Substrate specificity of avian influenza H5N1 neuraminidase

AIM: To characterise neuraminidase(NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus.METHODS: Avian influenza H5N1 strains from humans and birds were recruited for characterising their NA substrate specificity by using a modified commercial fluorescence Amplex Red assay. This method can identify the preference of α2,6-linked sialic acid or α2,3-linked sialic acid. Moreover, to avoid the bias of input virus, reverse genetic virus using NA gene from human isolated H5N1 were generated and used to compare with the seasonal influenza virus. Lastly, the substrate specificity profile was further confirmed by high-performance liquid chromatography(HPLC) analysis of the enzymatic product. RESULTS: The H5N1 NA showed higher activity on α2,3-linked sialic acid than α2,6-linked(P 0.0001). To compare the NA activity between the H5N1 and seasonal influenza viruses, reverse genetic viruses carrying the NA of H5N1 viruses and NA from a seasonal H3N2 virus was generated. In these reverse genetic viruses, the NA activity of the H5N1 showed markedly higher activity against α2,3-linked sialic acid than that of the H3N2 virus, whereas the activities on α2,6-linkage were comparable. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin(HA) with dual specificity showed reduced activity on α2,3-linkage. To confirm the substrate specificity profile, HPLC analytic of enzymatic product was performed. Similar to Amplex red assay, H5N1 virus showed abundant preference on α2,3-linked sialic acid.CONCLUSION: H5N1 virus maintains the avian specific NA and NA changes may be needed to accompany changes in HA receptor preference for the viral adaptation to humans.     

Latex sensitization and risk factors in female nurses in Thai governmental hospitals

Objectives

Exposure to latex allergens in latex gloves can cause occupational health problems in nurses, yet latex gloves are still widely used in Thai hospitals. Therefore, we conducted a study to determine the prevalence of latex sensitization in nurses and identify risk factors associated with sensitization.

Materials and Methods

A questionnaire, providing information on personal characteristics, ill-health, working conditions and symptoms related to latex product use, was administered to 363 female nurses working in two tertiary hospitals in southern Thailand. Latex sensitization was confirmed using a solid phase immunoassay to detect anti-latex IgE antibodies. Total glove protein levels were determined by using a modified Lowry method and latex aeroallergens by a competitive inhibition immunoassay.

Results

The overall prevalence of latex sensitization was 4.4%. Respiratory symptoms related to latex glove use were significantly associated with latex sensitization (OR = 5.5, 95% CI: 1.57–19). Total glove protein levels ranged 87.8–250.8 μg protein/dm². The prevalence of latex sensitization was higher (6.6% vs. 2.2%) in the hospital where gloves with higher protein levels (82–438 μg/g vs. 86–170 μg/g) were used. Furthermore, latex sensitization prevalence increased from 3% to 5% with increased average departmental aeroallergen concentrations.

Conclusions

Latex sensitization prevalence in Thai nurses was higher than previously reported. Respiratory exposure seems to play an important role, in addition to dermal exposure. If latex gloves cannot be replaced by non-latex alternatives, replacement with gloves with lower protein content should be considered.     

Inhibition of H5N1 highly pathogenic influenza virus by suppressing a specific sialyltransferase

Avian influenza viruses preferentially use α2,3-linked sialic acid as a receptor for binding and entry into target cells. The sialic acid is the terminal residue of various types of glycan. There are two major types of α2,3-linked sialic acid differing in the penultimate bond: Neu5Acα2-3Galβ1-3GalNAc and Neu5Acα2-3Galβ1-4GlcNAc. In the human airway, while Neu5Acα2-3Galβ1-3GalNAc is present only in alveolar epithelial cells, the Neu5Acα2-3Galβ1-4GlcNAc is expressed in both the upper and lower airway. Previous data showed preferential binding of hemagglutinin from H5N1 highly pathogenic influenza virus to Neu5Acα2-3Galβ1-4GlcNAc. We further show here that suppression of this sialic acid by siRNA against a sialyltransferase, ST3GAL4, can inhibit H5N1 avian influenza virus infection and that this gene is abundantly expressed in human pharynx, trachea and bronchus. These data suggest that the ST3GAL4 gene is responsible for biosynthesis of the viral receptor and may play a crucial role in infection of H5N1 avian influenza virus in humans.     

A serine-to-asparagine mutation at position 314 of H5N1 avian influenza virus NP is a temperature-sensitive mutation that interferes with nuclear localization of NP

We have generated a temperature-sensitive (ts) mutant from a human isolate of the H5N1 avian influenza virus by classical adaptation in cell culture. After 20 passages at low temperature, the virus showed a ts phenotype. The ts mutant also showed an attenuated phenotype after nasal inoculation in mice. Using reverse genetics, we generated reassortants carrying individual genomic segments of the wild-type and mutant viruses in an A/Puerto Rico/8/34 background, and found that the nucleoprotein (NP) gene could confer the ts phenotype. This mutant NP contains a serine-to-asparagine mutation at position 314 (S314N). The mutant NP protein showed a defect in nuclear localization at high temperature in mammalian cells.     

Case report: Increased viral receptor expression associated with high viral load and severe pneumonia in a young patient infected with 2009 H1N1 influenza a with no pre-existing conditions

A case of unusually high severity of influenza pneumonia leading to acute respiratory distress syndrome and death was investigated. This was a previously a healthy 28-year-old man with no underlying conditions, admitted to a hospital during the first wave of influenza pandemic in Thailand in July 2009. He had experienced high fever and influenza-like illness for 5 days before coming to the hospital. He developed acute respiratory distress syndrome and expired on day 7 after admission. In comparison to three other cases of influenza pneumonia in the same outbreak with known risk factors for severe influenza, such as pregnancy and diabetes mellitus, a much higher viral load was detected in the lungs of this patient despite antiviral treatment. In agreement with the high viral load, the lung specimens from this patient, but not the other three patients, showed a high expression of α-2,6-linked sialic acid by lectin staining. The gene responsible for the synthesis of this sialic acid was also found to be upregulated. The data indicated overexpression of the viral receptor as a potential mechanism for severe disease in some patients.