scholarly journals Reverse genetics approach towards understanding pathogenesis of H5N1 Hong Kong influenza A virus infection

2001 ◽  
Vol 356 (1416) ◽  
pp. 1841-1843 ◽  
Author(s):  
Masato Hatta ◽  
Gabriele Neumann ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Influenza A ◽  
Reverse Genetics ◽  
Gene Segment ◽  
Influenza Viruses ◽  
H5n1 Influenza ◽  
Influenza A Virus Infection ◽  
Cloned Cdna ◽  
First Time

In 1990, Palese and colleagues established a method (reverse genetics) that allowed one to generate influenza virus containing a gene segment derived from cloned cDNA. Although this method contributed tremendously to our understanding of influenza pathogenesis, the requirement of helper viruses limited its use in many experimental settings. Recently, we and others established systems for the generation of influenza viruses entirely from cloned cDNAs. These systems require only DNA cloning and transfection techniques, and can therefore be easily implemented by laboratories working in the fields of molecular biology and virology. Thus, for the first time, a system is now available that allows highly efficient generation of influenza virus without technical limitations. Using this technology, we generated the same strain of H5N1 influenza viruses that caused an outbreak in Hong Kong in 1997, killing six people.

scholarly journals Reemerging H5N1 Influenza Viruses in Hong Kong in 2002 Are Highly Pathogenic to Ducks

2004 ◽  
Vol 78 (9) ◽  
pp. 4892-4901 ◽  
Author(s):  
Katharine M. Sturm-Ramirez ◽  
Trevor Ellis ◽  
Barry Bousfield ◽  
Lucy Bissett ◽  
Kitman Dyrting ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
Neurological Dysfunction ◽  
Aquatic Birds ◽  
Antigenic Analysis ◽  
Highly Pathogenic ◽  
H5n1 Influenza

ABSTRACT Waterfowl are the natural reservoir of all influenza A viruses, which are usually nonpathogenic in wild aquatic birds. However, in late 2002, outbreaks of highly pathogenic H5N1 influenza virus caused deaths among wild migratory birds and resident waterfowl, including ducks, in two Hong Kong parks. In February 2003, an avian H5N1 virus closely related to one of these viruses was isolated from two humans with acute respiratory distress, one of whom died. Antigenic analysis of the new avian isolates showed a reactivity pattern different from that of H5N1 viruses isolated in 1997 and 2001. This finding suggests that significant antigenic variation has recently occurred among H5N1 viruses. We inoculated mallards with antigenically different H5N1 influenza viruses isolated between 1997 and 2003. The new 2002 avian isolates caused systemic infection in the ducks, with high virus titers and pathology in multiple organs, particularly the brain. Ducks developed acute disease, including severe neurological dysfunction and death. Virus was also isolated at high titers from the birds' drinking water and from contact birds, demonstrating efficient transmission. In contrast, H5N1 isolates from 1997 and 2001 were not consistently transmitted efficiently among ducks and did not cause significant disease. Despite a high level of genomic homology, the human isolate showed striking biological differences from its avian homologue in a duck model. This is the first reported case of lethal influenza virus infection in wild aquatic birds since 1961.

scholarly journals Influenza reverse genetics: dissecting immunity and pathogenesis

2014 ◽  
Vol 16 ◽  
Author(s):  
Siying Ye ◽  
Justin G. Evans ◽  
John Stambas
Keyword(s):  
Influenza Virus ◽  
Protein Function ◽  
Reverse Genetics ◽  
Influenza Virus Vaccine ◽  
Influenza Viruses ◽  
Host Pathogen Interactions ◽  
H5n1 Influenza ◽  
Host Pathogen ◽  
Cloned Cdna ◽  
Novel Therapeutic Strategies

Reverse genetics systems allow artificial generation of non-segmented and segmented negative-sense RNA viruses, like influenza viruses, entirely from cloned cDNA. Since the introduction of reverse genetics systems over a decade ago, the ability to generate ‘designer’ influenza viruses in the laboratory has advanced both basic and applied research, providing a powerful tool to investigate and characterise host–pathogen interactions and advance the development of novel therapeutic strategies. The list of applications for reverse genetics has expanded vastly in recent years. In this review, we discuss the development and implications of this technique, including the recent controversy surrounding the generation of a transmissible H5N1 influenza virus. We will focus on research involving the identification of viral protein function, development of live-attenuated influenza virus vaccines, host–pathogen interactions, immunity and the generation of recombinant influenza virus vaccine vectors for the prevention and treatment of infectious diseases and cancer.

scholarly journals Efficacy of Zanamivir against Avian Influenza A Viruses That Possess Genes Encoding H5N1 Internal Proteins and Are Pathogenic in Mammals

2001 ◽  
Vol 45 (4) ◽  
pp. 1216-1224 ◽  
Author(s):  
Irina A. Leneva ◽  
Olga Goloubeva ◽  
Robert J. Fenton ◽  
Margaret Tisdale ◽  
Robert G. Webster
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Avian Influenza ◽  
Influenza A ◽  
Influenza Viruses ◽  
Interspecies Transmission ◽  
H9n2 Influenza Virus ◽  
H5n1 Influenza ◽  
Genes Encoding ◽  
The Brain

ABSTRACT In 1997, an avian H5N1 influenza virus, A/Hong Kong/156/97 (A/HK/156/97), caused six deaths in Hong Kong, and in 1999, an avian H9N2 influenza virus infected two children in Hong Kong. These viruses and a third avian virus [A/Teal/HK/W312/97 (H6N1)] have six highly related genes encoding internal proteins. Additionally, A/Chicken/HK/G9/97 (H9N2) virus has PB1 and PB2 genes that are highly related to those of A/HK/156/97 (H5N1), A/Teal/HK/W312/97 (H6N1), and A/Quail/HK/G1/97 (H9N2) viruses. Because of their similarities with the H5N1 virus, these H6N1 and H9N2 viruses may have the potential for interspecies transmission. We demonstrate that these H6N1 and H9N2 viruses are pathogenic in mice but that their pathogenicities are less than that of A/HK/156/97 (H5N1). Unadapted virus replicated in lungs, but only A/HK/156/97 (H5N1) was found in the brain. After three passages (P3) in mouse lungs, the pathogenicity of the viruses increased, with both A/Teal/HK/W312/97 (H6N1) (P3) and A/Quail/HK/G1/97 (H9N2) (P3) viruses being found in the brain. The neuraminidase inhibitor zanamivir inhibited viral replication in Madin-Darby canine kidney cells in virus yield assays (50% effective concentration, 8.5 to 14.0 μM) and inhibited viral neuraminidase activity (50% inhibitory concentration, 5 to 10 nM). Twice daily intranasal administration of zanamivir (50 and 100 mg/kg of body weight) completely protected infected mice from death. At a dose of 10 mg/kg, zanamivir completely protected mice from infection with H9N2 viruses and increased the mean survival day and the number of survivors infected with H6N1 and H5N1 viruses. Zanamivir, at all doses tested, significantly reduced the virus titers in the lungs and completely blocked the spread of virus to the brain. Thus, zanamivir is efficacious in treating avian influenza viruses that can be transmitted to mammals.

scholarly journals Characterization of a Highly Pathogenic H5N1 Avian Influenza A Virus Isolated from Duck Meat

2002 ◽  
Vol 76 (12) ◽  
pp. 6344-6355 ◽  
Author(s):  
Terrence M. Tumpey ◽  
David L. Suarez ◽  
Laura E. L. Perkins ◽  
Dennis A. Senne ◽  
Jae-gil Lee ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Influenza Viruses ◽  
H5n1 Influenza Virus ◽  
Highly Pathogenic ◽  
H5n1 Influenza ◽  
Pathogenic H5n1

ABSTRACT Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans.

scholarly journals Cross-Reactive, Cell-Mediated Immunity and Protection of Chickens from Lethal H5N1 Influenza Virus Infection in Hong Kong Poultry Markets

2001 ◽  
Vol 75 (6) ◽  
pp. 2516-2525 ◽  
Author(s):  
Sang Heui Seo ◽  
Robert G. Webster
Keyword(s):  
T Cells ◽  
Influenza Virus ◽  
Hong Kong ◽  
Virus Infection ◽  
Cellular Immunity ◽  
Influenza Virus Infection ◽  
Influenza Viruses ◽  
H5n1 Influenza Virus ◽  
H9n2 Influenza Virus ◽  
H5n1 Influenza

ABSTRACT In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8+ T cells from inbred chickens (B2/B2) infected with an H9N2 influenza virus to naive inbred chickens (B2/B2) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8+ T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.

scholarly journals An R195K Mutation in the PA-X Protein Increases the Virulence and Transmission of Influenza A Virus in Mammalian Hosts

2020 ◽  
Vol 94 (11) ◽  
Author(s):  
Yipeng Sun ◽  
Zhe Hu ◽  
Xuxiao Zhang ◽  
Mingyue Chen ◽  
Zhen Wang ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Influenza A ◽  
Reverse Genetics ◽  
Inflammatory Responses ◽  
Gene Segment ◽  
Genetic Alterations ◽  
Influenza Viruses ◽  
Interspecies Transmission ◽  
Adaptive Mutation ◽  
Human Origin

ABSTRACT In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses. IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

scholarly journals International Laboratory Comparison of Influenza Microneutralization Assays for A(H1N1)pdm09, A(H3N2), and A(H5N1) Influenza Viruses by CONSISE

2015 ◽  
Vol 22 (8) ◽  
pp. 957-964 ◽  
Author(s):  
Karen L. Laurie ◽  
Othmar G. Engelhardt ◽  
John Wood ◽  
Alan Heath ◽  
Jacqueline M. Katz ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Incubation Time ◽  
Influenza A ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Influenza A Viruses ◽  
H5n1 Influenza ◽  
The World ◽  
International Laboratory ◽  
The Impact

ABSTRACTThe microneutralization assay is commonly used to detect antibodies to influenza virus, and multiple protocols are used worldwide. These protocols differ in the incubation time of the assay as well as in the order of specific steps, and even within protocols there are often further adjustments in individual laboratories. The impact these protocol variations have on influenza serology data is unclear. Thus, a laboratory comparison of the 2-day enzyme-linked immunosorbent assay (ELISA) and 3-day hemagglutination (HA) microneutralization (MN) protocols, using A(H1N1)pdm09, A(H3N2), and A(H5N1) viruses, was performed by the CONSISE Laboratory Working Group. Individual laboratories performed both assay protocols, on multiple occasions, using different serum panels. Thirteen laboratories from around the world participated. Within each laboratory, serum sample titers for the different assay protocols were compared between assays to determine the sensitivity of each assay and were compared between replicates to assess the reproducibility of each protocol for each laboratory. There was good correlation of the results obtained using the two assay protocols in most laboratories, indicating that these assays may be interchangeable for detecting antibodies to the influenza A viruses included in this study. Importantly, participating laboratories have aligned their methodologies to the CONSISE consensus 2-day ELISA and 3-day HA MN assay protocols to enable better correlation of these assays in the future.

scholarly journals Are Ducks Contributing to the Endemicity of Highly Pathogenic H5N1 Influenza Virus in Asia?

2005 ◽  
Vol 79 (17) ◽  
pp. 11269-11279 ◽  
Author(s):  
K. M. Sturm-Ramirez ◽  
D. J. Hulse-Post ◽  
E. A. Govorkova ◽  
J. Humberd ◽  
P. Seiler ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Virus Disease ◽  
Influenza Viruses ◽  
H5n1 Influenza Virus ◽  
Highly Pathogenic ◽  
Main Site ◽  
H5n1 Influenza ◽  
Pathogenic H5n1 ◽  
Virus Isolates

ABSTRACT Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.

scholarly journals Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes

2018 ◽  
Vol 92 (11) ◽  
pp. e00232-18 ◽  
Author(s):  
Carolien E. van de Sandt ◽  
Mark R. Pronk ◽  
Carel A. van Baalen ◽  
Ron A. M. Fouchier ◽  
Guus F. Rimmelzwaan
Keyword(s):  
Influenza Virus ◽  
T Lymphocytes ◽  
Virus Replication ◽  
Influenza A ◽  
Gene Segment ◽  
Influenza Viruses ◽  
Amino Acid Residues ◽  
Human Influenza ◽  
M Gene ◽  
Specific Ctls

ABSTRACT Influenza virus-specific CD8+ T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M158-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity. IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8+ T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity.

scholarly journals Prevalence of Antibodies against Seasonal Influenza A and B Viruses in Children in Netherlands

2011 ◽  
Vol 18 (3) ◽  
pp. 469-476 ◽  
Author(s):  
R. Bodewes ◽  
G. de Mutsert ◽  
F. R. M. van der Klis ◽  
M. Ventresca ◽  
S. Wilks ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Influenza Virus Infection ◽  
Population Based ◽  
Influenza Viruses ◽  
Serum Samples ◽  
Cross Sectional ◽  
Population Based Study ◽  
Influenza A H1n1 ◽  
First Time

ABSTRACTTo gain insight into the age at which children become infected with influenza viruses for the first time, we analyzed the seroprevalence of antibodies against influenza viruses in children 0 to 7 years of age in the Netherlands. Serum samples were collected during a cross-sectional population-based study in 2006 and 2007 and were tested for the presence of antibodies against influenza A/H1N1, A/H3N2, and B viruses representative of viruses present in previous influenza seasons using the hemagglutination inhibition assay. The seroprevalence of antibodies to influenza virus was higher in children 1 to 6 months of age than in children 7 to 12 months of age, which likely reflects the presence of maternally derived antibodies. The proportion of study subjects >1 year of age with detectable antibodies against influenza viruses gradually increased with age until they reached the age of 6 years, when they all had antibodies to at least one influenza A virus. These findings may have implications for the development of vaccination strategies aiming at the protection of young children against seasonal and/or pandemic influenza virus infection.

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