Host Cytokine Responses of Pigeons Infected with Highly Pathogenic Thai Avian Influenza Viruses of Subtype H5N1 Isolated from Wild Birds

Highly pathogenic avian influenza virus (HPAIV) of the H5N1 subtype has been reported to infect pigeons asymptomatically or induce mild symptoms. However, host immune responses of pigeons inoculated with HPAIVs have not been well documented. To assess host responses of pigeons against HPAIV infection, we compared lethality, viral distribution and mRNA expression of immune related genes of pigeons infected with two HPAIVs (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) isolated from wild birds in Thailand. The survival experiment showed that 25% of pigeons died within 2 weeks after the inoculation of two HPAIVs or medium only, suggesting that these viruses did not cause lethal infection in pigeons. Pigeon04 replicated in the lungs more efficiently than T.sparrow05 and spread to multiple extrapulmonary organs such as the brain, spleen, liver, kidney and rectum on days 2, 5 and 9 post infection. No severe lesion was observed in the lungs infected with Pigeon04 as well as T.sparrow05 throughout the collection periods. Encephalitis was occasionally observed in Pigeon04- or T.sparrow05-infected brain, the severity, however was mostly mild. To analyze the expression of immune-related genes in the infected pigeons, we established a quantitative real-time PCR analysis for 14 genes of pigeons. On day 2 post infection, Pigeon04 induced mRNA expression of Mx1, PKR and OAS to a greater extent than T.sparrow05 in the lungs, however their expressions were not up-regulated concomitantly on day 5 post infection when the peak viral replication was observed. Expressions of TLR3, IFNα, IL6, IL8 and CCL5 in the lungs following infection with the two HPAIVs were low. In sum, Pigeon04 exhibited efficient replication in the lungs compared to T.sparrow05, but did not induce excessive host cytokine expressions. Our study has provided the first insight into host immune responses of pigeons against HPAIV infection

Studies on the pathogenicity of duck tembusu virus strain KPS54A61 using mice and chickens

In Thailand, Flavivirus strain TMUV-KPS54A61 was isolated from mosquitoes and ducks. The pathogenicity of TMUV-KPS54A61 was tested in mice and chickens. The TCID50 of TMUV-KPS54A61 was estimated and a dilute was applied to groups A-D of adult chickens (105, 104, 103, 102 TCID50) and BALB/c mice using intracerebral inoculation. Young chickens were inoculated with 107TCID50 of TMUVKPS54A61. Adult chickens did not exhibit the clinical signs, while organ samples tested negative by RT-PCR for the genome of TMUV. On the other hand, groups A and B of BALB/c mice and young chickens showed clinical signs including anorexia, hunched posture, fluffy hair, diarrhea and retarded growth. Pathological changes observed including perivascular cuffing, multiple clusters of gliosis in cerebral and cerebellar. Necrosis of the liver cells and interstitial nephritis in the kidney were also found in young chickens, while the spleen and pancreas are unclear the pathological changes. Immunohistochemical staining of mouse spinal cord samples was positive for the virus protein. TMUV - KPS54A61 was detected in the serum, brain, liver, kidney, adrenal gland, pancreas and spinal cord by RT-PCR. Vero cells exhibited CPE after inoculation by the virus, which was isolated from the brain, spinal cord and kidneys. TMUV-KPS54A61 could maintain itself for a prolonged time in the brain, spinal cord and liver; therefore, it could be the target organs of virus and the TMUV - KPS54A61 could be pathogenic in young chickens and BALB/c mice

Haemosporidian Parasites of White-Breasted Waterhens (<i>Amaurornis phoenicurus</i>), with a Report and Molecular Characterization of <i>Haemoproteus gallinulae</i> in Thailand

Haemosporidian parasites are vector-borne parasites infecting terrestrial vertebrates as well as avian species, such as the White-breasted Waterhen, a Gruiformes waterbird found in lowlands near wetlands and distributed throughout Thailand. However, information regarding haemosporidia infection in this species is lacking. To establish regional information, 17 blood samples were collected from White-breasted Waterhens. Four haemoparasite lineages were identified in six blood samples: Haemoproteus gallinulae, Plasmodium collidatum, Plasmodium elongatum, and an unidentified Plasmodium species. H. gallinulae was characterized with morphological features in White-breasted Waterhens for the first time; the morphological characteristics were consistent with previous descriptions. H. gallinulae was more closely related to Haemoproteus species of Passeriformes birds than to those of Gruiformes birds. The Plasmodium parasites infecting these White-breasted Waterhens previously caused severe avian malaria in other host species. The unidentified Plasmodium species had rarely been documented, although it was reported in the Culex vector and was possibly associated with specialist parasites either as host or habitat. Our findings reveal multiple haemosporidian species reflecting the role of this avian host as a carrier of haemosporidians. This study offers species records and molecular materials that may provide critical information for further targeted research into these haemosporidia

Alleviation of soil acidification and modification of soil bacterial community by biochar derived from water hyacinth Eichhornia crassipes

Abstract The highly acid sulfate Rangsit soil series of Rangsit, Pathum-Thani district, Thailand poses a major problem for agriculture in the area. Water hyacinth is a naturally occurring weed that can grow aggressively, causing eutrophication and leading to many severe environmental impacts. Here, through the pyrolysis process, we convert water hyacinth to biochar and use it for acid soil amendment. We found the ratio between biochar, soil, and sand suitable for the cultivation of water convolvulus to be 50 g of biochar, 400 g of soil, and 100 g of sand (1:8:2). This soil mixture improved the pH of the soil from 4.73 to 7.57. The plant height of the water convolvulus grown in the soil mixture was the greatest at 20.45 cm and the plant weight with and without roots was greatest at 2.23 g and 2.52 g, respectively. Moreover, we demonstrated the dominance and high abundance of Bacillus among the community in soil with biochar amendment. Here we provide the first assessment of the appropriate amount of water hyacinth-derived biochar for mitigation of soil acidity and promotion of optimal water convolvulus growth. Moreover, biochar can optimally modify soil bacterial communities that benefit plant development

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response.

Influenza neuraminidase (NA) proteins expressed in TK- cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity. Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses

Generation of a pig induced pluripotent stem cell (piPSC) line from embryonic fibroblasts by incorporating LIN28 to the four transcriptional factor-mediated reprogramming: VSMUi001-D

Pig induced pluripotent stem cell (piPSC) line was generated from embryonic fibroblast cells using retroviral transduction approaches carrying human transcriptional factors: OCT4, SOX2, KLF4, c-MYC and LIN28. The generated piPSC line, VSMUi001-D, was positive for alkaline phosphatase activity and expressed the pluripotency associated transcription factors including OCT4, SOX2, NANOG and surface markers SSEA-1, all iPSC hallmarks of authenticity. Furthermore, VSMUi001-D exhibited a normal karyotype and formed embryoid bodies in vitro and teratomas in vivo. Upon cardiac differentiation, VSMUi001-D displayed spontaneous beating and expressed cardiomyocyte markers, like cardiac Troponin T

Inhibitory effect of anti-NA antisera against 3 virus concentrations (100, 25, and 10 TCID50) as determined by virus replication inhibition assay.

<p>Anti-H5N1 NA antisera modestly inhibit replication of reverse genetic (rg) virus carrying homologous NA and show moderate level of antibody titers that cross inhibit the H1N1pdm virus. On the other hand, anti-H1N1 NA antisera well inhibit replication of the H1N1pdm virus, but not cross inhibit rgH5N1 virus. Both kinds of anti-NA antisera do not inhibit rgPR8 virus and H3N2 virus. No significant difference in anti-NA antibody titers is found between pooled mouse sera collected at 4 weeks and 6 weeks p.i. (Mann-Whitney U test, <i>p</i> > 0.05). The geometric mean antibody titers are calculated. The error bars indicate standard deviations of 2 independent experiments for each lot of the test sera.</p

Cellular localization of NA proteins.

<p>The NA protein expressed in TK⁻ cells infected with rVac-H5N1 NA or rVac-H1N1 NA are present in cytoplasm, and in particular, on the cytoplasmic membrane as demonstrated by IFA confocal microscopy. TK⁻ cells infected with rVac-pSC11 as the virus control did not have a fluorescent signal.</p

Glycosylation of NA proteins.

<p>MWs of the cleavage products of NA protein after treatment with Peptide <i>N</i>-Glycosidase F (PNGase F) in comparison with the size of the untreated NA as demonstrated by WB assay. (A) H1N1pdm NA; (B) H5N1 NA.</p