METHODS: This retrospective study involved consecutive hospitalized patients with non-structural protein 1 (NS1) antigen positivity during an outbreak (Jan to April 2014). Multiplex RT-PCR was performed directly on NS1 positive serum samples to detect and determine the DENV serotypes. All PCR-positive serum samples were inoculated onto C6/36 cells. Multiplex PCR was repeated on the supernatant of the first blind passage of the serum-infected cells. Random samples of supernatant from the first passage of C6/36 infected cells were subjected to whole genome sequencing. Clinical and laboratory variables were compared between patients with and without DENV co-infections.
RESULTS: Of the 290 NS1 positive serum samples, 280 were PCR positive for DENV. Medical notes of 262 patients were available for analysis. All 4 DENV serotypes were identified. Of the 262 patients, forty patients (15.3 %) had DENV co-infections: DENV-1/DENV-2(85 %), DENV-1/DENV-3 (12.5 %) and DENV-2/DENV-3 (2.5 %). Another 222 patients (84.7 %) were infected with single DENV serotype (mono-infection), with DENV- 1 (76.6 %) and DENV- 2 (19.8 %) predominating. Secondary dengue infections occurred in 31.3 % patients. Whole genome sequences of random samples representing DENV-1 and DENV-2 showed heterogeneity amongst the DENVs. Multivariate analysis revealed that pleural effusion and the presence of warning signs were significantly higher in the co-infected group, both in the overall and subgroup analysis. Diarrhoea was negatively associated with co-infection. Additionally, DENV-2 co-infected patients had higher frequency of patients with severe thrombocytopenia (platelet count < 50,000/mm(3)), whereas DENV-2 mono-infections presented more commonly with myalgia. Elevated creatinine levels were more frequent amongst the co-infected patients in univariate analysis. Haemoconcentration and haemorrhagic manifestations were not higher amongst the co-infected patients. Serotypes associated with severe dengue were: DENV-1 (n = 9), DENV-2 (n = 1), DENV-3 (n = 1) in mono-infected patients and DENV-1/DENV-2 (n = 5) and DENV-1/DENV-3 (n = 1) amongst the co-infected patients.
CONCLUSION: DENV co-infections are not uncommon in a hyperendemic region and co-infected patients are skewed towards more severe clinical manifestations compared to mono-infected patients.
OBJECTIVE: A new variant of the equine fsh (efsh) gene was cloned, sequenced, and expressed in Pichia pastoris (P. pastoris) GS115 yeast expression system.
MATERIALS AND METHODS: The full-length cDNAs of the efshα and efshβ chains were amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA isolated from an Iranian Turkmen-thoroughbred horse's anterior pituitary gland. The amplified efsh chains were cloned into the pPIC9 vector and transferred into P. pastoris. The secretion of recombined eFSH using P. pastoris expression system was confirmed by Western blotting and immunoprecipitation (IP) methods.
RESULTS: The DNA sequence of the efshβ chain accession number JX861871, predicted two putative differential nucleotide arrays, both of which are located in the 3'UTR. Western blotting showed a molecular mass of 13 and 18 kDa for eFSHα and eFSHβ subunits, respectively. The expression of desired protein was confirmed by protein G immunoprecipitation kit.
CONCLUSIONS: eFSH successfully expressed in P. pastoris. These findings lay a foundation to improve ovulation and embryo recovery rates as well as the efficiency of total embryo-transfer process in mares.