Construction of Mutation Library in Haemophilus parasuis by Inserting Tn5 Transposon and the Screening of Attenuated Strain

[Objective] The study aimed to obtain attenuated strain of Haemophilus parasuis.[Method] Tn5 transposon technology was used to construct a library of mutants.Positive mutants were screened by kanamycin resistance.False positive was identified by PCR and then removed.Mice were infected to detect the virulence of mutants.The bionomics of attenuated strains were detected,too.[Result] The attenuated mutants showed similar reproductive activity to that of wild strain.The virulence of mutants was still stable after 30 passages.[Conclusion] This study provided foundation for exploring the virulence factors and pathogenic mechanism of HPS.

An Attenuated Strain of Cucumber Green Mottle Mosaic Virus as a Biological Control Agent against Pathogenic Viral Strains

Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, causes a severe disease of cucurbits. In the Moscow region of Russian Federation, the incidence of infection on cucumber plants in greenhouses is high;however, the virus is poorly studied. In this work, the full-length genomes of two pathogenic MC-1 and MC-2 strains of CGMMV isolated from cucumber plants grown in greenhouses in the Moscow region and the attenuated VIROG-43M strain were sequenced. Comparison of VIROG-43M nucleotide sequence with those of the pathogenic strains revealed three missense mutations. Their role in attenuation is discussed. For the first time, in a number of trials conducted under laboratory conditions and in commercial greenhouses, the efficiency of the attenuated VIROG-43M strain as a biocontrol agent for cucumber plant protection resulting in significant yield gain was demonstrated. Phylogenetic analysis with 83 full-length CGMMV coat protein genes isolated in 16 different countries showed that Russian strains are related to isolates from Spain, Greece, USA and Israel.

Effect of Attenuated Highly Pathogenic Pig Reproductive and Respiratory Syndrome(HP-PRRS) TJM-F92 Strain Vaccine on Immune Antibody Levels against Classical Swine Fever(CSF) and Foot-and-Mouth Disease(FMD)

Effects of attenuated highly pathogenic pig reproductive and respiratory syndrome（HP-PRRS）TJM-F92 strain vaccine on immune antibody level against classical swine fever（CSF）and foot-and-mouth disease（FMD）were studied from October 8 to November 12 in 2014,in order to optimize vaccination program of CSF,HP-PRRS and FMD and to provide scientific guidance for animal disease control and prevention work.The results showed that attenuated HP-PRRS（TJMF92 strain）vaccine had no significant effect on immune antibody level of hog cholera lapinized virus（HCLV,ST passage cell vaccine）attenuated vaccine and FMD-O inactivated vaccines（OZK/93 strain）,and single or combined use of three vaccines received good immunization effects.

A retrospective investigation on the phenotype and stability of the E-protein gene in Japanese Encephalitis (JE) virus strain SA14-14-2 used live-attenuated JE vaccine

To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years （1987-2001） were unchanged, in- cluding the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines sion noted. prepared are quite stable without any reversion noted.

Cryo-EM structures of infectious bursal disease viruses with different virulences provide insights into their assembly and invasion

Infectious bursal disease virus(IBDV)causes a highly contagious immunosuppressive disease in chickens,resulting in significant economic losses.The very virulent IBDV strain(vvIBDV)causes high mortality and cannot adapt to cell culture.In contrast,attenuated strains of IBDV are nonpathogenic to chickens and can replicate in cell culture.Although the crystal structure of T=1 subviral particles(SVP)has been reported,the structures of intact IBDV virions with different virulences remain elusive.Here,we determined the cryo-electron microscopy(cryo-EM)structures of the vvIBDV Gx strain and its attenuated IBDV strain Gt at resolutions of 3.3 Å and 3.2 Å,respectively.Compared with the structure of T=1 SVP,IBDV contains several conserved structural elements unique to the T=13 virion.Notably,the Nterminus of VP2,which is disordered in the SVP,interacts with the S_(F) strand of VP2 from its neighboring trimer,completing theβ-sheet of the S domain.This interaction helps to form a contact network by tethering the adjacent VP2 trimers and contributes to the assembly and stability of the IBDV virion.Structural comparison of the Gx and Gt strains indicates that H253 and T284 in the VP2 P domain of Gt,in contrast to Gx,form a hydrogen bond with a positively charged surface.This suggests that the combined mutations Q253 H/A284 T and the associated structural electrostatic features of the attenuated Gt strain may contribute to adaptation to cell culture.Furthermore,a negatively charged groove in VP2,containing an integrin binding IDA motif that is critical for virus attachment,was speculated to play a functional role in the entry of IBDV.