The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.
Animals ; China ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; pathogenicity ; Influenza in Birds ; virology ; Mice, Inbred BALB C ; Phylogeny ; Poultry

This study aims to investigate the virological impact of the stalk region and cysteine (C) in neuraminidase (NA) of influenza A/Anhui/1/05 (H5N1) and A/Ohio/07/2009 (H1N1) viruses. The NA of A/ Anhui/1/05 (H5N1), defined as AH N1, lacked 20 amino acids (including C, defined as s20) as compared with NA of A/Ohio/07/2009 (H1N1) (defined as 09N1). We deleted s20 of 09N1 to construct 09N1-s20, and inserted s20 into AH N1 to construct AH N1+s20. To investigate the impact of C on the biological function of NA, we deleted C in 09N1 to construct 09N1-C and inserted C into AH N1 to construct AH N1-C. The pseudo-type viral particle (pp) system was used to evaluate the impact of these mutants on virology. The combination of 09N1-C and 09H1 (defined as 09H1::09N1-C) showed an infectivity 8 times that of the wild type 09H1::09N1, while the infectivity of the combination of AH N1+C and AH H5 (defined as AH H5::AH N1+C) was much lower than that of the wild type AH H5::AH N1. The infectivity of the combination of 09N1-s20 and 09H1 (defined as 09H1::09N1-s20) was 4 times that of the wild type 09H1::09N1; the infectivity of the combination of AH N1+s20 and AH H5 (defined as AH H5:: AH N1+s20) was 1/7 that of the wild type AH H5::AH N1. The co-existence of 09N1-C and AH H5 displayed 6 times the infectivity of AH H5::09N1, while the infectivity of 09H1::AH N1+C was very low. Multimer analysis showed that in the wild type 09N1, the forms of NA were dimer > tetramer > monomer; the major component of NA in 09N1-C was monomer; in 09N1-s20, the forms of NA were monomer > dimer. AH N1 was mainly composed of monomer; in AH N1+s20, the forms of NA were dimer > monomer > tetramer; in AH N1+C, the forms of NA were dimer > tetramer. Deletion of C or s20 from 09N1 did not change the expression of NA. The study suggested that deletion of C from the stalk region of NA in A/Ohio/07/2009 (H1N1) increases infectivity. Insertion of C into NA's stalk region of A/ Anhui/1/05 (H5N1) significantly decreases infectivity. Cysteine deletion in the stalk region is important for the infectivity of A/Anhui/1/05 (H5N1) and A/Ohio/07/2009 (H1N1). It may interfere with the infectivity via changes in NA polymerization.
Amino Acid Motifs ; Humans ; Influenza A Virus, H1N1 Subtype ; chemistry ; enzymology ; genetics ; pathogenicity ; Influenza A Virus, H5N1 Subtype ; chemistry ; enzymology ; genetics ; pathogenicity ; Influenza, Human ; virology ; Neuraminidase ; chemistry ; genetics ; metabolism ; Viral Proteins ; chemistry ; genetics ; metabolism ; Virulence

Animals ; Humans ; Influenza A Virus, H5N1 Subtype ; immunology ; pathogenicity ; Poultry ; virology

The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. For a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identified to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.
Animals ; Birds ; Drug Resistance, Viral ; genetics ; Enzyme Inhibitors ; therapeutic use ; HEK293 Cells ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; genetics ; pathogenicity ; Influenza A Virus, H5N1 Subtype ; drug effects ; genetics ; pathogenicity ; Influenza in Birds ; drug therapy ; genetics ; virology ; Influenza, Human ; drug therapy ; genetics ; virology ; Mutagenesis, Site-Directed ; Neuraminidase ; antagonists & inhibitors ; genetics ; Oseltamivir ; administration & dosage

Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype have spread since 2003 in poultry and wild birds in Asia, Europe and Africa. In Korea, the highly pathogenic H5N1 avian influenza outbreaks took place in 2003/2004, 2006/2007 and 2008. As the 2006/2007 isolates differ phylogenetically from the 2003/2004 isolates, we assessed the clinical responses of chickens, ducks and quails to intranasal inoculation of the 2006/2007 index case virus, A/chicken/Korea/IS/06. All the chickens and quails died on 3 days and 3-6 days post-inoculation (DPI), respectively, whilst the ducks only showed signs of mild depression. The uninoculated chickens and quails placed soon after with the inoculated flock died on 5.3 and 7.5 DPI, respectively. Both oropharyngeal and cloacal swabs were taken for all three species during various time intervals after inoculation. It was found that oropharyngeal swabs showed higher viral titers than in cloacal swabs applicable to all three avian species. The chickens and quails shed the virus until they died (up to 3 to 6 days after inoculation, respectively) whilst the ducks shed the virus on 2-4 DPI. The postmortem tissues collected from the chickens and quails on day 3 and days 4-5 and from clinically normal ducks that were euthanized on day 4 contained the virus. However, the ducks had significantly lower viral titers than the chickens or quails. Thus, the three avian species varied significantly in their clinical signs, mortality, tissue virus titers, and duration of virus shedding. Our observations suggest that duck and quail farms should be monitored particularly closely for the presence of HPAIV so that further virus transmission to other avian or mammalian hosts can be prevented.
Animals ; Antibodies, Viral/blood ; Brain/virology ; *Chickens ; *Coturnix ; *Ducks ; Heart/virology ; Influenza A Virus, H5N1 Subtype/*pathogenicity ; Influenza in Birds/epidemiology/transmission/*virology ; Kidney/virology ; Korea/epidemiology ; Lung/virology ; Virus Shedding

OBJECTIVETo evaluate the risk of human infection after the outbreak of avian influenza H5N1 in animals, and probe the possibility for virus transmission.

METHODSBy means of field epidemiological study, molecular epidemiology, serology and emergency surveillance, persons who had ever closely contacted with sick or dead poultry were observed. While, the RT-PCR and gene sequencing method were used to detect H5 nucleic acid from environmental swabs from 4 epidemic spots, and hemagglutination inhibition assay was also used to detect H5 antibody.

RESULTSOf 22 environmental swabs detected from 4 epidemic spots, one was positive for H5 nucleic acid, and the homogeneity was 95.9% as compared with H5N1 virus A/China/GD01/2006 (H5N1) found in Guangzhou in 2006 by gene sequence analysis. 62 environmental swabs from live poultry stalls of food markets near epidemic spot were detected negative. Six of 68 blood samples of contacts were positive for H9 antibody, and all were negative for H5 antibody. 68 throat swabs of contacts were detected negative for H5 nucleic acid. No close contact was found abnormal after 7 days medical observation. 337 influenza-like cases were reported in emergency surveillance, and no suspicious case was found.

CONCLUSIONThe current outbreak of H5N1 avian influenza in water fowls has not yet caused further transmission, and human avian influenza case has not been observed. It indicates that the ability of H5N1 virus to transmit to human is not strong yet, and the risk of human infection for H5N1 is still low.

Animals ; Antibodies, Viral ; blood ; China ; epidemiology ; Disease Outbreaks ; Ducks ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; pathogenicity ; Influenza in Birds ; epidemiology ; transmission ; Influenza, Human ; epidemiology ; transmission ; Risk Assessment

INTRODUCTIONWith the potential threat of an avian influenza (AI) pandemic, healthcare workers (HCWs) are expected to play important roles, and they encounter significant stress levels from an expected increase in workload. We compared the concerns, perceived impact and preparedness for an AI pandemic between HCWs working in public primary care clinics and a tertiary healthcare setting.

MATERIALS AND METHODSAn anonymous, self-administered questionnaire was given to 2459 HCWs working at 18 public polyclinics (PCs) and a tertiary hospital (TH) in Singapore from March to June 2006. The questionnaire assessed work-related and non-work-related concerns, perceived impact on personal life and work as well as workplace preparedness.

RESULTSWe obtained responses from 986 PC and 873 TH HCWs (response rate: 74.6% and 76.7%). The majority in both groups were concerned about the high AI risk from their occupation (82.7%) and falling ill with AI (75.9%). 71.9% accepted the risk but 25.5% felt that they should not be looking after AI patients with 15.0% consider resigning. HCWs also felt that people would avoid them (63.5%) and their families (54.1%) during a pandemic. The majority expected an increased workload and to feel more stressed at work. For preparedness, 74.2% felt personally prepared and 83.7% felt that their workplaces were prepared for an outbreak. TH HCWs were more likely to be involved in infection-control activities but the perception of infection-control preparedness in both groups was high (>80.0%).

CONCLUSIONSHCWs in both public primary and tertiary healthcare settings felt prepared, personally and in their workplaces, for a pandemic. Their main concerns were risks of falling ill from exposure and the possibility of social ostracism of themselves and their families. Preparedness levels appeared high in the majority of HCWs. However, concerns of HCWs could affect their overall effectiveness in a pandemic and should be addressed by incorporating strategies to manage them in pandemic planning.

Adolescent ; Adult ; Aged ; Animals ; Attitude of Health Personnel ; Birds ; Disaster Planning ; Disease Outbreaks ; Female ; Humans ; Infectious Disease Transmission, Patient-to-Professional ; prevention & control ; Influenza A Virus, H5N1 Subtype ; pathogenicity ; Influenza in Birds ; transmission ; Influenza, Human ; epidemiology ; prevention & control ; transmission ; virology ; Male ; Middle Aged ; Primary Health Care ; Singapore

OBJECTIVETo explore histopathologic and ultrastructural characteristics of human avian influenza (AI) infection and related etiological pathogenesis.

METHODSPostmortem lung and heart samples were collected from the patient who died of avian influenza virus infection on November 29, 2003 in China. Light and electron microscopy, immunohistochemistry and histochemistry were used to investigate the pathological changes.

RESULTSThe main pathological findings included extensive pulmonary consolidation, hemorrhage, pulmonary edema and local hemorrhagic infarct. The lamina of alveoli and bronchioles were abundantly filled with protein-rich fluid, erythrocytes, fibrin and cell debris admixed with many neutrophilis, macrophages, lymphocytes and a few of monokaryon and multinuclear giant cells. Hyaline membranes were formed. Local pulmonary tissues were heavily damaged by hemorrhage and necrosis. Alveolar septum was disintegrated. Mesenchymal edema with a few of macrophages infiltration of heart was found. Electron microscopy showed the avian influenza A virus-like particles (type C and type A) of 80 - 120 nm diameter and envelopes in the cytoplasm of pneumocytes and endothelial cells.

CONCLUSIONSFatal pneumonia associated with highly pathogenic avian influenza A virus (H5N1) infection leads to extensive pulmonary consolidation, edema and marked hemorrhagic necrosis and inflammation. Electron microscopy can identify avian influenza A virus-like particles. The findings may offer an important theoretical basis for clinical diagnosis and treatment.

Animals ; Autopsy ; methods ; Birds ; China ; Humans ; Influenza A Virus, H5N1 Subtype ; pathogenicity ; ultrastructure ; Influenza A virus ; classification ; Influenza in Birds ; pathology ; Influenza, Human ; diagnostic imaging ; pathology ; surgery ; virology ; Microscopy, Electron ; Ultrasonography ; Virulence Factors

OBJECTIVETo identify histopathologic changes of major organs and to correlate clinical symptoms in patients infected by avian influenza H5N1.

METHODSAutopsy study was performed in two patients died of avian influenza HSN1 infection, following conventional protocols and strict safety procedures. Tissue samples from all major organs of two cases and lung samples of one case were collected and fixed in 4% formaldehyde. Histopathologic changes were evaluated by light microscope.

RESULTSDiffuse alveolar damage (DAD) of the lung was seen in both cases. Lesions at various stages of development were seen involving different areas of the lung. At the early stages, the lungs exhibited exudative changes, including capillary congestion, necrosis of alveolar epithelial cells, and intra-alveolar edema. Hyaline membranes were prominent and diffusely distributed along alveoli. In the middle-late stages of the disease, the lungs exhibited proliferative and fibrotic changes, including proliferation of pneumocytes and bronchial epithelium, fibrosis of the interstitium and alveolar spaces. Lung biopsy tissue of one case showed DAD and interstitial fibrosis in a background of bronchiectasis. Lymph nodes and spleens showed quantity reduction of lymphocytes and active hemophagocytosis. Other changes in major organs included interstitial carditis in one case and acute renal tubular necrosis in one case. In one case, the brain showed edema with cytoplasmic eosinophilia, loss of structure, axon welling and focal necrosis around ventricle. Multiple foci of trophoblastic necrosis with dystrophic calcification were observed in placenta of one pregnant patient. Acute necrotizing deciduitis was found focally. Sections of fetal lung showed edema and scattered interstitial neutrophils were consistent with acute interstitial pneumonitis.

CONCLUSIONSThe respiratory tract is the major target of avian influenza A H5N1 virus infection. The changes of DAD in the lungs resulted in hypoxia, leading to multiple organ failure and death.

Adult ; Alveolar Epithelial Cells ; pathology ; Animals ; Birds ; Fatal Outcome ; Female ; Humans ; Influenza A Virus, H5N1 Subtype ; pathogenicity ; Influenza in Birds ; pathology ; physiopathology ; virology ; Influenza, Human ; pathology ; Male ; Pregnancy ; Pulmonary Fibrosis ; etiology ; pathology

C57BL/6 mice were inoculated intranasally (50 microl) with serial 10-fold dilution of HAB/01 H5N1 virus. Three and five days later, three mice of each group were euthanized. Lung injury was assessed by observation of lung histopathology, virus titers and MCD50 were also measured. Our data showed that H5N1 viral infection in mice resulted in mainly epithelial injury and interstitial pneumonia, featuring significant weight loss, dramatically increased lung wet weight:body weight ratio, inflammatory cellular infiltration, alveolar and interstitial edema, hemorrhage in lungs with high virus titers, and MCD50 was 10(-6.5)/ 0.05 mL. These results suggested that a mouse model of H5N1 viral infection was successfully established which may benefit study of H5N1 avian influenza virus and pathogenic mechanism of host.
Animals ; Brain ; pathology ; virology ; Disease Models, Animal ; Female ; Humans ; Influenza A Virus, H5N1 Subtype ; pathogenicity ; Influenza, Human ; pathology ; virology ; Liver ; pathology ; virology ; Lung ; pathology ; virology ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Spleen ; pathology ; virology