Proteomic Comparison of Three Wild-Type Pseudorabies Virus Strains and the Attenuated Bartha Strain Reveals Reduced Incorporation of Several Tegument Proteins in Bartha Virions.

Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily and the causative agent of Aujeszky's disease in pigs. Driven by the large economic losses associated with PRV infection, several vaccines and vaccine programs have been developed. To this day, the attenuated Bartha strain, generated by serial passaging, represents the golden standard for PRV vaccination. However, a proteomic comparison of the Bartha virion to wild-type (WT) PRV virions is lacking. Here, we present a comprehensive mass spectrometry-based proteome comparison of the attenuated Bartha strain and three commonly used WT PRV strains: Becker, Kaplan, and NIA3. We report the detection of 40 structural and 14 presumed nonstructural proteins through a combination of data-dependent and data-independent acquisition. Interstrain comparisons revealed that packaging of the capsid and most envelope proteins is largely comparable in-between all four strains, except for the envelope protein pUL56, which is less abundant in Bartha virions. However, distinct differences were noted for several tegument proteins. Most strikingly, we noted a severely reduced incorporation of the tegument proteins IE180, VP11/12, pUS3, VP22, pUL41, pUS1, and pUL40 in Bartha virions. Moreover, and likely as a consequence, we also observed that Bartha virions are on average smaller and more icosahedral compared to WT virions. Finally, we detected at least 28 host proteins that were previously described in PRV virions and noticed considerable strain-specific differences with regard to host proteins, arguing that the potential role of packaged host proteins in PRV replication and spread should be further explored. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha-an attenuated strain created by serial passaging-represents an exceptional success story in alphaherpesvirus vaccination. Here, we used mass spectrometry to analyze the Bartha virion composition in comparison to three established WT PRV strains. Many viral tegument proteins that are considered nonessential for viral morphogenesis were drastically less abundant in Bartha virions compared to WT virions. Interestingly, many of the proteins that are less incorporated in Bartha participate in immune evasion strategies of alphaherpesviruses. In addition, we observed a reduced size and more icosahedral morphology of the Bartha virions compared to WT PRV. Given that the Bartha vaccine strain elicits potent immune responses, our findings here suggest that differences in protein packaging may contribute to its immunogenicity. Further exploration of these observations could aid the development of efficacious vaccines against other alphaherpesvirus vaccines such as HSV-1/2 or EHV-1.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha Hyperactivates Plasmacytoid Dendritic Cells by Generating Large Amounts of Cell-Free Virus in Infected Epithelial Cells.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus and the causative agent of Aujeszky's disease. Successful eradication campaigns against PRV have largely relied on the use of potent PRV vaccines. The live attenuated Bartha strain, which was produced by serial passaging in cell culture, represents one of the hallmark PRV vaccines. Despite the robust protection elicited by Bartha vaccination, very little is known about the immunogenicity of the Bartha strain. Previously, we showed that Bartha-infected epithelial cells trigger plasmacytoid dendritic cells (pDC) to produce much higher levels of type I interferons than cells infected with wild-type PRV. Here, we show that this Bartha-induced pDC hyperactivation extends to other important cytokines, including interleukin-12/23 (IL-12/23) and tumor necrosis factor alpha (TNF-α) but not IL-6. Moreover, Bartha-induced pDC hyperactivation was found to be due to the strongly increased production of extracellular infectious virus (heavy particles [H-particles]) early in infection of epithelial cells, which correlated with a reduced production of noninfectious light particles (L-particles). The Bartha genome is marked by a large deletion in the US region affecting the genes encoding US7 (gI), US8 (gE), US9, and US2. The deletion of the US2 and gE/gI genes was found to be responsible for the observed increase in extracellular virus production by infected epithelial cells and the resulting increased pDC activation. The deletion of gE/gI also suppressed L-particle production. In conclusion, the deletion of US2 and gE/gI in the genome of the PRV vaccine strain Bartha results in the enhanced production of extracellular infectious virus in infected epithelial cells and concomitantly leads to the hyperactivation of pDC. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha has been and still is critical in the eradication of PRV in numerous countries. However, little is known about how this vaccine strain interacts with host cells and the host immune system. Here, we report the surprising observation that Bartha-infected epithelial porcine cells rapidly produce increased amounts of extracellular infectious virus compared to wild-type PRV-infected cells, which in turn potently stimulate porcine plasmacytoid dendritic cells (pDC). We found that this phenotype depends on the deletion of the genes encoding US2 and gE/gI. We also found that Bartha-infected cells secrete fewer pDC-inhibiting light particles (L-particles), which appears to be caused mainly by the deletion of the genes encoding gE/gI. These data generate novel insights into the interaction of the successful Bartha vaccine with epithelial cells and pDC and may therefore contribute to the development of vaccines against other (alphaherpes)viruses.

Alphaherpesvirus-induced activation of plasmacytoid dendritic cells depends on the viral glycoprotein gD and is inhibited by non-infectious light particles.

Plasmacytoid dendritic cells (pDC) are important innate immune cells during the onset of viral infections as they are specialized in the production of massive amounts of antiviral type I interferon (IFN). Alphaherpesviruses such as herpes simplex virus (HSV) or pseudorabies virus (PRV) are double stranded DNA viruses and potent stimulators of pDC. Detailed information on how PRV activates porcine pDC is lacking. Using PRV and porcine primary pDC, we report here that PRV virions, so-called heavy (H-)particles, trigger IFNα production by pDC, whereas light (L-) particles that lack viral DNA and capsid do not. Activation of pDC requires endosomal acidification and, importantly, depends on the PRV gD envelope glycoprotein and O-glycosylations. Intriguingly, both for PRV and HSV-1, we found that L-particles suppress H-particle-mediated activation of pDC, a process which again depends on viral gD. This is the first report describing that gD plays a critical role in alphaherpesvirus-induced pDC activation and that L-particles directly interfere with alphaherpesvirus-induced IFNα production by pDC.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha K61: A Brief Review on the Knowledge Gathered During 60 Years of Research.

Pseudorabies virus (PRV) is a member of the alphaherpesvirus subfamily of the herpesviruses and is the causative agent of Aujeszky's disease in pigs, causing respiratory, neurological, and reproductive symptoms. Given the heavy economic losses associated with Aujeszky's disease epidemics, great efforts were made to develop efficacious vaccines. One of the best modified live vaccines to this day is the attenuated Bartha K61 strain. The use of this vaccine in extensive vaccination programs worldwide has assisted considerably in the eradication of PRV from the domesticated pig population in numerous countries. The Bartha K61 strain was described in 1961 by Adorján Bartha in Budapest and was obtained by serial passaging in different cell cultures. Ever since, it has been intensively studied by several research groups, for example, to explore its efficacy as a vaccine strain, to molecularly and mechanistically explain its attenuation, and to use it as a retrograde neuronal tracer and as a vector vaccine. Given that the Bartha K61 vaccine strain celebrates its 60th birthday in 2021 with no sign of retirement, this review provides a short summary of the knowledge on its origin, characteristics, and use as a molecular tool and as a vaccine.