| Japanese encephalitis (JE) is a zoonotic infectious disease which is prevalent in Asia andPacific areas widely and it affects human health and causes a fundamental harmful effect onanimal husbandry, especially livestock industry. Its pathogen, Japanese encephalitis virus(JEV), is a typical arbovirus and mosquitoes play an important role in transmission this virus.It was reported that there are more than60kinds of animals can be infected with JEV innature, most of them have no obvious clinical symptoms after infection, but high level ofJapanese encephalitis virus neutralizing antibodies can be detected in serum.1. E.coli-expressed domain III of Japanese encephalitis virus envelope protein、preparationand identification of rabbit anti-JEV E protein polyclonal antibodyOne of its structure protein, envelope protein is mainly consisted of three domains, thedomain III(312~411amino acids) which is the important region for induction of neutralizingantibodies fold on the surface of virus independently and form immuoglobulin-like pattern. Inthis experiment, we expressed prokaryoticly the pET-32a-EIII protein, immuned New Zealandwhite rabbit after purification, and prepared the rabbit anti-E protein of JEV polyclonalantibody. Its titer is1:256000in enzyme-linked immunosorbent assay(ELISA). Afteridentification, this antibody can react with recombinant protein obviously throughWestern-blot, and revealed a single band with molecular weight of53KD approximately（Eprotein) when reaction with concentrated JEV. In indirect immune fluorescence assay(IFA)，this polyclonal antibody reactive with JEV in BHK-21cells, and reveal green fluorescence incytoplasm, the control groups have no special fluorescence. The PRNT50(plaque reductionneutralization test) of this polyclonal antibody is1:160. Taken together, this antibody canreact with recombinant protein and JEV envelope glycoprotein very well and it has a certainamount of neutralizing antibody against JEV. 2. Dynamics of the neutralizing antibodies against JEV infected companion dogsIt was reported that it has high sero-positive and high level of neutralizing antibodiesagainst JEV among dogs after infected with virus in foreign. Companion dogs are close tohuman and there are more and more individuals, especially unban resident, chose to breedcompanion dogs in recent years, so companion dogs as sentinel animal to monitor the riskpeople infected with JEV has important values. A total of253sera were collected monthlyfrom companion dogs with different ages during May2011to Apr.2012and detected JEVneutralizing antibodies. The overall positive rate of JEV neutralizing antibodies in companiondogs was28.1%, with significant seasonal variation. The positive rate for the months (Marchto May) before and for the months (October to December) after JE epidemic season (June toSeptember) was16.3%and43.4%, respectively, showing a statistically significant differencebetween the two groups (χ~2=14.432, p<0.01). The positive rate for dogs younger and olderthan one year old was24.1%and36.1%, respectively, a statistically significant difference hasbeen detected between the two groups (χ~2=3.952, p<0.05). The positive rate for the male andfemale companion dogs was32.5%and24.6%, respectively, no statistical significance hasbeen found (χ~2=1.860, p>0.05). The results showed that companion dogs were infected withJEV seriously, especially in the JE epidemic season, and companion dogs can serve as asentinel animal to assess the risk of human infection by JEV.After that, two beagles were intraperitoneally and subcutaneously inoculated with5×10~6and10~7plaque-forming units (PFU) of JEV(SH-JEV01) respectively, and no clinicalsigns or increase in body temperature were observed during the observation period. Wecollected the dogs’ blood, separated serum and detected JEV neutralizing antibody titer. It canbe detected in the4thday and reach to high level in the30thday after challenge. |
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