Ching-Ho Wang

Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

ABSTRACT PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.

Galectin-1 Binds to Influenza Virus and Ameliorates Influenza Virus Pathogenesis

ABSTRACT Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant (Kd ) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.

S1 and N Gene Analysis of Avian Infectious Bronchitis Viruses in Taiwan

Abstract The disease caused by infectious bronchitis virus (IBV) produces great economic loss for the poultry industry. The purpose of this study is to investigate the molecular epidemiology of IBV in Taiwan. An old IBV strain isolated in 1964 and another 31 strains isolated from 1991 to 2003 were selected for N-terminal S1 gene analysis. Based on their phylogenetic tree, 13 strains were selected for sequencing the entire S1 and partial nucleocapsid (N) genes. The results indicated that Taiwanese IBV strains could be divided into two distinct lineages, Taiwan Group I and Taiwan Group II, with one Massachusetts strain and one Chinese strain. No recombination was found between H120 and the Taiwanese strains in the S1 gene. However, the S1 gene showed a noticeably higher divergence than the N gene. The phylogenetic trees constructed from the S1 and N genes indicate that intergenic recombination has occurred. Since most local strains are in Taiwanese clusters, developing vaccines from local strains is necessary for IBV control in Taiwan.

Rapid and specific influenza virus detection by functionalized magnetic nanoparticles and mass spectrometry

BackgroundThe timely and accurate diagnosis of specific influenza virus strains is crucial to effective prophylaxis, vaccine preparation and early antiviral therapy. The detection of influenza A viruses is mainly accomplished using polymerase chain reaction (PCR) techniques or antibody-based assays. In conjugation with the immunoassay utilizing monoclonal antibody, mass spectrometry is an alternative to identify proteins derived from a target influenza virus. Taking advantage of the large surface area-to-volume ratio, antibody-conjugated magnetic nanoparticles can act as an effective probe to extract influenza virus for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and on-bead mass spectrometric analysis.ResultsIron oxide magnetic nanoparticles (MNP) were functionalized with H5N2 viral antibodies targeting the hemagglutinin protein and capped with methoxy-terminated ethylene glycol to suppress nonspecific binding. The antibody-conjugated MNPs possessed a high specificity to H5N2 virus without cross-reactivity with recombinant H5N1 viruses. The unambiguous identification of the captured hemagglutinin on magnetic nanoparticles was realized by SDS-PAGE visualization and peptide sequence identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS).ConclusionsThe assay combining efficient magnetic separation and MALDI-MS readout offers a rapid and sensitive method for virus screening. Direct on-MNP detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provided high sensitivity (~103 EID50 per mL) and a timely diagnosis within one hour. The magnetic nanoparticles encapsulated with monoclonal antibodies could be used as a specific probe to distinguish different subtypes of influenza.

Development of attenuated vaccines from Taiwanese infectious bronchitis virus strains

Abstract Due to variations in serotypes among different strains of avian infectious bronchitis viruses (IBV), vaccination of chicks with imported vaccines fails to protect them from IBV infections in Taiwan. Therefore, we develop attenuated vaccines from local strains in Taiwan. A Taiwan Group I (TW I) strain was passaged 74 times through specific pathogen-free (SPF) chicken embryonated eggs, and then tested in SPF chickens. The attenuated vaccine was not pathogenic in 1-week-old chicks, had a neutralization index (NI) of greater than 4.4 and efficacy of 90% when inoculated birds were challenged with a field IBV strain. Similar results were obtained for a vaccine made from a Taiwan Group II IBV strain. Additionally, the TW I attenuated vaccine strain had no reversion to virulence after five back passages in chicks. In conclusion, these attenuated vaccines have potential for controlling local Taiwanese IBV infections in chickens.

Genetic diversity and correlation with feline infectious peritonitis of feline coronavirus type I and II: A 5-year study in Taiwan

Abstract The outcomes of feline coronavirus (FCoV) infection vary greatly from asymptomatic or mild enteric infection to fatal feline infectious peritonitis (FIP). On the basis of in vitro neutralization tests, FCoVs can be divided into two serotypes. To explore the correlation between different types of FCoV and FIP, clinical specimens collected from 363 naturally infected cats during 2003–2007 were analyzed. Amplification of a portion of the S gene from the FCoV was performed and a total of 222 cases were differentiated. Among them, 197 (88.7%) cats were type I-positive, 13 (5.9%) were type II-positive, and 12 (5.4%) were positive for both types. Irrespective of the predominance of type I FCoV infection in Taiwan, type II FCoV demonstrated a significantly higher correlation with FIP (p <0.01). Analysis of partial S gene sequences of the local type I and II FCoVs strains revealed that type I viruses were more genetically divergent (6.2–11.7%) than type II viruses (0.6–3.2%) within the 5-year study period. The higher genetic diversity of type I FCoVs might be due to the larger infected cat population and to the long period of viral persistence in asymptomatic cats in comparison to type II viruses.

Identification of Taiwan and China-like recombinant avian infectious bronchitis viruses in Taiwan

Abstract Infectious bronchitis virus (IBV) infections in poultry cause great economic losses to the poultry industry worldwide. The emergence of viral variants complicates disease control. The IBV strains in Taiwan were clustered into two groups, Taiwan group I and Taiwan group II, based on the S1 gene. A variant was previously identified and showed a distinct S1 gene homology with other local strains. This study investigated the 3′ 7.3kb genome of eight Taiwan strains isolated from 1992 to 2007. The genes of interest were directly sequenced. Sequence analyses were performed to detect any recombination event among IBVs. The results demonstrated that all of the examined viruses maintained the typical IBV genome organization as 5′-S-3a-3b-E-M-5a-5b-N-UTR-3′. In the phylogenetic analyses, various genes from one strain were clustered into separate groups. Moreover, frequent recombination events were identified in the Simplot analyses among the Taiwan and China CK/CH/LDL/97I-type strains. Putative crossover sites were located in the S1, S2, 3b, M genes and the intergenic region between the M and 5a genes. All of the recombinants showed chimeric IBV genome arrangements originated from Taiwan and China-like parental strains. Field IBVs in Taiwan undergo genetic recombination and evolution.

Isolation and characterization of potentially pathogenic H5N2 influenza virus from a chicken in Taiwan in 2008.

Abstract During the surveillance of avian influenza, an H5N2 influenza A virus was isolated from a cloacal swab sample of an apparently healthy chicken in Taiwan in October 2008. It was found that the HA of the virus had a pair of dibasic amino acid residues at the cleavage site, which might be a marker of highly pathogenic avian influenza virus. However, the intravenous pathogenicity index of the isolate was 0.89, indicating that the virus was approaching high pathogenicity in chickens. Virus isolation was negative in 2916 birds from 146 farms in a 3-km radius around the farm where the virus was isolated. Genetic analysis of the eight segments of the isolate indicated that the isolated virus was a reassortant whose HA and NA gene segments belonged to the American lineage and internal genes to the Eurasian lineage.

Experimental Selection of Virus Derivatives with Variations in Virulence from a Single Low-Pathogenicity H6N1 Avian Influenza Virus Field Isolate

Abstract SUMMARY. A mixture of viruses with variations in virulence is likely present within a low-pathogenicity avian influenza virus (LPAIV) population. An H6N1 AIV was isolated from a field case showing 3.8% weekly mortality and a 33% egg production drop in Taiwan. The pathologic lesions included proventricular hemorrhage and urate deposition in the kidneys and on visceral organs. From the field isolate, a clone (2838N) that caused no lesions or death was obtained using limit dilution in chicken embryos and a clone (2838V) that caused renal lesions and death was obtained using contact infection in chicks. Both clones were inoculated intranasally in 4-week-old specific pathogen free (SPF) chickens to test their virulence. Renal urate deposition was found in chickens inoculated with 2838V but not in chickens inoculated with 2838N. In situ hybridization, polymerase chain reaction, and virus isolation were used to confirm the spread of 2838V from the respiratory tract to the renal tissue. We found that contact infection in chickens is a good method to obtain a more virulent clone from a heterogeneous virus population.

Evolution of infectious bronchitis virus in Taiwan: Characterisation of RNA recombination in the nucleocapsid gene

Abstract Avian infectious bronchitis virus (IBV) belongs to the Coronaviridae family and causes significant economic loss in Taiwan (TW), even in flocks that have been extensively immunised with Massachusetts (Mass)-serotype vaccines. Phylogenetic analysis of all non-structural and most structural genes shows that TW IBV is genetically distinct from the US strain and more similar to Chinese (CH) IBV. In contrast, the nucleocapsid (N) gene of TW IBV presents phylogenetic incongruence. RNA recombination at the 5′ end of the N gene between TW and US IBV is shown to be responsible for this discordance. Surprisingly, the recombinant N gene is found in all of tested TW IBV isolates, suggesting that a recombination event gave origin to a founder lineage. Our data indicate that RNA recombination in the recombinant 5′ end of the N gene may have caused the emergence of the current IBV population in Taiwan.