunized with CAV-2-HA.
Keywords: tiger; H5N1 influenza virus; evolution; interspecies transmission;
recombinant; canine adenovirus type 2; hemagglutinin
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