Bovine respiratory disease complex (BRDC) is a comprehensive disease in cattle caused by various viral and bacterial infections. Among them, bovine herpesvirus type I (BoHV−1) and bovine viral diarrhea virus (BVDV) play important roles and have caused huge financial losses for the cattle industry worldwide. At present, vaccines against BRDC include trivalent attenuated BoHV−1, BVDV−1, and BVDV−2 live vaccines, BoHV−1 live attenuated vaccines, and BoHV−1/BVDV bivalent live attenuated vaccines, which have limitations in terms of their safety and efficacy. To solve these problems, we optimized the codon of the BVDV−1 E2 gene, added the signal peptide sequence of the BoHV−1 gD gene, expressed double BVDV−1 E2 glycoproteins in tandem at the BoHV−1 gE gene site, and constructed a BoHV−1 genetics-engineered vectored vaccine with gE gene deletion, named BoHV−1 gE/E2−Linker−E2⁺ and BoHV−1 ΔgE. This study compared the protective effects in BoHV−1, BoHV−1 ΔgE, BoHV−1 gE/E2−Linker−E2⁺, and BVDV−1 inactivated antigen immunized guinea pigs and calves. The results showed that BoHV−1 gE/E2−Linker−E2⁺ could successfully induce guinea pigs and calves to produce specific neutralizing antibodies against BVDV−1. In addition, after BoHV−1 and BVDV−1 challenges, BoHV−1 gE/E2−Linker−E2⁺ can produce a specific neutralizing antibody response against BoHV−1 and BVDV−1 infections. Calves immunized with this type of virus can be distinguished as either vaccinated animals (gE^-) or naturally infected animals (gE⁺). In summary, our data suggest that BoHV−1 gE/E2−Linker−E2⁺ and BoHV−1 ΔgE have great potential to prevent BVDV−1 or BoHV−1 infection.

Bovine herpesvirus type I (BoHV-1) is an important pathogen that causes respiratory disease in bovines. The disease is prevalent worldwide, causing huge economic losses to the cattle industry. Gene-deficient vaccines with immunological markers to distinguish them from wild-type infections have become a mainstream in vaccine research and development. In order to knock out the gE gene BoHV-1, we employed the CRISPR/Cas9 system. Interesting phenomena were observed at the single guide RNA (sgRNA) splicing site, including gene insertion, gene deletion, and the inversion of 5′ and 3′ ends of the sgRNA splicing site. In addition to the deletion of the gE gene, the US9 gene, and the non-coding regions of gE and US9, it was found that the US4 sequence, US6 sequence, and part of the US7 sequence were inserted into the EGFP sgRNA splicing site and the 3′ end of the EGFP sequence was deleted. Similar to the BoHV-1 parent, the BoHV-1 mutants induced high neutralizing antibodies titer levels in mice. In summary, we developed a series of recombinant gE-deletion BoHV-1 samples using the CRISPR/Cas9 gene editing system. The mutant viruses with EGFP⁺ or EGFP⁻ will lay the foundation for research on BoHV-1 and vaccine development in the future.

Nanobodies (VHHs, ~12-15 kDa) are single-domain antibody fragments valued for diagnostic applications, yet their engineering is frequently limited by poor solubility and aggregation. This study used the aggregation-prone anti-VirB4 nanobody a_b5 as a model to establish a two-stage solubility optimization strategy: rational single-point mutagenesis, then AI-driven de novo design. Consensus sequence analysis and CamSol prediction converged on glycine 54; G54A and G54D mutations reduced the Z-average hydrodynamic diameter by ~31% and ~32%, respectively, but particle sizes remained in the hundreds of nanometers, revealing an inherent ceiling for single-point mutagenesis. To surpass this limit, a modular AI pipeline integrating AlphaFold3 hotspot analysis, RFdiffusion backbone generation, dual-pathway ProteinMPNN sequence design (Vanilla and Soluble), AlphaFold3 ipTM screening, and 100-ns molecular dynamics was constructed. Among 780 designed sequences across 10 nanobody-antigen pairs, B5S achieved a CamSol solubility score of 3.007 (480% increase over wild-type 0.518). DLS confirmed a Z-average diameter of 6.084 nm with 43.4% monomer peak; B5S was solubly expressed in E. coli while the Vanilla counterpart B5V precipitated. Direct ELISA confirmed dose-dependent binding of B5S to VirB4 with apparent K_D ~856.2 nmol/L. These results demonstrate that AI-driven holistic redesign can simultaneously optimize binding interface quality and macromolecular solubility.