Disease replication kinetics were measured by infecting confluent BHK-21 and DEF cells in six-well plates at a multiplicity of illness (MOI) of 0

Disease replication kinetics were measured by infecting confluent BHK-21 and DEF cells in six-well plates at a multiplicity of illness (MOI) of 0.01. the positively charged fundamental amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the effectiveness of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K Rabbit Polyclonal to CFLAR mutation improved viral neutralization level of sensitivity to the early immune sera. Collectively, our findings provide the 1st evidence that a fundamental amino acid substitution at E367 strongly effects the and illness FLT3-IN-1 of TMUV. IMPORTANCE Outbreaks of Tembusu disease (TMUV) illness have caused huge economic deficits in the production of home waterfowl since the disease was first identified in China in 2010 2010. To control TMUV illness, a live-attenuated vaccine candidate of TMUV was developed in our earlier study, but the mechanisms of virulence attenuation are not fully recognized. Here, we found that the Thr-to-Lys substitution at E367 is definitely a crucial determinant of TMUV virulence attenuation in ducks. We shown the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide fresh insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines. (1), which was originally isolated from mosquitoes in Malaysia in 1955 (2). The 1st recognized disease caused by TMUV was reported in chicken flocks in Perak State, Malaysia, in 2000 and was characterized by encephalitis and retarded growth with hyperglycemia (3). In 2010 2010, a panzootic outbreak of TMUV illness occurred in home duck and goose flocks throughout the waterfowl-producing regions of China, leading to huge economic deficits for the poultry market (4,C6). Recently, this disease was also observed in duck flocks in Southeastern Asian countries (7, 8). Currently, TMUV illness seems to be probably one of the most important flavivirus diseases of poultry, influencing almost all varieties of home ducks and geese (9,C14), with sporadic instances in chickens (11, 15, 16). In laying flocks, TMUV offers been shown to elicit higher pathogenic effects and long-term illness in the ovaries of laying ducks, as characterized by ovarian hemorrhage, ovaritis, and follicle atresia with severe egg drop (4,C6, 17). While the medical indications and mortality of young parrots are age dependent, younger parrots are more susceptible to fatal illness and severe lesions in the brain, heart, and spleen cells and thus display a higher mortality rate (18, 19). Despite the unique medical syndromes observed in ducks infected with TMUV, the genetic basis for the pathogenesis of the disease in ducks is definitely poorly recognized. Furthermore, a remarkable characteristic of TMUV is that the disease can be efficiently transmitted via FLT3-IN-1 the aerosol route and direct contact, in addition to mosquito-borne transmission (20). Therefore, outbreaks of TMUV illness can occur throughout the year, and disease can spread rapidly among duck flocks (7). This is quite different from additional vector-borne flavivirus infections. TMUV consists of a positive-sense single-stranded RNA genome of approximately 10.9?kb in length encoding a single large precursor polyprotein. The polyprotein is definitely predicted to be FLT3-IN-1 proteolytically processed into three structural proteins (capsid [C], precursor membrane [prM], and envelope glycoprotein [E]), and seven nonstructural (NS) proteins, much like those of additional flaviviruses (1, 21). The E protein of flavivirus, the major surface protein of the viral particle, takes on an important part in host-specific adaptation, cell tropism, disease attachment, and membrane fusion with target cells (22,C25) and is the dominating target of neutralizing antibodies (26). It has been reported that a mutation(s) in E protein could modulate viral antigenicity, stability, and pathogenesis (27,C31). For example, three residues in E protein website III (EDIII) of yellow fever disease (YFV) 17D enhanced binding to glycosaminoglycans (GAGs), inhibited disease spread in extraneural cells, and reduced virulence in mice (24). In addition, the Glu-to-Lys mutation at residue 138 of the E protein of Japanese encephalitis disease (JEV) enhanced susceptibility to heparin inhibition and strongly attenuated viral neurovirulence and neuroinvasiveness in IFNRC/C mice (28, 32). Studies.