Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

Two novel recombinant porcine reproductive and respiratory syndrome viruses belong to sublineage 3.5 originating from sublineage 3.2.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an agent of porcine reproductive and respiratory syndrome (PRRS), which causes substantial economic losses to the swine industry. PRRSV displays rapid variation, and five lineages coexist in mainland China. Lineage 3 PRRSVs emerged in mainland China in 2005 and prevailed in southern China after 2010. In the present study, two lineage 3 PRRSV strains, which are named SD110-1608 and SDWH27-1710, were isolated from northern China in 2017. To explore the characteristics and origins of the two strains, we divided lineage 3 into five sublineages (3.1-3.5) based on 146 open reading frame (ORF) 5 sequences. Both strains and the strains isolated from mainland China were classified into sublineage 3.5. Lineage 3 PRRSVs isolated from Taiwan and Hong Kong were classified into sublineages 3.1-3.3 and sublineage 3.4, respectively. Recombination analysis revealed that SD110-1608 and SDWH27-1710 were derived from recombination of QYYZ (major parent strain) and JXA1 (minor parent strain). Sequence alignment showed that SD110-1608 and SDWH27-1710 shared a 36-aa insertion in Nsp2 with QYYZ isolated from Guangdong Province in 2010. Based on the evolutionary relationship among GP2a, GP3, GP4, GP5 and N proteins between sublineages 3.2 (FJ-1) and 3.5 (FJFS), we speculated that sublineage 3.5 (mainland China) originated from sublineage 3.2 (Taiwan, China). This study provides important information regarding the classification and transmission of lineage 3 PRRSVs.

Emergence of novel porcine reproductive and respiratory syndrome viruses (ORF5 RFLP 1-7-4 viruses) in China.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) pose a serious threat to the porcine industry of China, and the importation of novel strain(s) makes it challenging to control these viruses. Several NADC30-like PRRSV outbreaks have occurred in mainland China since 2013. In the current study, we report two novel PRRSVs, designated LNWK96 and LNWK130, which belong to lineage 1 and are closely related to US strains with ORF5 restriction fragment length polymorphism (RFLP) 1-7-4. The two viruses had a 100-aa deletion in the nsp2 gene corresponding to positions 328-427 in the VR-2332 strain, which was consistent with most of the ORF5 RFLP 1-7-4 viruses. Recombination analyses indicated that both viruses derived from the recombination of 1-7-4 isolates and ISU30 or NADC30, which were isolated in the United States. Taken together, these results demonstrate the emergence of ORF5 RFLP 1-7-4-like (NADC34-like) PRRSVs in China for the first time.

Adaptions of field PRRSVs in Marc-145 cells were determined by variations in the minor envelope proteins GP2a-GP3.

The recent rapid evolution of PRRSVs has resulted in certain biological characteristic changes, such as the fact that an increasing number of field PRRSVs can be isolated from PAMs but not from Marc-145 cells. In this study, we first isolated Marc-145-unadaptive field PRRSV strains from PAMs; sequence analysis showed that these PRRSVs belong to the HP-PRRSV (lineage 8) branch or NADC30-Like (lineage 1) branch. We further found major variations in ORF2-4 regions. To explore the viral adaptation mechanisms in detail, we constructed a full-length cDNA clone of MY-376, a Marc-145-unadaptive PRRSV. Construction of serially chimeric viruses of HuN4-F112 (a Marc-145-adaptive strain) and MY-376 demonstrated that variation in the minor envelope protein (GP2a and GP3) complex is a main determinant of PRRSV tropism for Marc-145 cells.

Characterization of two newly emerged isolates of porcine reproductive and respiratory syndrome virus from Northeast China in 2013.

A newly emerged porcine reproductive and respiratory syndrome virus (PRRSV) that has caused severe reproductive losses in sows appeared in some regions of China in 2013. To explore the biology of this new PRRSV and understand more fully genetic diversity in PRRSV isolates from China, the complete genome of the two 2013 Chinese isolates, designated HLJA1 and HLJB1, were analyzed. Genomic sequence analysis showed that HLJA1 and HLJB1 shared 88.6-98.3% nucleotide identity with genotype 2 (North American type, NA-type) isolates, but only 61.1% with the genotype 1 (European type, EU-type) isolate of Lelystad virus, indicating that both these isolates belong to the NA-type PRRSV genotype. Phylogenetic analysis showed that the NA-type PRRSV isolates formed three subgroups (1, 2 and 3); representatives of these subgroups are VR-2332, CH-1a and HUN4, respectively. HLJA1 and HLJB1 belong to subgroup 2. Analysis of NSP2 revealed that HLJA1 has a 48-amino acid deletion at positions 473-480 and 482-521, unlike other HP-PRRSV isolates, while HLJB1 has only a 1-amino acid deletion at position 481 compared with CH-1a. Interestingly, HLJA1 replicated in PAM cells but not in MARC-145 cells, whereas HLJB1 replicated in both cell types. The neutralizing antibody titer of pig hyperimmune sera against HUN4 was significantly higher than that of HLJA1 or HLJB1. Additionally, genetic variability in GP5 and GP3 proteins and in the novel ORF5a protein was evident. In addition to elucidating the genetic relationships between PRRSV isolates, our results suggest that Chinese PRRSV will remain a pandemic virus.

Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012.

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61-vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.

Expression of short peptide by an improved isocaudamer tandem repeat strategy.

An improved isocaudamer tandem repeat strategy for the production of short functional peptide was demonstrated in the study. The coding sequence of short peptide was codon optimized, and two isocaudamers were induced into the end of coding sequence. By re-cutting with isocaudamers and re-ligating, the coding sequence of short peptide in the expression vector was increased in a multiple manner (21, 22, 23, 24, 25 …….). In the present study, an 8 amino-acidresidue peptide of porcine reproductive and respiratory syndrome virus (PRRSV) was effectively expressed in 8 copies and 16 copies by this approach, then the proteins in 8 copies and 16 copies were used to generate antibody against this epitope in rabbits. The results showed that PRRSVs were well recognized by the antibody in indirect immunofluorescence assay. The technology using isocaudamer to insert multiple tandem repeats in the vector provides an important approach for the studies of small molecule peptides.

Highly pathogenic porcine reproductive and respiratory syndrome virus GP5 B antigenic region is not a neutralizing antigenic region.

In 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) caused great economic losses emerged in China and continues to be a threat for the pig industry. B antigenic region (AR) ((37)SHL/FQLIYNL(45)) of GP5 was considered to be a major linear neutralizing AR in PRRSV classical strains. However, peptide-purified antibodies against this AR did not neutralize PRRSV in a recent report. Compared with classical PRRSV, one amino acid mutation (L/F(39)→ I(39)) was found in B AR of HP-PRRSV. To study the ability of B AR of HP-PRRSV to induce neutralizing antibody (NA) in vitro and in vivo, rabbit antisera against B AR with and without the mutation and pig hyperimmune sera with high titer of NAs against HP-PRRSV were prepared. Immunofluorescence assays (IFA) showed that the two rabbit antisera both had reactivity to classical PRRSV CH-1a and HP-PRRSV HuN4 with no observable difference in IFA titer. However, antisera did not have neutralizing activity against classical PRRSV CH-1a and HP-PRRSV HuN4. No correlation was observed between the levels of anti-B AR peptide antibodies and NAs in pig hyperimmune sera that were detected by indirect ELISA and virus neutralization, respectively. B AR peptide-specific serum antibodies had no neutralizing activity and, GST-B fusion protein could not inhibit neutralization of NAs in pig hyperimmune sera. Based on these findings, we conclude that B AR of HP-PRRSV is not a neutralizing AR of HP-PRRSV GP5.