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Objective This study was conducted to enable on-line prediction of primal and commercial cut weights in Korean slaughter pigs by AutoFom III, which non-invasively scans pig carcasses early after slaughter using ultrasonic sensors. Methods A total of 162 Landrace, Yorkshire, and Duroc (LYD) pigs and 154 LYD pigs representing the yearly Korean slaughter distribution were included in the calibration and validation dataset, respectively. Partial least squares (PLS) models were developed for prediction of the weight of deboned shoulder blade, shoulder picnic, belly, loin, and ham. In addition, AutoFom III’s ability to predict the weight of the commercial cuts of spare rib, jowl, false lean, back rib, diaphragm, and tenderloin was investigated. Each cut was manually prepared by local butchers and then recorded. Results The cross-validated prediction accuracy (R2cv) of the calibration models for deboned shoulder blade, shoulder picnic, loin, belly, and ham ranged from 0.77 to 0.86. The R2cv for tenderloin, spare rib, diaphragm, false lean, jowl, and back rib ranged from 0.34 to 0.62. Because the R2cv of the latter commercial cuts were less than 0.65, AutoFom III was less accurate for the prediction of those cuts. The root mean squares error of cross validation calibration (RMSECV) model was comparable to the root mean squares error of prediction (RMSEP), although the RMSECV was numerically higher than RMSEP for the deboned shoulder blade and belly. Conclusion AutoFom III predicts the weight of deboned shoulder blade, shoulder picnic, loin, belly, and ham with high accuracy, and is a suitable process analytical tool for sorting pork primals in Korea. However, AutoFom III’s prediction of smaller commercial Korean cuts is less accurate, which may be attributed to the lack of anatomical reference points and the lack of a good correlation between the scanned area of the carcass and those traits.

Several viruses have been found to encode a deubiquitinating protease (DUB). These viral DUBs are proposed to play a role in regulating innate immune or inflammatory signaling. In human cytomegalovirus (HCMV), the largest tegument protein encoded by UL48 contains DUB activity, but its cellular targets are not known. Here, we show that UL48 and UL45, an HCMV-encoded inactive homolog of cellular ribonucleotide reductase (RNR) large subunit (R1), target receptor-interacting protein kinase 1 (RIP1) to inhibit NF-κB signaling. Transfection assays showed that UL48 and UL45, which binds to UL48, interact with RIP1 and that UL48 DUB activity and UL45 cooperatively suppress RIP1-mediated NF-κB activation. The growth of UL45-null mutant virus was slightly impaired with showing reduced accumulation of viral late proteins. Analysis of a recombinant virus expressing HA-UL45 showed that UL45 interacts with both UL48 and RIP1 during virus infection. Infection with the mutant viruses also revealed that UL48 DUB activity and UL45 inhibit TNFα-induced NF-κB activation at late times of infection. UL48 cleaved both K48- and K63-linked polyubiquitin chains of RIP1. Although UL45 alone did not affect RIP1 ubiquitination, it could enhance the UL48 activity to cleave RIP1 polyubiquitin chains. Consistently, UL45-null virus infection showed higher ubiquitination level of endogenous RIP1 than HA-UL45 virus infection at late times. Moreover, UL45 promoted the UL48-RIP1 interaction and re-localization of RIP1 to the UL48-containing virion assembly complex. The mouse cytomegalovirus (MCMV)-encoded DUB, M48, interacted with mouse RIP1 and M45, an MCMV homolog of UL45. Collectively, our data demonstrate that cytomegalovirus-encoded DUB and inactive R1 homolog target RIP1 and cooperatively inhibit RIP1-mediated NF-κB signaling at the late stages of HCMV infection.

Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that covalently conjugates protein. Protein modification by ISG15 (ISGylation) is known to inhibit the replication of many viruses. However, studies on the viral targets and viral strategies to regulate ISGylation-mediated antiviral responses are limited. In this study, we show that human cytomegalovirus (HCMV) replication is inhibited by ISGylation, but the virus has evolved multiple countermeasures. HCMV-induced ISG15 expression was mitigated by IE1, a viral inhibitor of interferon signaling, however, ISGylation was still strongly upregulated during virus infection. RNA interference of UBE1L (E1), UbcH8 (E2), Herc5 (E3), and UBP43 (ISG15 protease) revealed that ISGylation inhibits HCMV growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. A viral regulator pUL26 was found to interact with ISG15, UBE1L, and Herc5, and be ISGylated. ISGylation of pUL26 regulated its stability and inhibited its activities to suppress NF-κB signaling and complement the growth of UL26-null mutant virus. Moreover, pUL26 reciprocally suppressed virus-induced ISGylation independent of its own ISGylation. Consistently, ISGylation was more pronounced in infections with the UL26-deleted mutant virus, whose growth was more sensitive to IFNβ treatment than that of the wild-type virus. Therefore, pUL26 is a viral ISG15 target that also counteracts ISGylation. Our results demonstrate that ISGylation inhibits HCMV growth at multiple steps and that HCMV has evolved countermeasures to suppress ISG15 transcription and protein ISGylation, highlighting the importance of the interplay between virus and ISGylation in productive viral infection.

Viral deubiquitinases (DUBs) regulate cellular innate immunity to benefit viral replication. In human cytomegalovirus (HCMV), the UL48-encoded DUB regulates innate immune responses, including NF-k B signaling. Although UL48 DUB is known to regulate its stability via auto-deubiquitination, its impact on other viral proteins is not well understood. In this study, we investigated the role of UL48 DUB in regulating the ubiquitination of viral proteins by comparing the levels of ubiquitinated viral peptides in cells infected with wild-type virus and DUB active-site mutants using mass spectrometry. We found that ubiquitinated peptides were increased in DUB mutant virus infection for 90% of viral proteins, with the innermost tegument proteins pp150 (encoded by UL32) and pUL48 itself being most significantly affected. The highly deubiquitinated lysine residues of pUL48 were mapped within its N-terminal DUB domain and the nuclear localization signal. Among them, the arginine substitution of lysine 2 (K2R) increased pUL48 stability and enhanced viral growth at low multiplicity of infection, indicating that K2 auto-deubiquitination has a role in regulating pUL48 stability. pUL48 also interacted with pp150 and increased pp150 expression by downregulating its ubiquitination. Furthermore, we found that, unlike the wild-type virus, mutant viruses expressing the UL48 protein with the DUB domain deleted or DUB active site mutated contain higher levels of ubiquitin conjugates, including the ubiquitinated forms of pp150, in their virions. Collectively, our results demonstrate that UL48 DUB mainly acts on the innermost tegument proteins pp150 and pUL48 itself during HCMV infection and may play a role in protecting virions from the inclusion of ubiquitin conjugates. IMPORTANCE Herpesviruses encode highly conserved tegument proteins that contain deubiquitinase (DUB) activity. Although the role of viral DUBs in the regulation of host innate immune responses has been established, their roles in the stability and function of viral proteins are not well understood. In this study, we performed a comparative analysis of the levels of ubiquitinated viral peptides between wild-type and DUB-inactive HCMV infections and demonstrated that the innermost tegument proteins pp150 and pUL48 (DUB itself) are major targets of viral DUB. We also show that ubiquitinated viral proteins are effectively incorporated into the virions of DUB mutant viruses but not the wild-type virus. Our study demonstrates that viral DUBs may play important roles in promoting the stability of viral proteins and inhibiting the inclusion of ubiquitin conjugates into virions. [ABSTRACT FROM AUTHOR]

Vaccination is the most efficient method for infectious disease prevention. Parenteral injections such as intramuscular, intradermal, and subcutaneous injections have several advantages in vaccine delivery, but there are many drawbacks. Thus, the development of a new vaccine delivery system has long been required. Recently, microneedles have been attracting attention as new vaccination tools. Microneedle is a highly effective transdermal vaccine delivery method due to its mechanism of action, painlessness, and ease of use. Here, we summarized the characteristics of microneedles and the possibilities as a new vaccine delivery route. [ABSTRACT FROM AUTHOR]

Background: The A20 ubiquitin-editing enzyme is a target of nuclear factor kappa B (NF-κB) and also plays a key role in regulating the NF-κB signaling pathway. NF-κB activity is increased during human cytomegalovirus (HCMV) infection and HCMV appears to be adapted to this change. To better understand the regulation of NF-κB signaling during HCMV infection, we investigated how A20 expression is controlled during HCMV infection. Methods: The expression level of A20 in human fibroblast cells infected with HCMV or UV-inactivated virus (UV-HCMV) was measured by immunoblot analysis, cell staining, and quantitative real-time PCR. Changes of histone modifications on the A20 promoter were determined by chromatin immunoprecipitation assays. Lentiviral vectors were used to knockdown A20 in fibroblast cells. Results: A20 expression was increased at early times after HCMV infection. This increase of the A20 protein level was promoted by viral gene expression under low viral load conditions. The viral IE1 protein, which is known to activate NF-κB, increased the A20 promoter activity through the upstream NF-κB sites in reporter assays, suggesting that IE1 is at least partly involved in A20 induction. Analysis of A20 expression with a high viral load demonstrated that the A20 regulation by HCMV was biphasic; both A20 protein and mRNA levels were increased at the early stage of infection, but decreased at the late stage. Under high viral load conditions, A20 upregulation was more profound with UV-HCMV than with HCMV, indicating a role of the viral gene product(s) in limiting A20 induction. Consistently, more histone modifications for euchromatin were found on the A20 promoter during UV-HCMV infection than with HCMV infection. A20 knockdown by shRNA reduced HCMV growth. Conclusion: These results suggest that the biphasic regulation of A20 expression may be important for productive HCMV infection. [ABSTRACT FROM AUTHOR]

DNA-dependent activator of interferon regulatory factor (DAI) acts as a cytosolic B-form DNA sensor that induces type I interferons. However, DAI is not required for DNA sensing in certain cell types due to redundancy of the DNA sensing system. Here, we investigated the effect of DAI on herpes simplex virus 1 (HSV-1) infection in HepG2 hepatocellular carcinoma cells. DAI transcription was induced after gamma interferon (IFN-γ) treatment or HSV-1 infection. HSV-1 replication was enhanced by DAI knockdown, and ectopic DAI expression repressed viral replication in a manner requiring the Zβ and D3 domains, but not the Zα domain. This activity of DAI was more prominent at low multiplicity of infection (MOI) and correlated with the reduced expression of viral immediate-early genes. Consistently, DAI repressed the activation of ICP0 promoter in reporter gene assays. DAI knockdown did not affect the BDNA-mediated IFN-β transcription and IRF3 activation, and overexpression of DAI and RIP1 did not enhance NF-κB activation by BDNA treatment, demonstrating that DAI is not essential for the B-DNA-mediated IFN production in HepG2 cells. DAI colocalized with ICP0 in a subset of nuclear and cytoplasmic foci in infected cells and interacted with ICP0 in coimmunoprecipitation assays. The anti- HSV-1 effect of DAI was not observed in ICP0-deleted mutant virus infection at a high MOI in HepG2 cells and mouse embryonic fibroblasts. Degradation of IFI16 and PML by ICP0 was enhanced in infection of DAI-knockdown cells. Collectively, these results demonstrate that DAI can suppress HSV-1 growth independent of DNA sensing through mechanisms involving suppression of viral genomes and regulation of ICP0. [ABSTRACT FROM AUTHOR]

This study was conducted to evaluate the relationship among market weight, slaughter age, yield grade, and primal cut yield in Hanwoo. A total of 403 Hanwoo (Korean native cattle) was assessed for carcass traits such as carcass cold weight, backfat thickness, ribeye area, dressing percentage, yield index, and marbling score. The production yield of the individual major primal cuts of Hanwoo beef was also measured. Carcass cold weight, ribeye area, and backfat thickness, which affect meat quality increased with increased market weight (p < 0.05). The production yield of the ten major primal cuts also increased with increased market weight (p < 0.05). In terms of slaughter age, carcass cold weight, ribeye area, and backfat thickness all increased from 25 months to 28–29 months, and the production yield of all prime cuts also increased with increasing slaughter age. According to the meat yield grade, carcass cold weight and backfat thickness increased from grade A to grade C, although the ribeye area was not affected. The combined findings of the study suggest that slaughtering Hanwoo at the weight of 651–700 kg and 701–750 and age of 28.23 and 29.83 months could be desirable to achieve the best quality and quantity grade of Hanwoo beef. However, the positive correlation of carcass cold weight and backfat thickness, and the negative correlation of the yield index according to primal cuts yield indicated that it is necessary to couple the slaughtering management of cattle with improved genetic and breeding method of Hanwoo to increase the production yield of the major prime cuts of Hanwoo beef.