Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate’s protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development.

Murine gammaherpesvirus 68 replicates in the alveolar epithelium and induces an inflammatory infiltrate in the lung, following intranasal challenge, and is cleared 10 and 13 days after infection by a T-cell-dependent mechanism. In order to understand the development of the immune response to this virus and how leukocyte trafficking to the lung is regulated, chemokine expression during MHV-68 infection was examined in lung tissue using an RNase protection assay. Expression of RANTES, eotaxin, MIP-1 alpha, MIP-1 beta, IP-10, and MCP-1 was upregulated by day 7 after infection. Chemokine concentrations in lung lavage fluid were also determined by ELISA. MCP-1, RANTES, MIP-1 alpha, eotaxin, and KC were upregulated during MHV-68 infection. Most of these chemokines have been reported to be chemoattractants for either activated T cells or monocytes, which are the major cellular components of the inflammatory infiltrate induced by the virus. Upregulated expression of the corresponding receptors for the chemokines, including CCR1, CCR2, CCR3, CCR5, and CXCR3, coincided with the development of the inflammatory infiltrate. The chemokine levels peaked at around day 7 after infection, coinciding with peak viral titers and slightly preceding maximal T cell infiltration. In vitro chemotaxis assays confirmed that lung lavage fluid from MHV-68-infected mice had chemotactic activity, which was partially blocked by antibodies to IP-10 and RANTES. These observations suggest that the chemokines detected play an important role in regulating leukocyte trafficking to the lungs during MHV-68 infection.

Swine pseudorabies (PR) re-emerged in Bartha-vaccinated pig herds and caused death of millions of piglets in China since the later part of 2011. We isolated a novel pseudorabies virus (PRV), named HNX strain, from the brain of abortion fetuses to diagnose the disease. To reveal the genomic organization and characterize the HNX strain, the complete genomes of HNX and Fa strain, an isolate in the 1960s, were sequenced and analyzed. The genomic size of HNX and Fa strains were 142,294 and 141,930 nt, respectively, with corresponding G + C contents of 73.56 and 73.70 %. The two strains consistently possessed 70 open reading frames. In addition, comparative genomic analysis between HNX and Bartha strains was performed to understand the possible reason of immune failure. The major virulence-associated genes of HNX strain had slight changes, whereas glycoprotein B and glycoprotein C genes of HNX strain had 73 mutations; the homology at the whole genomic level between HNX and Bartha strains was 90.6 %. Genome-wide comparison between HNX and Fa strains indicated that the strains shared about 96.4 % of homology and clustered in a separate Chinese isolate group; the two strains are also distant from the isolates from other countries. Similarity plot and bootscanning analysis of complete genome sequences of nine PRV strains, including HNX and Fa, four newly Chinese strains, and three traditional reference strains, revealed that non-recombination events occurred in the HNX strain. The PRV HNX strain with genomic variations might contribute to the PR outbreak in China since the later part of 2011.

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The complete genome sequence of a novel pseudorabies virus, strain HNB, isolated from a dead weaned pig in China, was determined using next-generation sequencing. The viral genome sequence of HNB shared 90.6% nucleotide similarity with that of the traditional vaccine strain, the Bartha strain.