PCR Detecting Types For Infectious Hypodermal And Hematopoietic Necrosis Virus (IHHNV) And Pathogenicity Analysis

Infectious hypodermal and hematopoietic necrosis virus is one of themajor epidemic viruses infecting penaeid shrimp. The virus was classifiedinto the subfamily Densoirinae, genus Brevidensovirus, as Penaeusstylirostris densovirus (PstDNV).Its host spread all over the worldcontaining wild and farmed penaeid shrimp.For Litopenaeus stylirostris,themortality is up to90%.And furthermore, this virus also causes chronicinfections called runt deformity syndrome (RDS) in P. monodon,Litopenaeus vannamei and Marsupenaeus japonicas,affecting the growthof penaeid shrimp.The infected shrimp can infect others or descendantthrough horizontal or vertical way.IHHNV is the smallest penaeid shrimpvirus with20to22nm sizes and non-enveloped icosahedral virions andpossesses approximately4.0kb of linear ssDNA.The genome containspartially overlapping open reading frames(ORFs),encoding non-structuralprotein1,non-structural2and capsid protein，respectively.Genebank hasincluded different geographical origins,including AF218266,AY362548,AF273215,EF633688,AY355306,AY355307,Y355308,AY3625 47and so on.Currently, there found two genotypes of IHHNV: infection (type1andtype2) and non-infection (type3A and type3B).And the second one is thatIHHNV gene segment is inserted into penaeid shrimp genome. OIErecommended by5primer (389F/R,392F/R,77012F/77353R,309F/R�'�MG831F/R) to be used to detect different genotypes of IHHNV.Foreign scholars have turned to study IHHNV earlier thanChina. Some studies of IHHNV reported more limited in China.In recentyears, we still don’t know the injury of IHHNV in farmed shrimp.What weknow about its harmfulness is confined to part of the information whendesease outbreaks.Therefore, some samples collected from different areasand time are dectected through usual PCR in order to know prevalencestatus of IHHNV in farmed shrimp in China. The post larvae of L.vannamei are fed with diseased shrimp tissues in order to acquire data ofIHHNV pathogenicity.The research contents include：1. Preliminary screening of different genetic types of domestic IHHNVAccording to the National Standards, four IHHNV primers (389F/R、392F/R、77012F/77353R、309F/R) recommended by OIE had been used forIHHNV testing of prawns collected in different locations in China duringMay2011and July2012. Four genetic types had been obtained through PCR. IHHNV has been detected in litopenaeus vannamei, penaeusmonodon, fenneropenaeus chinensis and furrowed prawn, but not inMorphological or white shrimp. Litopenaeus vannamei shows the highestpositive rate, while fenneropenaeus chinensis has the lowest positive rate.Compared with prawn sample from2012, the sample from2011shows ahigher positive rate. And the positive rate of sample from Eastern China ishigher than that of sample from Northern and Southern China. Besides, partof the collected plankton and crabs were also detected andthere was nopositive resultsdetected. This research provides scientific explanation forthe popularity of IHHNV in China as well as the basic information forfurther research of IHHNV genotype.2. Whole genome amplification of IHHNVWe selected eight shrimp samples which were detected strong positiveresult by4sets of primer pairs. The primer pairs designed in conservedsequences researching on IHHNV complete genome sequence (Genebank:AF218266) were used to amplify IHHNV conserved sequence. Throughconstructing phylogeny tree, we found that Penaeus monodon collected inShanghai showed more diversity in eight detected samples. Using cRACEmethod to amplify the flanking sequences of the gene, we got a3797bpsequence of IHHNV genome. The gene fragment had highly similarity withHawaii strain. This result provides scientific basis for pathogenicity of IHHNV infecting shrimps and molecular epidemic evaluation.3. Analysis of pathogenicity of IHHNVPosition feed containing infection-type IHHNV has been diluted intogradient concentration. Then the feed was fed to healthy prawns. Thedeaths of prawns were recorded. The results show that collective death wasfound in the group fed on original position feed as well as the group fed on10-time diluted feed. Only one prawn survived in each group. Within othergroups fed on100-time,1000-time or10000-time diluted feed, one groupfed on normal feed and one was blank control group, and the resultsshowed that the prawns die slowly. But no obvious difference was observed.Meanwhile, real-time fluorescent quantitative PCR was used to detect themultiplication of IHHNV in prawn body, in order to test the pathogenicityof this virus.