Full Length Genome Sequencing And Immunogenicity Of A GE/GI Deleted Virus From A Highly Virulent Pseudorabies Virus Strain From China

Pseudorabies (PR), caused by pseudorabies virus (PRV), is an economically important infectious disease. It causes fatal encephalitis in newborn pigs, respiratory disorders in growing-fattening pigs, and reproductive failure in sows. By using the marker vaccines that lack of the nonessential gE and an accompanying differentiating infected from vaccinated animals (DVID) serologic test, the United States and some European countries had eradicated PR from domestic pig populations. In China, anti-viral vaccination has contributed effectively to its control during the past 30 years. However, at the end of 2011, a severe PRV outbreak took place in many PRV vaccinated pigs farms and spread quickly in the pig herds in China and caused great economic losses to the swine industry, which being characterized by fatal encephalitis in newborn pigs and reproductive failure in sows. In addition, grower-finisher pigs as well as newborn pigs were also infected and died during the present epidemic period, which suggests that current vaccine strains provide poor protection against the novel epidemic strains.In this study, we isolated 5 wild-type PRV isolates from an immunized pig farm in southern-eastern of China. One of them, ZJ01 strain, was selected and the pathogenesis and antigenicity were characterized. And the genome sequence of ZJ01 was determined by Ion-Torrent sequencing. Meanwhile, a BAC infectious clone of a novel PRV strain ZJ01 was constructed. And an inactivated gE/gI deleted PRV vaccine was prepared. And it was found that it could provide protection against ZJ01 fatal challenge in swine. These findings facilitate us to mine the molecular mechanisms underlying their highly virulent and antigenic variants of those newly PRV isolates and develop a new vaccine to control this disease in the future. The contents of this study contain five parts as following:1. Isolation and identification a highly virulent pseudorabies virus from ChinaPseudorabies virus (PRV) is an important agent that threatens the swine industry all over the world. Pseudorabies has been controlled efficiently in China for many years by vaccination. However, it suddenly broke out in many pig herds characterised by abortion in pregnant sows and severe neurological disorders in piglets at the end of 2011. In this study, a systematic investigation, ranging from virus isolation, genetic and pathology to immunogenicity analysis, was carried out with the aim of understanding pathogenesis features of the novel PRV isolates. Twenty-nine of 38 tissue samples from 13 farms in 4 provinces in diagnosed as wild-type PRV infection by PCR in East-southern of China in 2012 to 2013. Five positive tissue samples were collected and homogenized with Dulbecco’s modified Eagles medium (DMEM). The supernatant was inoculated to BHK-21 cells and incubated for 3 days for CPE. The viruses were collected and plaque purified for three cycles, and confirmed by PCR and electron microscope observation. The sequence analysis of the gE and gC genes of 5 isolates from 4 different provinces showed that all isolates belonged to a relatively independent cluster and contained 2 and 7 amino acid insertions in gE and gC genes, respectively. Mice and rabbits were challenged with PRV ZJ01. The results showed that mice and rabbits were exhibited central nervous signs and died. PRV isolate ZJ01 also cause 100% morbidity and 100% mortality both in 14 and 80-days old pigs. Next, fifteen 10-week-old specific-pathogen-free piglets were randomly assigned to three groups of five piglets each and housed separately. Pigs in Group 1 and Group 2 were challenged intranasally with 1 mL 1070TCID50/mL ZJO1 and LA strain, respectively. Pathological analysis showed that ZJO1 had 100% mortality, while traditional virulent PRV LA strain resulted in no deaths in experimental infection of 10-week-old specific-pathogen-free piglets. Combined with gross lesion examination, histopathological observations and quantitative real-time polymerase chain reaction, ZJO1 exhibited more severe pathogenicity in adult pigs compared with LA. In conclusion, these findings indicated that ZJO1 could define as a novel highly virulent PRV isolate.2. Identify the antigenic characteristics of a novel highly-virulent PRV ZJO1 strainTo further indentify the antigenic characteristics, we designed a cross serum-virus neutralization assay, the results showed that the serum antibodies to the commercial PRV vaccines (Bartha-K61, Bucharest, and HB-98) had significantly lower neutralizing activity to ZJO1 isolate, comparing with those to the original vaccine strains. But the neutralizing titers of anti-ZJO1 serum to ZJ01 were similar to those to the vaccine strains. The antigenic relatedness R between ZJO1 and vaccine strains was 0.378-0.455. Moreover, in the vaccination test, high levels of NT antibodies against LA were determined in the pigs that received Bartha-K61 vaccine before ZJO1 challenge. However, the inactivated vaccinated pigs developed significantly higher ZJO1-specific NT responses (p<0.05) than pigs vaccinated with Bartha-K61 vaccine after immunization. After ZJO1 challenge, administration of the inactivated vaccine rather than Bartha-K61 provided complete protection against lethal ZJO1 challenge by evaluating the clinical signs, gross lesion examination, histopathological observations and quantitative real-time polymerase chain reaction. These findings indicated that ZJO1 is a highly pathogenic PRV isolate with antigenic variants.3. Full length genome sequencing of a highly virulent pseudorabies virus strain ZJO1In this study, we report the deep genome sequencing of a highly virulent PRV strain ZJO1 isolated from piglets with clinical signs in China. The genome sequence of ZJO1 was derived from purified virion DNA and was determined by Ion-Torrent sequencing and Sanger sequencing (for gap closing). The full length of the genome of ZJO1 was determined about 141029 bp and encoding 69 ORFs with an overall G+C content of 73.5%. BamHI RFLP patterns of PRV ZJO1 strain matched the predicted fragment size.Comparison results indicated that the main coding proteins genes of ZJO1 exhibited mutations, deletion, or insertions, compared with those of Kaplan, Becker and Bartha-K61 strains. For example, ZJO1 and other recent isolates circulating in China have a 1-amion acid (D) insertion in aa position 46 of gE and have a specific mutation in aa position 418 of gE (V>A); regarding gI, ZJO1 has specific mutations in nucleotide positions 29 (V>A),238-246, and 340 (N>T) compared with other PRV strains; it was found that the novel PRV strain ZJO1 has a 3-amino acid insertion (+SPG) between positions 75 and 77, and it has many mutations compared with other PRV strains, except Chinese classical PRV strains Ea and HNYD-2008-China, meanwhile, ZJO1 and other novel Chinese strains also have some specific mutations in amino acids 59-126 and 540=734;ZJO1 and other novel PRV strains in China have a 7-amino acid insertion in the gC protein between amino acids 63 and 69 (+AAASTPA) compared with the classical PRV strains Becker and Kaplan; a 7-amino acid insertion and two 1-amino acid deletions were found in the UL2 and UL12 sequences of ZJO1, respectively; by comparing the ZJO1 UL50 sequence with those of strains Bartha-K61, Kaplan, Becker, and Ea (a traditional isolate of China), we found that ZJO1 had an insertion at amino 22, as well as several unique mutations; IEI80 also exhibited mutations, deletion, or insertions. Moreover, many mutations located in regulatory sequences of UL30, UL34, UL39, UL14, EPO, IE180, UL1, US2 and US6 gene between PRV ZJO1 and the reference sequences. Phylogenetic tree analysis based on the four full-length genomes revealed that ZJO1 belong to a new tightly clustered branch. This study will facilitates us to mine the molecular mechanisms underlying their highly virulent and antigenic variants of those newly PRV isolates in China.4. Generation and characterization of a bacterial artificial chromosome infectious clone of a novel pseudorabies virus isolate ZJO1We report on the generation of an infectious bacterial artificial chromosome (BAC) clone of the novel PRV strain ZJO1 and the virulence of the gE/gI-negative mutant of ZJO1. For generation of the PRV BAC, we insert mini-F vector sequences by homologous recombination in lieu of Us7 (gI) and Us8 (gE) of PRV ZJO1. Circular DNA of the resulting in recombinant virus vZJO1-GFPΔgE/gI was electroporated into Escherichia coli and a full-length PRV BAC clone (pZJ01) was recovered. Transfection of pZJ01 into BHK-21 cells PRV-specific plaques exhibiting green autofluorescence. And then, a gE/gI-negative mutant (vZJO1ΔgE/gI) and a revertant virus (vZJ01AgE/gI-R) were constructed by co-transfection of pZJ01 with related sequences. Plaque area measurements showed vZJ01ΔgE/gI plaques were decreased those of parental ZJ01 virus or revertant virus vZJ01ΔgE/gI-R. There was no significant difference between PRV with respect to virus titers determined infected cells and infected-cell supernatants. Mice and rabbits were challenged intranasally with PRV ZJ01 gE/gI deleted mutant vZJ01ΔgE/gI and its parental strain ZJ01, respectively. The results showed that gE/gl null mutant prolonged the survival time of mice and rabbits. Ten 60-day-old piglets were randomly assigned to two groups of five piglets each and housed separately. Pigs in Group 1 and Group 2 were challenged intranasally with 1 mL107.0TCID50/mL ZJO1 and vZJO1AgE/gl strain, respectively. The results showed that ZJO1 had 100% mortality, while vZJO1ΔgE/gl resulted in no deaths in experimental infection of piglets. Combined with gross lesion examination, histopathological observations, ZJO1 exhibited more severe pathogenicity in pigs compared with vZJ01AgE/gl. In other words, the virulence of the ZJ01 gE/gl deleted mutant vZJ01 AgE/gl had decreased when compared to ZJO1. This BAC clone facilitates us to mine the molecular mechanisms underlying their highly virulent and antigenic variants of those newly PRV isolates.5. Immune protection of an inactivated PRV gE/gI deleted strain vaccine from high virulent PRV ZJO1To study the immunogenicity features of PRV ZJ01 gE/gI deleted mutant, vZJO1ΔgE/gI was inactivated by formalin and an inactivated vaccine was prepared by homogenized with an oil adjuvant. The protective efficacy of this inactivated vaccine was examined in piglets and compared with that of a live Bartha-K61 vaccine. The result showed that the inactivated vZJ01ΔgE/gI vaccine generated significantly higher ZJO1-specific NT responses (p<0.05) than pigs vaccinated with Bartha-K61 vaccine after immunization. Meanwhile, no anti-gE antibody was developed in all vaccinated pigs. After fatal ZJ01 challenge, all five animals in the inactivated vZJ01ΔgE/gl vaccine group survived without exhibiting any clinical sings, but two of five animals exhibited central nervous signs in the Bartha-K61 group. Meanwhile, all the non-vaccinated control animals died at 7 days post-challenge. All the animals from the inactivated vaccine group displayed slighter clinical signs, got lower viral DNA levels in tissues, and gain higher RDWG than the no vaccine control group. It indicated that the inactivated vZJ01ΔgE/gI vaccine provided higher protection against lethal ZJ01 challenge than live Bartha-K61 vaccine. It should be a promising vaccine candidate for controlling the variant strains of PRV now circulating in China.In summary, wild-type PRV had spread widely in the Southern of China since late 2011. Our pathological results showed that one of the pandemic isolates, PRV ZJ01, was an isolate of highly virulence. Combined the cross serum-virus neutralization assay and the vaccination test, we found that ZJ01 is a highly pathogenic PRV isolate with antigenic variants. Based on the complete genome sequence, ZJ01 belong to a new tightly clustered branch. Meanwhile, the constructed BAC clone of PRV ZJ01 will facilitates us to mine the molecular mechanisms underlying their highly virulent and antigenic variants of those newly PRV isolates. The inactivated vZJ01 AgE/gI vaccine is a promising vaccine candidate for controlling the variant strains of PRV now circulating in China in the future.