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The hemagglutinin (HA) genes of 12 H9N2 influenza virus strains isolated from chickens in Mainland China during the period 1995-2002 were genetically analyzed. All the isolates possessed the same amino acid motif -R-S-S-R/G-L- at the cleavage site of HA. Except for the conserved amino acids, as is the case in the other avian influenza viruses, located in the receptor binding site, all of the 12 isolates possessed N at amino acid position 183; A, T, or V at position 190; K at position 137, whereas the representative strains of the other lineage (except Dk/HK/Y280/97-like lineage) virus of H9N2 viruses had H, E, and R at these positions respectively. These could be considered as the partial molecular markers of the H9 viruses isolated from chickens in Mainland China. Phylogenetic analyses showed HA genes of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. No A/quail/Hong Kong/Gl/97-like virus was found in chicken, population since the outbreak of H9N2 influenza in Mainland China in 1992. The available evidence indicates that HA genes of H9 influenza virus circulating in Mainland China during the past years were well conserved.

The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995–2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63–65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.

During 2000-2007, 218 influenza viruses of 28 different combinations of HA (H1-H13) and NA (N1-N9) subtypes were isolated from fecal samples of free-flying water birds at two distant lakes in Hokkaido, Japan. Phylogenic analysis of the matrix (M) genes of 67 strains, selected on the basis of their subtype combinations, revealed that A/duck/Hokkaido/W95/2006 (H10N8) was a reassortant whose M and NA genes [corrected] belonged to North American non-gull-avian and the other six [corrected] genes to Eurasian non-gull-avian lineages. The M genes of other 65 strains belonged to Eurasian non-gull-avian and the one to Eurasian-gull lineages. The M genes of 65 strains were grouped into three different sublineages, indicating that influenza viruses circulating in different populations of free-flying water birds have evolved independently in nature.

To improve the sensitivity of a kit, ESPLINE INFLUENZA A&B for rapid diagnosis of influenza by detecting influenza A or B virus specific nucleoproteins (NP), the ESPLINE INFLUENZA A&B-N was developed by using newly established monoclonal antibodies (MAbs) to the respective NP molecule. MAbs FVA2-11 and FrB1-03 recognize the epitope on the amino acid region 59-130aa of the NP molecule of influenza A virus, and that on the region 72-191aa of the NP of influenza B virus, respectively. The new kit detected influenza A and B virus antigens with a detection limit of 10(2.0)-10 (2.7) pfu/test, which is 4-1000 times higher than that of the original kit. Importantly, this kit detected each of influenza A viruses of the known hemagglutinin (HA) subtypes (H1-H15) including the H5N1 viruses recently isolated from human and avian sources in Asia. The kit also detected all of the 15 representative influenza B virus strains tested. The ESPLINE INFLUENZA A&B-N is thus a rapid and highly sensitive and specific kit for the diagnosis of either influenza A or B virus infections.

Outbreaks of H5N1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry and resulted in human deaths in Thailand and Viet Nam in 2004. Rapid typing and subtyping of H5N1 viruses, especially from clinical specimens, are desirable for taking prompt control measures to prevent the spread of the disease. Here, we developed a set of oligonucleotide primers able to detect, type and subtype H5 and N1 influenza viruses in a single step multiplex reverse transcription-polymerase chain reaction (RT-PCR). RNA was extracted from allantoic fluid or from specimens with guanidinium isothiocyanate reagent. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A, subtype H5 and N1 in a single reaction system under identical conditions. The amplified DNA fragments were analyzed by agarose gel electrophoresis. All the H5N1 viruses tested in the study and the experimental specimens presented three specific bands by the method established here. The results presented here suggest that the method described below is rapid and specific and, therefore, could be valuable in the rapid detection of H5N1 influenza viruses in clinics.

H9N2 subtype avian influenza viruses are widespread in domestic poultry. Genetic analysis indicated that three lineages of H9N2 viruses have been established in Eurasia and only one lineage is present on chicken farms in mainland China. Here, NS1 genes of eight H9N2 chicken influenza viruses, isolated in mainland China during 1998–2002, were completely sequenced and phylogenetically analyzed. By comparison, the homology of the NS1 of the A/chicken/Neimenggu/ZH/02 (Ck/NM/ZH/02) strain had a high identity (93.8%) with that of A/chicken/Korea/323/96 (Ck/Kor/323/96), which is an A/duck/Hong Kong/Y439/97 (Dk/HK/Y439/97)-like virus. NS1 peptides of seven strains possessed 217 amino acids, while that of the strain Ck/NM/ZH/02 coded 230 amino acids. Except for the amino acid at position 225, the additional amino acid sequence (13 AAs) of NS1 of Ck/NM/Zh/02 at the carboxy-terminus is identical with that of Ck/Kor/323/96. Phylogenetic analysis showed that seven of the tested strains belong to the A/duck/Hong Kong/Y280/97 (DK/HK/Y280/97)-like lineage, while the NS1 gene of Ck/NM/Zh/02 belongs to the Dk/HK/Y439/97-like lineage and has a close relationship with that of the Ck/Kor/323/96-like viruses. Therefore, although most of the H9N2 influenza viruses circulating on chicken farms in mainland China belong to the DK/HK/Y280/97-like lineage, the present results indicate that the other two of the three H9N2 lineage viruses also circulate in the chicken population in mainland China.

A highly sensitive immuno-PCR method specific for the detection of antigens of and antibodies to bovine herpesvirus 1 was established. This assay attained a high sensitivity of up to 10(7.0) times higher than that of enzyme-linked immunosorbent assay (ELISA) or PCR for antigen detection and 10(5.0) times higher than that of ELISA for antibody detection.

H2 influenza virus caused a pandemic in 1957 and has the possibility to cause outbreaks in the future. To assess the evolutionary characteristics of H2 influenza viruses isolated from migratory ducks that congregate in Hokkaido, Japan, on their flyway of migration from Siberia in 2001, we investigated the phylogenetic relationships among these viruses and avian and human viruses described previously. Phylogenetic analysis showed that the PB2 gene of Dk/Hokkaido/107/01 (H2N3) and the PA gene of Dk/Hokkaido/95/01 (H2N2) belonged to the American lineage of avian virus and that the other genes of the isolates belonged to the Eurasian lineage. These results indicate that the internal protein genes might be transmitted from American to Eurasian avian host. Thus, it is further confirmed that interregional transmission of influenza viruses occurred between the North American and Eurasian birds. The fact that reassortants could be generated in the migratory ducks between North American and Eurasian avian virus lineage further stresses the importance of global surveillance among the migratory ducks.