UL54 Gene Research Articles

A recombinant pseudorabies virus surface - displaying the classical swine fever E2 protein induces specific antibodies rapidly

Pseudorabies virus (PRV) and classical swine fever virus (CSFV) are both economically important pathogens threatening the pig industry in many countries. The triple-gene-deleted variant of PRV, herein referred to as rPRVTJ-delgE/gI/TK, has exhibited pronounced efficacy and safety profiles. This underscores its viability as a prospective vaccine vector. However, the generation of specific anti-E2 antibodies necessitates elevated immunization doses and extended durations when the extracellular domain of the E2 protein of CSFV is secreted via the recombinant rPRVTJ-delgE/gI/TK vector. To enhance the presentation of exogenous antigens by antigen-presenting cells (APCs), we engineered the E2 protein expressed on the surface of PRV particles in this study. The recombinant virus expressing the E2 protein with a heterogonous transmembrane domain was generated in the backbone of rPRVTJ-delgE/gI/TK and designated as rPRVTJ-UL44-E2. The E2 gene was fused to the 3′ terminus of the UL44 gene utilizing P2A, a self-cleaving peptide sequence. The electron microscopy showed that the E2 protein was anchored on the surface of the viral particles of rPRVTJ-delgE/gI/TK-E2. The insertion of the E2 gene did not alter the native biological characteristics of the viral vector. Rabbits immunized with 107 median tissue culture infective doses (TCID50) of rPRVTJ-UL44-E2 exhibited a rapid seroconversion to anti-E2 specific antibodies within 7 days post-immunization (dpi). All the rabbits immunized with the rPRVTJ-UL44-E2 had generated antibodies specific to E2 prior to the administration of the booster immunization. However, the immunized rabbits were not protected from the CSFV C-strain challenge. Nevertheless, this strategy has notably achieved rapid induction of E2-specific non-neutralizing antibodies. These findings provide insights that the design of rPRVTJ-UL44-E2 requires optimization, thereby indicating a promising avenue for augmenting vaccine-induced immune responses.

Impact of the interaction between herpes simplex virus 1 ICP22 and FACT on viral gene expression and pathogenesis.

Facilitates chromatin transcription (FACT) interacts with nucleosomes to promote gene transcription by regulating the dissociation and reassembly of nucleosomes downstream and upstream of RNA polymerase II (Pol II). A previous study reported that herpes simplex virus 1 (HSV-1) regulatory protein ICP22 interacted with FACT and was required for its recruitment to the viral DNA genome in HSV-1-infected cells. However, the biological importance of interactions between ICP22 and FACT in relation to HSV-1 infection is unclear. Here, we mapped the minimal domain of ICP22 required for its efficient interaction with FACT to a cluster of five basic amino acids in ICP22. A recombinant virus harboring alanine substitutions in this identified cluster led to the decreased accumulation of viral mRNAs from UL54, UL38, and UL44 genes, reduced Pol II occupancy of these genes in MRC-5 cells, and impaired HSV-1 virulence in mice following ocular or intracranial infection. Furthermore, the treatment of mice infected with wild-type HSV-1 with CBL0137, a FACT inhibitor currently being investigated in clinical trials, significantly improved the survival rate of mice. These results suggested that the interaction between ICP22 and FACT was required for efficient HSV-1 gene expression and pathogenicity. Therefore, FACT might be a potential therapeutic target for HSV-1 infection.IMPORTANCEICP22 is a well-known regulatory factor of HSV-1 gene expression, but its mechanism(s) are poorly understood. Although the interaction of FACT with ICP22 was reported previously, its significance in HSV-1 infection is unknown. Given that FACT is involved in gene transcription, it is of interest to investigate this interaction as it relates to HSV-1 gene expression. To determine a direct link between the interaction and HSV-1 infection, we mapped a minimal domain of ICP22 required for its efficient interaction with FACT and generated a recombinant virus carrying mutations in the identified domain. Using the recombinant virus, we obtained evidence suggesting that the interaction between ICP22 and FACT promoted Pol II transcription from HSV-1 genes and viral virulence in mice. In addition, CBL0137, an inhibitor of FACT, effectively protected mice from lethal HSV-1 infection, suggesting FACT might be a potential target for the development of novel anti-HSV drugs.

Diagnosis of Human Cytomegalovirus Drug Resistance Mutations in Solid Organ Transplant Recipients-A Review.

Human cytomegalovirus (HCMV) infection may be asymptomatic in healthy individuals but can cause severe complications in immunocompromised patients, including transplant recipients. Breakthrough and drug-resistant HCMV infections in such patients are major concerns. Clinicians are first challenged to accurately diagnose HCMV infection and then to identify the most effective antiviral drug and determine when to initiate therapy, alter drug dosage, or switch medication. This review critically examines HCMV diagnostics approaches, particularly for immunocompromised patients, and the development of genotypic techniques to rapidly diagnose drug resistance mutations. The current standard method to identify prevalent and well-known resistance mutations involves polymerase chain reaction amplification of UL97, UL54, and UL56 gene regions, followed by Sanger sequencing. This method can confirm clinical suspicion of drug resistance as well as determine the level of drug resistance and range of cross-resistance with other drugs. Despite the effectiveness of this approach, there remains an urgent need for more rapid and point-of-care HCMV diagnosis, allowing for timely lifesaving intervention.

A New Duplication in 668 to 672 Sites of UL54 Gene in Cytomegalovirus Ganciclovir Resistant Liver Transplanted Patients

Background: In liver transplant recipients, human Cytomegalovirus (CMV) infection is a significant concern. Ganciclovir is the preferred medication for treating widespread CMV infections. In some cases, patients might require high doses of treatment for CMV infections that are resistant to ganciclovir, although liver transplant patients who have received extended ganciclovir and valganciclovir prophylaxis have reported infrequent cases of ganciclovir-resistant CMV infections. Mutations in the UL54 gene, responsible for encoding deoxyribonucleic acid (DNA) polymerase, can result in resistance. Objectives: In this study, the focus was on UL54 mutations and their association with ganciclovir resistance. Methods: In this study, 23 liver transplant recipients who were admitted to the transplant departments of Namazi and Abu Ali Sina hospitals were examined. Cytomegalovirus infection was then confirmed in them using the quantitative real-time polymerase chain reaction (PCR) method. The UL54 mutations were found after electrophoresis using the nested PCR method, and the PCR products were subsequently sequenced using the Sanger method. Sequence analysis and locating of UL54 mutations were performed using Finch software (version 1.4.0). Results: After sequencing 52 samples from 23 patients, 25 mutations in the UL54 gene were identified in 9 patients who were CMV-infected, occurring at a median of 32 days after transplant. These mutations, including S655L (10/9, 40%), N685S (8/9, 32%), F669L (4/9, 16%), A688V (2/9, 8%), and the novel AK124703.1: p.V668-G672dup (1/9, 4%), were detected in 9 liver transplant recipients over a median period of 2 years in the UL54 gene. Furthermore, a phylogenetic analysis was conducted to investigate the origins of these mutations in CMV isolated from the Iranian population. Conclusions: Considering that treatment with the drug ganciclovir has led to resistance mutations, particularly the new AK124703.1:p.V668-G672dup mutation, and inefficiency in treatment, it is necessary to determine drug-resistant CMV strains and closely monitor these patients. This includes determining viral load, assessing response to treatment, and identifying non-response at regular intervals until the viral load is completely eradicated in order to ensure the effectiveness of the treatment.

Emodin as an Inhibitor of PRV Infection In Vitro and In Vivo.

Pseudorabies (PR) is an acute and severe infectious disease caused by pseudorabies virus (PRV). Once the virus infects pigs, it is difficult to eliminate, resulting in major economic losses to the global pig industry. In addition, reports of human infection with PRV suggest that the virus is a potential threat to human health; thus, its significance to public health should be considered. In this paper, the anti-PRV activities of emodin in vitro and in vivo, and its mechanism of action were studied. The results showed that emodin inhibited the proliferation of PRV in PK15 cells in a dose-dependent manner, with an IC50 of 0.127 mg/mL and a selection index of 5.52. The addition of emodin at different stages of viral infection showed that emodin inhibited intracellular replication. Emodin significantly inhibited the expression of the IE180, EP0, UL29, UL44, US6, and UL27 genes of PRV within 48 h. Emodin also significantly inhibited the expression of PRV gB and gD proteins. The molecular docking results suggested that emodin might form hydrogen bonds with PRV gB and gD proteins and affect the structure of viral proteins. Emodin effectively inhibited the apoptosis induced by PRV infection. Moreover, emodin showed a good protective effect on PRV-infected mice. During the experimental period, all the control PRV-infected mice died resulting in a survival rate of 0%, while the survival rate of emodin-treated mice was 28.5%. Emodin also significantly inhibited the replication of PRV in the heart, liver, brain, kidneys and lungs of mice and alleviated tissue and organ damage caused by PRV infection. Emodin was able to combat viral infection by regulating the levels of the cytokines TNF-α, IFN-γ, IL-6, and IL-4 in the sera of infected mice. These results indicate that emodin has good anti-PRV activity in vitro and in vivo, and is expected to be a new agent for the prevention and control of PRV infection.

Duck plague virus tegument protein vp22 plays a key role in the secondary envelopment and cell-to-cell spread

Duck plague virus (DPV) is one of the major infectious and fatal diseases of geese, ducks, and other wild waterfowl. The DPV UL49 gene product VP22 is one of the most abundant tegument proteins. However, the role of the DPV VP22 is enigmatic to be clarified. In this study, we found deletion of the UL49 gene resulted in reduced viral growth curve and smaller plaque size in duck embryo fibroblast (DEF) cells, confirming that DPV VP22 is required for efficient viral growth in vitro. In addition, deletion of the UL49 gene inhibited the secondary envelopment of the virus, the release of viral particles, and the spread of viruses between cells. Our study signified the importance of VP22 for DPV secondary envelopment, release, cell-to-cell spread, and accumulation of viral RNA. These findings provide a basis for further study of the function of VP22 in DPV or other herpesviruses.

A preliminary study on the UL97 gene of cytomegalovirus isolated from patients suspected of ganciclovir resistance in Sri Lanka

Background: Human cytomegalovirus (HCMV) is an opportunistic virus causing infections in immunocompromised patients. The widespread use of ganciclovir to treat CMV infections has led to the development of ganciclovir resistance. To date, no studies have been reported in Sri Lanka on mutations in viral genes detected from CMV identified by PCR. No Sri Lankan studies have been reported to date of ganciclovir resistance associated CMV gene mutations. Objective: To screen the CMV UL97 gene identified in a cohort of patients who did not respond to ganciclovir for resistance conferring mutations. Methodology: A retrospective study was carried out on suspected ganciclovir resistant patients who had static or increased levels of CMV viraemia treated with ganciclovir for 2-3 weeks to determine mutations of UL97 gene. DNA of CMV was extracted; Codons 439 – 645 of the UL97 gene was amplified by PCR only from patients who did not respond to ganciclovir and two fragments of 354bp (1317-1671) and 285bp (1650-1935) were sequenced. Results: Twelve of the 340 patients with CMV infection had static or increased levels of CMV viraemia despite being treated with ganciclovir for 2-3 weeks. No mutation was found in the UL97 gene detected from the 12 patients which could confer ganciclovir resistance. However, there was an incidental finding of a single nucleotide change from thymine to cytosine at the 1368 nucleotide position of the UL97 gene which was detected in all 12 patients. Conclusion: No mutation was found in the UL97 gene from CMV patients in this preliminary study which could confer ganciclovir resistance. UL54 gene of CMV should be considered in addition to UL97 gene in future studies as mutations reported in both genes are known to contribute to ganciclovir resistance.

Establishment of an In Vitro Model of Pseudorabies Virus Latency and Reactivation and Identification of Key Viral Latency-Associated Genes.

Alphaherpesviruses infect humans and most animals. They can cause severe morbidity and mortality. The pseudorabies virus (PRV) is a neurotropic alphaherpesvirus that can infect most mammals. The PRV persists in the host by establishing a latent infection, and stressful stimuli can induce the latent viruses to reactivate and cause recurrent diseases. The current strategies of antiviral drug therapy and vaccine immunization are ineffective in eliminating these viruses from the infected host. Moreover, overspecialized and complex models are also a major obstacle to the elucidation of the mechanisms involved in the latency and reactivation of the PRV. Here, we present a streamlined model of the latent infection and reactivation of the PRV. A latent infection established in N2a cells infected with the PRV at a low multiplicity of infection (MOI) and maintained at 42 °C. The latent PRV was reactivated when the infected cells were transferred to 37 °C for 12 to 72 h. When the above process was repeated with a UL54-deleted PRV mutant, it was observed that the UL54 deletion did not affect viral latency. However, viral reactivation was limited and delayed. This study establishes a powerful and streamlined model to simulate PRV latency and reveals the potential role of temperature in PRV reactivation and disease. Meanwhile, the key role of the early gene UL54 in the latency and reactivation of PRV was initially elucidated.

Antiviral activity of mangiferin from the rhizome of Anemarrhena asphodeloides against herpes simplex virus type 1

Objective: To evaluate the antiviral activity of pure compounds against herpes simplex virus type 1 (HSV-1) from the rhizome of Anemarrhena asphodeloides. Methods: Bioassay-guided isolation was conducted to separate the active compound and its chemical structure was elucidated by spectral analysis. In vitro antiviral efficacy of active compound was detected by Cell Counting Kit-8 assay, plaque reduction assay, and fluorescence observation. RT-PCR was used to determine the viral load and the cytokine-related gene expression after HSV-1 infection. In vivo study was also conducted to further determine antiviral efficacy of an active compound against HSV-1. Results: An active compound was isolated and elucidated as mangiferin. Mangiferin significantly inhibited the replication of HSV-1 in Vero cells with a half maximal inhibitory concentration (IC50) of 64.0 mg/L. Time-of-addition and time-of-removal assays demonstrated that mangiferin could effectively inhibit the replication of HSV-1 in the early stage (8 h). UL12, UL42, and UL54 gene expression levels of HSV-1 in the 64 mg/L mangiferin-treated group were markedly reduced as compared with the HSV-1 group (P<0.01). Fluorescence observation showed that mangiferin attenuated the mitochondrial damage maintaining ΔΨm induced by HSV-1 in Vero cells. The expression of inflammatory factors TNF-α, IL- 1β, and IL-6 was remarkably increased in the virus-infected group as compared with that in the normal group (P<0.05), the levels of these inflammatory factors dropped after treatment with mangiferin. Mangiferin significantly decreased the viral load and attenuated the HSV-1-induced up-regulation of TNF-α, IL1β, and IL-6. The relative protection rate of HSV-1-infected mice could reach up to 55.5% when the concentration of mangiferin was 4 g/kg. Conclusions: Mangiferin exhibits promising antiviral activity against HSV-1 in vitro and in vivo and could be a potential antiviral agent for HSV-1.

Research Note: Duck plague virus pUL48 is a late protein that plays an important role in viral replication

Duck plague virus (DPV) pUL48 is a homologous of herpes simplex virus VP16, and some studies have shown that VP16 is essential for viral replication and proliferation, but there are few studies on DPV pUL48. Therefore, in order to study the function of pUL48 protein, we constructed a UL48-deleted mutant (DPV-BAC-∆UL48) that completely reemoved the UL48 gene from the DPV BAC genome and the revertant virus (DPV-BAC-∆UL48R) by using the 2-step red recombination system. Compared with the parental virus (DPV-BAC) and the revertant virus, the titer of UL48-deleted mutant was reduced by more than 38.2%, and the efficiency of producing infectious virions was significantly reduced. In addition, the average size of plaques produced by UL48-deleted mutant was about 30% smaller than that of the parental and revertant viruses, suggesting that pUL48 protein affected the cell-to-cell transmission of DPV. Finally, pharmacological inhibition assay showed that pUL48 is a late protein of DPV. In this study, we found that UL48, as a late gene, plays an important role in viral replication by affecting the formation of DPV infectious virion, virus cell-to-cell transmission, and viral genome transcription, which may provide some help for the study of the function of DPV pUL48 protein and the prevention and control of DPV.

Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells.

Herpes simplex virus type 1 (HSV-1) is a common virus of mankind and HSV-1 infections are a significant cause of blindness. The current antiviral treatment of herpes infection relies on acyclovir and related compounds. However, acyclovir resistance emerges especially in the long term prophylactic treatment that is required for prevention of recurrent herpes keratitis. Earlier we have established antiviral siRNA swarms, targeting sequences of essential genes of HSV, as effective means of silencing the replication of HSV in vitro or in vivo. In this study, we show the antiviral efficacy of 2´-fluoro modified antiviral siRNA swarms against HSV-1 in human corneal epithelial cells (HCE). We studied HCE for innate immunity responses to HSV-1, to immunostimulatory cytotoxic double stranded RNA, and to the antiviral siRNA swarms, with or without a viral challenge. The panel of studied innate responses included interferon beta, lambda 1, interferon stimulated gene 54, human myxovirus resistance protein A, human myxovirus resistance protein B, toll-like receptor 3 and interferon kappa. Our results demonstrated that HCE cells are a suitable model to study antiviral RNAi efficacy and safety in vitro. In HCE cells, the antiviral siRNA swarms targeting the HSV UL29 gene and harboring 2´-fluoro modifications, were well tolerated, induced only modest innate immunity responses, and were highly antiviral with more than 99% inhibition of viral release. The antiviral effect of the 2’-fluoro modified swarm was more apparent than that of the unmodified antiviral siRNA swarm. Our results encourage further research in vitro and in vivo on antiviral siRNA swarm therapy of corneal HSV infection, especially with modified siRNA swarms.

P1617: CLINICAL SIGNIFICANCE OF CYTOMEGALOVIRUS DRUG RESISTANCE GENE MUTATIONS IN CYTOMEGALOVIRUS INFECTION AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background: Cytomegalovirus (CMV) infection is the most important complication after allo-HSCT and threatens the prognosis of patients seriously. Currently, there are limited data on CMV drug-resistant gene mutations in CMV infection after transplantation in China. Aims: In order to further understand the expression of CMV drug-resistant gene mutations in patients with poor efficacy of CMV infection in single center tertiary hematology hospital in china. Methods: The clinical data of 41 patients with CMV infection after allo-HSCT were retrospectively analyzed from January 2019 to July 2021 in transplantation Center of Hematology Hospital, Chinese Academy of Medical Sciences, due to refractory and probable refractory CMV infection, who were tested for CMV resistance gene mutation. The current recommended definitions are based on Guidelines for the management of cytomegalovirus infection from ECIL 7. A total of 21 patients with AML, 8 patients with ALL, 6 patients with AA, 5 patients with MDS and 1 patients with Fanconi anemia are included. Donors were HLA haploid-identical related (N = 36), HLA-identical siblings and un-related (N = 5). Informed consent was obtained. Results: ① Of All 41 patients included, 1 patient (2.4%) had UL97 mutation, 19 patients (46.3%) had UL54 mutation, no patients occur simultaneously with UL54 and UL97 mutation. The overall mutation frequency was 48.8% (20/41), and all mutation types were missense mutation. ② UL54 gene were detected 8 cases of T691S missense mutation (8/41, 19.5%), 3 cases of M827I missense mutation (3/41, 7.3%) and 1 case of A786V missense mutation (1/41, 2.4%). All above these 3 mutation sites had been reported, but the drug resistance significance was unknown. There were 1 site mutation (A543S) of UL97 gene and 19 sites mutation of UL54 gene that had not been reported. The most common mutation site is the T691S mutation in UL54 gene. In all the 41 patients, only one patient exhibited a mutation with definite anti-CMV drug resistance significance: Q578H mutation of UL54 gene, and finally was successfully treated with twice CMV-CTL infusions (figure). ③ Compared with patients without UL54 mutation, patients with UL54 mutation strains had a higher proportion of CMV-DNA re positive rate in 10 days after remission of DNAemia (42.1% vs.9.5%, χ2=5.647, P=0.028), and hadthe significantly longer complete clearance time of CMV DNAemia (40 days vs. 24 days, Z=2.198, P=0.027). ④ Compared with those patients without CMV gene mutation, the patients with T691S mutation had a higher incidence in terms of CMV-DNA turned re positive in 10 days and 14 days after remission of DNAemia (71.4% vs. 19.0%, P=0.008; 71.4% vs. 9.5%, P=0.001; 71.4% vs. 9.5%, P=0.001, respectively). The median time of CMV-DNA reaching the peak was significantly later (14 days vs. 10 days, P=0.016). The median clearance time of CMV DNAemia was significantly longer (56 days vs. 24 days, P=0.002), and the differences were statistically significant. Compared with the non-T691s UL54 mutation, the patients with T691S mutation also had similar results. Image:Summary/Conclusion: CMV gene missense mutations were detected in 48.8% of patients infected with CMV after transplantation, most of which occurred in UL54 gene and T691S mutation is a common missense mutation. The incidence of missense mutations with clear drug resistance was low (2.4%) and might be induced by antiviral therapy. The median clearance time of CMV DNAemia in UL54 mutated patients was significantly longer, and the proportion of CMV-DNA turned re positive was increased, which might be caused by T691S mutation in UL54 gene.

Efficacy of an HSV-1 Neuro-Attenuated Vaccine in Mice Is Reduced by Preventing Viral DNA Replication.

We previously isolated an HSV-1 mutant, KOS-NA, that contains two non-synonymous mutations in UL39. One of the mutations, resulting in an R950H amino acid substitution in ICP6, renders KOS-NA severely neuro-attenuated and significantly reduces HSV-1 latency. Vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated eye diseases even at a very low immunizing dose, indicating its utility as a vaccine scaffold. Because KOS-NA contains a neuro-attenuating mutation in a single gene, we sought to improve its safety by deleting a portion of the UL29 gene whose protein product, ICP8, is essential for viral DNA replication. Whereas KOS-NA reduced replication of HSV-1 challenge virus in the corneal epithelium and protected mice against blepharitis and keratitis induced by the challenge virus, KOS-NA/8- and an ICP8- virus were significantly less efficacious except at higher doses. Our results suggest that the capacity to replicate, even at significantly reduced levels compared with wild-type HSV-1, may be an important feature of an effective vaccine. Means to improve safety of attenuated viruses as vaccines without compromising efficacy should be sought.

Analysis of Novel Drug-Resistant Human Cytomegalovirus DNA Polymerase Mutations Reveals the Role of a DNA-Binding Loop in Phosphonoformic Acid Resistance.

The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance.

Cytomegalovirus Infection and the Implications of Drug-Resistant Mutations in Pediatric Allogeneic Hematopoietic Stem Cell Transplant Recipients: A Retrospective Study from a Tertiary Hospital in China

IntroductionDrug-resistant cytomegalovirus (CMV) infection remains a challenge in the management of pediatric recipients of hematopoietic stem cell transplantation (HSCT). In this study, we retrospectively reviewed the clinical data on pediatric recipients of HSCT and identified known and unknown drug-resistant CMV variants.MethodsA total of 221 children underwent allogeneic HSCT between October 2017 and November 2019 at Shenzhen Children’s Hospital; of these, 35 patients were suspected of having drug-resistant CMV infections and were tested for drug-resistant mutations in the UL97 and UL54 genes by Sanger sequencing.ResultsMutations in UL97 or UL54, or in both, were detected in 11 patients. Most of these mutations have not been previously reported. The UL97 mutation (A582V) was detected in only one patient who also harbored two UL54 mutations (T760X and R876W). One patient with both the G604S and T691A mutations in the UL54 gene died of CMV pneumonia. We investigated the risk factors associated with the development of drug-resistant CMV infection. Patients in whom both the donor and recipient had positive CMV serostatuses were less likely to have drug-resistant mutations (Fisher’s exact test, p < 0.05).ConclusionNewly and previously detected CMV mutations in UL97 and UL54 may be associated with the development of drug-resistant CMV infection. The detection of these mutations may provide guidance for the management of post-transplant CMV infections.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40121-021-00452-4.

The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from -0.8 log10 to +0.8 log10, the majority of dPCR results were between -0.2 log10 to +0.2 log10. Only 4 out of 45 results exceeded this interval with the maximum deviation of -0.542 log10. In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than ±0.2 log10 from the target value, while more than 95% deviated less than ±0.4 log10 from the target value. Evaluation of intra- and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing.

Molecular Epidemiology of Cytomegalovirus UL97 and UL54 variants in Taiwan.

The antiviral resistance of cytomegalovirus (CMV) infections is associated with mutations in the CMV UL54 and UL97 gene regions and is a serious problem in immunocompromised patients. However, the molecular epidemiology of UL54 and UL97 in Taiwan is unclear. We conducted a retrospective study of patients with CMV infections between January and December 2016 in two tertiary hospitals, one regional hospital in Taiwan. CMV DNAemia was confirmed by elevated CMV DNA titers. Then the regions of the UL54 and UL97 mutations were amplified by PCR and sequenced. Of 729 patients with CMV syndrome, 112 CMV DNAemia patients were enrolled. Twelve novel variants in UL54 (P342S, S384F, K434R, S673F, T754M, R778H, C814S, M827I, G878E, S880L, E888K, and S976N) and one novel variant in UL97 (M615T) were discovered. UL97 antiviral resistance mutations (L595S, M460I, and M460V) were found in four patients (3.6%). In the drug resistance strains, the mutation events occurred after 83-150 days of therapy, and drug resistance was also observed in these patients. The following high frequency variants were observed: D605E in UL97 and A885T, N898D, V355A, N685S, and A688V in UL54. The results demonstrate that the positive rate of CMV DNAemia was 15.3% (112/729) among the patients with clinical CMV infection symptoms. The proportion of antiviral resistance CMV strains within CMV DNAemia patients was 3.6%. With the information of polymorphism incidence in the UL54 and UL97 patients from our study, determination of the genetic profile of UL54 and UL97 among immunocompromised populations with refractory CMV infection is recommended.

UL28 and UL33 homologs of Marek\u2019s disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells

Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek’s disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.

Newly detected mutations in the Meq oncogene and molecular pathotyping of very virulent Marek\u2019s disease herpesvirus in Tunisia

Marek's disease (MD) is a contagious avian viral disease that is responsible for large economic losses to farmers. The disease is caused by Marek's disease virus (species Gallid alphaherpesvirus 2), which causes neurological lesions, immune suppression, and tumor proliferation of lymphoid cells that invade a large number of organs and tissues. Despite widespread vaccination, Marek's disease virus (MDV), has shown a continuous increase in its virulence and has acquired the ability to overcome immune responses induced by vaccines. In the present study, the oncogenic serotype MDV-1 was detected by real-time PCR in DNA samples extracted from organs developing tumor infiltrations. Identification of the pathotype based on a 132-bp tandem repeat and sequencing and phylogenetic analysis of the Meq gene and its encoded protein allowed classification of the isolated viruses as "very virulent", with two new and unique mutations in the Meq gene resulting in amino acid substitutions. Sequencing of pp38, vIl-8, UL1 and UL44 genes did not reveal any new mutations that were characteristic of the Tunisian isolates or correlated with virulence. These results raised concerns about the ability of HVT and CVI988 vaccines, which are currently used in Tunisia and other countries, to protect chickens against highly virulent virus strains.

The Role of VP16 in the Life Cycle of Alphaherpesviruses.

The protein encoded by the UL48 gene of alphaherpesviruses is named VP16 or alpha-gene-transactivating factor (α-TIF). In the early stage of viral replication, VP16 is an important transactivator that can activate the transcription of viral immediate-early genes, and in the late stage of viral replication, VP16, as a tegument, is involved in viral assembly. This review will explain the mechanism of VP16 acting as α-TIF to activate the transcription of viral immediate-early genes, its role in the transition from viral latency to reactivation, and its effects on viral assembly and maturation. In addition, this review also provides new insights for further research on the life cycle of alphaherpesviruses and the role of VP16 in the viral life cycle.