Search

The covalent attachment of ubiquitin and ubiquitin-like polypeptides to cellular and viral proteins regulates numerous processes that enable virus infection, viral genome replication, and the spread of viruses to new hosts. The importance of these protein modifications in the regulation of the life cycle of herpesviruses is underscored by the discovery that all known members of this virus family encode at least one protease that specifically recognizes and disassembles ubiquitin conjugates. The structural and functional characterization of the viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infection and increasing evidence suggests that the viral deconjugases may also play a role in malignant transformation.

Economic importance of common carp (Cyprinus carpio L.) increases every year. Viral diseases are major threat for carp aquaculture and cause significant economic losses. Koi herpesvirus (KHV) is one of the most serious carp diseases. Current study is focused on confirmation of possible differences in early immune response to KHV depending on level of resistance. Class I interferon signalling, complement cascade and cell-mediated cytotoxicity are hypothesized as major mechanisms of early innate immune response against KHV. Different breeds of common carp show distinct level of resistance to KHV. Two breeds of common carp with completely different susceptibility to KHV were chosen for current research: amur wild carp (AS) as highly resistant and koi carp (KOI) as very susceptible breed. KHV infection caused no mortalities, but the viral load in selected tissues increased during infection. Levels of expressions of chosen genes was examined using qRT-PCR and overall change in protein expression profiles was analysed by mass spectrometry. Significant differences in immune response between AS and KOI were detected mostly at the level of protein expression. Although cell-mediated cytotoxicity showed minimal influence during KHV infection, many immune response parameters related to class I interferon signalling pathway and complement cascade were increased earlier during KHV infection in AS comparing to KOI.

The aim of this study was to describe the prevalence, characteristics and outcome of critically burn patients with pulmonary HSV reactivation.Retrospective, single-center cohort study in a burn critical care unit in a tertiary center, including all consecutive severely burn patients with bronchoalveolar lavage performed for pneumoniae suspicion and screened for HSV from January 2013 and April 2017. We used logistic regression to identify factors associated with HSV reactivation and outcomes.94 patients were included, mean age was 51 (39-64) years; median total body surface area burned was 36 (25-54)% and ICU mortality 38%. Fifty-five patients (59%) had pulmonary HSV reactivation and 30 (55%) were treated with acyclovir. Patients with HSV reactivation were more severely ill with higher SOFA score at admission compared to patient without HSV reactivation (6 [3-8] vs. 2 [1-4], p0.0001 respectively). In multivariate analysis, sex, SOFA score at admission and smoke inhalation were significantly associated with HSV reactivation. Only septic shock was associated with 90-day mortality when HSV reactivation was not.Pulmonary HSV reactivation is frequent among severely ill burn patients. Initial severity and smoke inhalation are risk factors. Antiviral treatment was not associated with outcome.

Human herpes simplex viruses (HSVs) belong to the Herpesviridae family. At present, no vaccine or curative treatment is available for the prevention of HSV infections. Here, we review the cell surface receptors that are recognized by HSV's glycoprotein B, glycoprotein C, glycoprotein D, and the glycoprotein H – glycoprotein L complex to facilitate entry into host cells. These receptors include heparan sulfate (HS), herpesvirus entry mediator (HVEM), and nectin-1/-2, 3-O-sulfated heparan sulfate (3-OS HS).

Epstein-Barr virus (EBV), discovered in 1964, is a double-stranded DNA virus belonging to the Herpesviridae family. EBV has a lymphoid tropism with transforming capacities using different oncogenic viral proteins. This virus has two replication cycles: a lytic cycle mainly occuring during primary infection and a latent cycle allowing viral persistence into host memory B cells. More than 90% of adults are seropositive for EBV worldwide, with a past history of asymptomatic or mild primary infection. EBV infection can sometimes cause life-threatening complications such as hemophagocytic lymphohistiocytosis, and lead to the development of lymphoproliferative disorders or cancers. Risk factors associated with these phenotypes have been recently described through the study of monogenic primary immune deficiencies with EBV susceptibility. We here review the virological and immunological aspects of EBV infection and EBV-related complications with an overview of current available treatments.

Background Ultra high-risk (UHR) criteria were introduced to identify people at imminent risk of developing psychosis. To improve prognostic accuracy, additional clinical and biological risk factors have been researched. Associations between psychotic disorders and infections with Toxoplasma gondii and Herpesviridae have been found. It is unknown if exposure to those pathogens increases the risk of transition to psychosis in UHR cohorts. Methods We conducted a long-term follow-up of 96 people meeting UHR criteria, previously seen at the Personal Assessment and Crisis Evaluation (PACE) clinic, a specialized service in Melbourne, Australia. Transition to psychosis was assessed using the Comprehensive Assessment of the At-Risk Mental State (CAARMS) and state public mental health records. The relationship between IgG antibodies to Herpesviridae (HSV-1, HSV-2, CMV, EBV, VZV) and Toxoplasma gondii and risk for transition was examined with Cox regression models. Results Mean follow-up duration was 6.46 (±3.65) years. Participants who transitioned to psychosis (n = 14) had significantly higher antibody titers for Toxoplasma gondii compared to those who did not develop psychosis (p = 0.03). After adjusting for age, gender and year of baseline assessment, seropositivity for Toxoplasma gondii was associated with a 3.6-fold increase in transition hazard in multivariate Cox regression models (HR = 3.6; p = 0.036). No significant association was found between serostatus for Herpesviridae and risk of transition. Conclusions Exposure to Toxoplasma gondii may contribute to the manifestation of positive psychotic symptoms and increase the risk of transitioning to psychosis in UHR individuals.

Human herpesviruses are double-stranded DNA viruses that are classified into nine species. More than 90% of adults are ever infected with one or more herpesviruses. The symptoms of infection with different herpesviruses are diverse ranging from mild or asymptomatic infections to deadly diseases such as aggressive lymphomas and sarcomas. Timely and accurate detection of herpesvirus infection is critical for clinical management and treatment. In this study, we established a single-tube nonuple qPCR assay for detection of all nine herpesviruses using a 2-D multiplex qPCR method with a house-keeping gene as the internal control. The novel assay can detect and distinguish different herpesviruses with 30 to 300 copies per 25 µL single-tube reaction, and does not cross-react with 20 other human viruses, including DNA and RNA viruses. The robustness of the novel assay was evaluated using 170 clinical samples. The novel assay showed a high consistency (100%) with the single qPCR assay for HHVs detection. The features of simple, rapid, high sensitivity, specificity, and low cost make this assay a high potential to be widely used in clinical diagnosis and patient treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12250-021-00354-2.

Summary Intracellular epidermal inclusions were detected within histological sections of skin biopsies from two panther chameleons (Furcifer pardalis) with chronic cheilitis. Transmission electron microscopy (TEM) confirmed the abundant presence of icosahedral intracytoplasmic and intranuclear viral particles in infected keratinocytes, with an average diameter of 120–125 nm, consistent with herpesviruses (HVs). TEM also revealed the presence of virions in intercellular spaces and keratinocyte nuclei and features suggestive of capsid assembly, nuclear egress with primary envelopment and anterograde transport leading to virion assembly and release. Polymerase chain reaction (PCR) primers targeting a conserved region of herpesvirus DNA-dependent DNA polymerase were used to amplify and sequence a product from a nested HV PCR performed on skin biopsies of both chameleons. Comparative sequence analysis indicates that the virus detected in both chameleons was a novel member of the Alphaherpesvirinae, which we refer to as chamaeleonid herpesvirus 1 (chamHV 1). Based on the identical findings in both chameleons, we consider chamHV 1 to be a candidate aetiological agent of cheilitis in panther chameleons. This is the first report of skin lesions in a chameleon species associated with HV infection.

Poxviruses and herpesviruses encode secreted versions of cytokine receptors as a unique strategy to evade the host immune response. Recent advances in the field have shown the great impact of some of these proteins in immune modulation and viral pathogenesis, and have uncovered unique properties of these viral proteins not found in the cellular counterparts. These modifications inspired by viruses lead to improved immune modulatory activity of the soluble cytokine receptors, information that has been used to develop more efficient therapeutics to treat inflammatory conditions.

Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen of goldfish (Carassius auratus) and Prussian carp (Carassius auratus gibelio) causing herpesviral hematopoietic necrosis. Our previous study revealed that CyHV-2 can persistently infect the kidney and spleen of goldfish that recovered from a primary infection. In this study, we tried to identify the cells persistently infected with the virus in surviving fish and investigated virus reactivation in the survivors injected with immunosuppressants, namely dexamethasone (Dex) and cyclosporine A (CsA). Virus DNA was detected from the monocytes that were isolated from the trunk kidney of the asymptomatic survivors, suggesting that monocytes/macrophages are major cells that may be persistently infected with CyHV-2. A significant increase of virus DNA levels was detected in the group injected with Dex at 10 and 21 days post-injection (dpi). In the fish group injected with CsA, the virus DNA level was the same as that in the control group at 10 dpi but increased in some organs at 21 dpi. Compared with Dex-injected fish at 10 dpi, the group injected with both Dex and CsA showed a greater increase in virus DNA levels. The gene expression of phagocytosis-associated genes, major histocompatibility complex (MHC) class II and p47phox, and anti-virus antibody levels increased in the CsA group due to virus reactivation in the infected cells but not in the Dex and Dex & CsA groups, indicating that Dex effectively suppressed monocyte/macrophage function and antibody production. In addition, recombinant interferon γ (IFNγ) supplementation in the kidney leukocyte culture that was isolated from survivors showed a reduction of virus DNA. CsA may inhibit T-helper 1 (Th1) cells and consequently IFNγ production, causing a synergetic effect with Dex on virus reactivation. The results suggest that the activity of monocytes/macrophages stimulated by IFNγ can relate to virus latency and reactivation in asymptomatic virus carriers.

Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1β, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.

Viruses infect their human hosts by a series of interactions between viral and host proteins, indicating that detailed knowledge of such virus-host interaction interfaces are critical for our understanding of viral infection mechanisms, disease etiology and the development of new drugs. In this review, we primarily survey human host-virus interaction data that are available from public databases following the standardized PSI-MS format. Notably, available host-virus protein interaction information is strongly biased toward a small number of virus families including herpesviridae, papillomaviridae, orthomyxoviridae and retroviridae. While we explore the reliability and relevance of these protein interactions we also survey the current knowledge about viruses functional and topological targets. Furthermore, we assess emerging frontiers of host-virus protein interaction research, focusing on protein interaction interfaces of hosts that are infected by different viruses and viruses that infect multiple hosts. Finally, we cover the current status of research that investigates the relationships of virus-targeted host proteins to other comorbidities as well as the influence of host-virus protein interactions on human metabolism.

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a β-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, β-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that β-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, β-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in β-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of β-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

Background Herpesviridae infections incur significant morbidity and indirect effects on mortality among allogeneic haematopoietic cell transplant (allo-HCT) recipients. Objectives To study the effects of antiviral prevention strategies among haemato-oncological individuals undergoing allo-HCT. Data sources Cochrane Central Register of Controlled Trials, MEDLINE, Embase and LILACS. We further searched for conference proceedings and trial registries. Study eligibility criteria Randomized controlled trials (RCTs). Participants Adults with haematological malignancy undergoing allo-HCT. Interventions Antiviral prophylaxis versus no treatment/placebo or pre-emptive treatment and pre-emptive treatment versus prophylaxis with the same agent. Methods Random-effects meta-analysis was conducted computing pooled risk ratios (RR) with 95% CI and the inconsistency measure (I2). The certainty of the evidence was appraised by GRADE. Results We included 22 RCTs. Antiviral prophylaxis reduced all-cause mortality (RR 0.83, 95% CI 0.7–0.99; 15 trials, I2 = 0%), cytomegalovirus (CMV) disease (RR 0.54, 95% CI 0.34–0.85; n = 15, I2 = 20%) and herpes simplex virus (HSV) disease (RR 0.29, 95% CI 0.2–0.43; n = 13, I2 = 18%) compared with no treatment/placebo or pre-emptive treatment, all with high-certainty evidence. Furthermore, antivirals reduced HSV infection, CMV pneumonitis, CMV infection and varicella zoster virus disease. Anti-CMV prophylaxis (+/– pre-emptive treatment) compared with pre-emptive treatment alone reduced non-significantly all-cause mortality (RR 0.78, 95% CI 0.6–1.02; n = 8, I2 = 0%), CMV disease (RR 0.47, 95% CI 0.23–0.97; n = 9, I2 = 30%) and HSV disease (RR 0.41, 95% CI 0.24–0.67; n = 4, I2 = 0%) with high-certainty evidence, as well as CMV and HSV infections. Antiviral prophylaxis did not result in increased adverse event rates overall or more discontinuation due to adverse events. Conclusions Antiviral prophylaxis directed against herpesviruses is highly effective and safe, reducing mortality, HSV and CMV disease, as well as herpesvirus reactivations among allo-HCT recipients. Anti-CMV prophylaxis is more effective than pre-emptive treatment alone with respect to HSV and CMV disease and infection.

Introduction Placental viral infections are associated with fetal inflammation and adverse pregnancy outcomes. However, there have been limited studies on how placental macrophages in the villous and adjacent fetal umbilical endothelial cells respond to a viral insult. This study aimed to evaluate the communication between Hofbauer cells (HBCs) and human umbilical vein endothelial cells (HUVECs) during a viral infection. Methods HBCs were either uninfected or infected with the γ-herpesvirus, MHV-68, and the conditioned medium (CM) collected. HUVECs were exposed to HBC CM and the levels of the pro-neutrophilic response markers: IL-8; E-selectin; intercellular adhesion molecule 1 (ICAM-1); and vascular adhesion molecule 1 (VCAM-1) measured by ELISA and qPCR. The role of HBC-derived IL-1β was investigated using an IL-1β blocking antibody (Ab) or IL-1 receptor antagonist (IL-1Ra). Results MHV-68 infection of HBCs induced a significant increase in IL-1β secretion. CM from infected HBCs induced HUVEC expression of IL-8, E-selectin, VCAM-1, ICAM-1 mRNA, and secretion of IL-8. The HUVEC response to the CM of MHV-infected HBCs was inhibited by a neutralizing IL-1β Ab and by IL-1Ra. Discussion Virally-induced HBC IL-1β activates HUVECs to generate a pro-neutrophilic response. This novel cell-cell communication pathway may play an important role in the genesis of fetal inflammation associated with placental viral infection.

Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail.

7 Pág., The family Trichechidae (order Sirenia) comprises three species African (Trichechus senegalenses), West Indian (T. manatus), WIM)], and the Amazonian manatees (T. inunguis, AMM). Whereas WIM inhabits both riverine and coastal systems in the western Atlantic, AMM is the only exclusively freshwater sirenian, endemic to the Amazon River Basin. The study of infectious agents is essential to species conservation, especially considering that both species are classified as Vulnerable by the IUCN Red List and as Endangered by the Brazilian Red List. The current knowledge about viral agents in sirenians is scarce. Herpesviruses and adenovirus are DNA viruses able to infect and cause disease in a wide range of hosts. Herein, we used panPCR protocols to survey herpesvirus and adenovirus in blood samples of wild WIM (n = 23) and AMM (n = 26) under human care in Brazil. Herpesvirus DNA was detected in one juvenile female WIM (1/23; 4.3%; 95% CI -4.7 – 13.3) from Ceará state and in four AMM (two juvenile females, a juvenile male, and an adult female; 4/26; 15.4%; 95% CI 0.5 – 30.3) from Amazonas state. The two different gammaherpesvirus DNA polymerase sequence types identified (one per species, a sequence type in a WIM and another one in three AMM) were highly similar (99% nucleotide identity) to Trichechid herpesvirus 1, reported in West Indian manatees of Florida (USA), and 100% identical when translated into amino acids. A herpesviral glycoprotein B sequence was identified in two AMM. None of the samples was positive to adenovirus. To the authors’ knowledge, this is the first herpesvirus detection in manatees from South America, expanding the herpesvirus geographical range, and the first in WIM and AMM worldwide. Our findings suggest (i) that West Indian and Amazonian manatees are possibly the natural hosts of the detected herpesvirus, and (ii) coevolution of that gammaherpesvirus with Trichechus. Future studies are necessary to characterize the obtained virus and elucidate potential pathological effects (if any) in these species., We thank the staff of AQUASIS and INPA for their logistical and technical support. We thank the Sao Paulo Research Foundation (FAPESP), the Brazilian National Council for Scientific and Technological Development (CNPq), and the Coordination for the Improvement of Higher Education Personnel (CAPES) for their support. Ana Carolina Ewbank and Samira Costa-Silva receive PhD Grants from FAPESP (process numbers 2018/20956–0 and 2020/12434–9). Aricia Benve nuto and Roberta Zamana Ramblas receive PhD Grants from CNPq [process numbers 141868/2019–8 and 165364/2018–1]. Carlos Sacristan and Irene Sacristan receive Juan the la Cierva incorporacion fellowships under process numbers IJC2020–046019-I and FJC2020–046311–1, respectively. Jose Luiz Catao-Dias is the recipient of a professorship from CNPq (304999–18). This study was approved by the Ethical Committee in Animal Research of the School of Veterinary Medicine and Animal Sciences, University of Sao Paulo (Process number 7151291019). The samples used in this study were collected in full compliance with specific federal permits issued by the Brazilian Ministry of Environment and approved by the Biodiversity Information and A.C. Ewbank et al.

International audience; The Pacific oyster Crassostrea gigas is established in the marine intertidal zone, experiencing rapid and highly dynamic environmental changes throughout the tidal cycle. Depending on the bathymetry, oysters face oxygen deprivation, lack of nutrients, and high changes in temperature during alternation of the cycles of emersion/ immersion. Here we showed that intertidal oysters at a bathymetry level of 3 and 5 m delayed by ten days the onset of mortality associated with Pacific Oyster Mortality Syndrome (POMS) as compared to subtidal oysters. Intertidal oysters presented a lower growth but similar energetic reserves to subtidal oysters but induced proteomic changes indicative of a boost in metabolism, inflammation, and innate immunity that may have improved their resistance during infection with the Ostreid herpes virus. Our work highlights that intertidal harsh environmental conditions modify host-pathogen interaction and improve oyster health. This study opens new perspectives on oyster farming for mitigation strategies based on tidal height.

Cyprinid herpesvirus 2 (CyHV-2) is the causative pathogen of herpesviral haematopoietic necrosis disease, which has caused huge economic losses to aquaculture industry in China. In this study, nine truncated CyHV-2 membrane glycoproteins (ORF25, ORF25C, ORF25D, ORF30, ORF124, ORF131, ORF136, ORF142A, ORF146) and a GFP reporter protein were respectively expressed using baculovirus surface displaying system. Western blot showed that the proteins were successfully packaged in the recombinant virus particles. In baculovirus transduced gibel carp kidney cells, the target proteins were expressed and displayed on the fish cell surface. Healthy gibel carp were immunized by immersion with the recombinant baculoviruses and the fish treated with phosphate-buffered saline (PBS) were served as mock group. The expression of interleukin-11 (IL-11), interferon α (IFNα) and a complement component gene C3 were significantly up-regulated in most experimental groups, and interferon γ (IFNγ) expression in some groups were also induced after immunization. Subsequently, the immunized gibel carp were challenged by intraperitoneal injection of CyHV-2 virus. All the immunized groups exhibited reduced mortality after CyHV-2 challenge. In the groups immunized with baculoviruses displaying and expressing ORF25, ORF25C and ORF146, the relative percentage survival values reached 83.3%, 87.5% and 70.8%, respectively. Our data suggested that baculovirus-displayed ORF25, ORF25C and ORF146 could be potential vaccine candidates for the prevention of CyHV-2 infection in gibel carp.

Aquaculture is one of the world's most important and fastest growing food production sectors, with an average annual growth of 5.8% during the period 2001-2016. Common carp (Cyprinus carpio) is one of the main aquatic species produced for human consumption and is the world's third most produced finfish. Koi carp, on the other hand, are grown as a popular ornamental fish. In the late 1990s, both of these sectors were threatened by the emergence of a deadly disease caused by cyprinid herpesvirus 3 (CyHV-3; initially called koi herpesvirus or KHV). Since then, several research groups have focused their work on developing methods to fight this disease. Despite increasing knowledge about the pathobiology of this virus, there are currently no efficient and cost-effective therapeutic methods available to fight this disease. Facing the lack of efficient treatments, safe and efficacious prophylactic methods such as the use of vaccines represent the most promising approach to the control of this virus. The common carp production sector is not a heavily industrialized production sector and the fish produced have low individual value. Therefore, development of vaccine methods adapted to mass vaccination are more suitable. Multiple vaccine candidates against CyHV-3 have been developed and studied, including DNA, bacterial vector, inactivated, conventional attenuated and recombinant attenuated vaccines. However, there is currently only one vaccine commercially available in limited regions. The present review aims to summarize and evaluate the knowledge acquired from the study of these vaccines against CyHV-3 and provide discussion on future prospects.

Cyprinid herpesvirus II (CyHV-2) is highly contagious and pathogenic to Carassius auratus gibelio (gibel carp), causing enormous economic losses in aquaculture in Yancheng city, Jiangsu province, China; however, to date, there is no effective way to protect C. auratus gibelio from CyHV-2 infection. In this study, a recombinant baculovirus vector vaccine, BacCarassius-D4ORFs, containing a fused codon-optimized sequence D4ORFs comprising the ORF72 (region 1–186 nt), ORF66 (region 993–1197 nt), ORF81 (region 603–783 nt) and ORF82 (region 85–186 nt) genes of CyHV-2, driven by a Megalobrama amblycephala β-actin promoter, was constructed. Then, qPCR, Western blotting and immunofluorescence assays showed that the fused gene D4ORFs was successfully delivered and expressed in fish cells or tissues by transduction with BacCarassius-D4ORFs. The fused gene D4ORFs could not be detected by PCR in the C. auratus gibelio injected with BacCarassius-D4ORFs after 7 weeks. Specific antibody against ORF72 could be detected in the serum of vaccinated C. auratus gibelio by injection with BacCarassius-D4ORFs. Furthermore, when C. auratus gibelio were vaccinated with BacCarassius-D4ORFs via the oral or injection route, followed by challenge with CyHV-2, the relative survival rate of immunized C. auratus gibelio reached 59.3% and 80.01%, respectively. These results suggested that BacCarassius-D4ORFs has the potential to be used as a vector-based vaccine for the prevention and treatment of disease caused by CyHV-2 infection.

Horses are often used as blood donors for commercial horse serum (HS) production and to manufacture biologicals. HS is an alternative for fetal bovine serum (FBS) used as a supplement for cell culture and vaccine production. Furthermore, HS is also frequently obtained in order to produce antisera toxins and pathogens. The advent of high-throughput sequencing (HTS) has promoted changes in virus detection, since previous knowledge of targets is not required. Thus, the present study aimed to describe the virome of five different batches of commercial HS from New Zealand (three batches) and Brazil and the United States (one batch each) using HTS. Each HS pool were processed and sequenced using an Illumina MiSeq platform. Sequences-related to viruses belonging to the Flaviviridae, Herpesviridae, and Parvoviridae families were detected. Particularly, equine hepacivirus (EqHV), equine pegivirus (EPgV), and Theiler's disease-associated virus (TDAV) were more frequent found in the batches analyzed. The presence of viral genomes in cell culture sera illustrates that these commercial sera can contain a mixture of different viruses and, therefore, can be regarded as potentially infectious for susceptible hosts. Moreover, the innocuity of commercial HS is important for the efficiency and security of diagnostics and the production of biological products.

Type I interferons, as a class of multipotent cytokines, play a key role in host antiviral immune responses. In this study, a type I IFN coding gene of gibel carp, Carassius auratus gibelio, CagIFNa was cloned and sequenced. The full-length cDNA sequence of CagIFNa consists of 724 nucleotides that encode a predicted protein of 183 amino acids. CagIFNa has two highly conserved cysteine residues in the deduced protein, which is mostly conserved in the fish group I type I IFNs. CagIFNa was identified as a member of the IFNa subgroup of group I type I IFNs by phylogenetic analysis. CagIFNa transcripts were detected in all investigated tissues with higher levels in the liver, intestine, spleen and head kidney of gibel carp. Following injection with Cyprinid herpesvirus 2 (CyHV-2), CagIFNa gene expression was significantly inhibited in the spleen but delayed and then increased in head kidneys. Similarly, while CagIFNa expression was rapidly induced in gibel carp brain (GiCB) cells by poly I:C stimulation and its high induction level was delayed following CyHV-2 infection. CagIFNa overexpression in GiCB cells drastically reduced virus CPE and titer. Furthermore, several genes associated with type I IFN signaling pathway including IRF3, IRF7, IRF9, STAT1, Mx1 and PKR were induced in GiCB cells overexpressing CagIFNa upon CyHV-2 infection. These results show that CagIFNa plays a role in antiviral immune system in gibel carp.

The aim of this review was to provide an updated overview of drug-induced hypersensitivity syndrome (DiHS)/drug reaction with eosinophilia and systemic symptoms (DRESS). Several new insights have been made, particularly with regards to the diagnosis, pathogenesis and care of some important complications and sequelae. The indication of herpesvirus reactivations in diagnosis in the assessment of disease severity is now better specified. Nevertheless, because fatal complications and autoimmune sequelae have been under-recognized, there is a clear need to identify effective parameters for assessing disease severity and predicting prognosis of the disease in the early phase. In this regard, we have established a scoring system that can be used to monitor severity, predict prognosis and stratify the risk of developing severe complications including fatal cytomegalovirus (CMV) disease. Regulatory T cells are likely to be central to the mechanism and would represent potential targets for therapeutic approaches that can ameliorate inflammatory responses occurring at the acute phase while preventing the subsequent development of harmful outcomes, such as CMV disease and autoimmune diseases. Keywords: Corticosteroids, Drug-induced hypersensitivity syndrome (DiHS), Drug reaction with eosinophilia and systemic symptoms (DRESS), Herpesviruses, Regulatory T cells

The aim of the study was to determine the seroprevalence of feline panleukopenia virus (FPV), feline herpesvirus type 1 (FHV-1) and feline calicivirus (FCV) in stray colony cats from Milan, Italy. Cats were divided in groups based on age, gender, reproductive status, health status and colony of origin. Blood samples were tested with an in-clinic ELISA test. The possible presence of a link between the antibody titre or the presence of seropositive results and the independent variables (age, gender, reproductive status, health status and colony location) was assessed by means of multinomial and univariate logistic regression models, respectively. Seroprevalence of 85.4% was reported for FCV. The diffusion of the other two pathogens in the cat population was much lower compared to FCV, with 45.7% and 37.1% seroprevalence observed for FPV and FHV-1, respectively. An increase of antibody titres from kitten to senior was generally observed for the three pathogens. Age was a statistically significant variable for FHV-1, with senior cats significantly associated with higher antibody titres and higher percentages of seropositive animals compared to younger age groups. Neutered cats had significantly higher antibody titres and showed significantly higher FHV-1 seroprevalences compared to sexually intact cats. Colonies from two of the nine administrative districts of Milan showed significantly higher FPV seroprevalences compared to the others. No other significant differences were observed. Our results, based on cats belonging to 70 different colonies located in urban areas far from each other, suggest that the three viruses circulate in the feline population of stray cats in Milan. The feline calicivirus represents the most common circulating pathogen, as observed also in other studies worldwide. Finally, our results suggest that stray cats may be not adequately protected against FPV, FHV-1 and FCV and vaccination could be a possible strategic solution, especially for FPV.

Discovering new therapeutics for human viral diseases is important for combatting emerging infectious viruses and omnipresent circulating viruses as well as those that can become resistant to the drugs we currently have available. The innate host defense peptide (HDP) repertoire present in animals is a wealth of potential antimicrobial agents that could be mined to meet these needs. While much of the body of research regarding HDPs is in the context of bacteria, there is increasing evidence that they can be an effective source for antivirals. Peptides can be identified in a number of ways, including eco-conservation-minded approaches. Those shown to have antiviral properties can be modified to exhibit desired properties as the relationship between structure and function is elucidated and then developed into therapeutics for human use. This review looks at the discovery and therapeutic potential of HDPs for human viral infections.

We developed a computational approach called Domain-architecture Aware Inference of Orthologs (DAIO) for the analysis of protein orthology by combining phylogenetic and protein domain-architecture information. Using DAIO, we performed a systematic study of the proteomes of all human Herpesviridae species to define Strict Ortholog Groups (SOGs). In addition to assessing the taxonomic distribution for each protein based on sequence similarity, we performed a protein domain-architecture analysis for every protein family and computationally inferred gene duplication events. While many herpesvirus proteins have evolved without any detectable gene duplications or domain rearrangements, numerous herpesvirus protein families do exhibit complex evolutionary histories. Some proteins acquired additional domains (e.g., DNA polymerase), whereas others show a combination of domain acquisition and gene duplication (e.g., betaherpesvirus US22 family), with possible functional implications. This novel classification system of SOGs for human Herpesviridae proteins is available through the Virus Pathogen Resource (ViPR, www.viprbrc.org).

Herpesvirus (HV) infections in cetaceans are frequently associated with skin and mucosal lesions. Although HV infections have been reported worldwide, their occurrence in southern Atlantic marine mammals is still poorly understood. We tested skin, oral and genital mucosal beta-actin PCR-positive samples from 109 free-ranging Brazilian cetaceans using a universal herpesvirus DNA polymerase PCR. Herpesvirus-positive skin samples from a Guiana dolphin (Sotalia guianensis), a dwarf sperm whale (Kogia sima), a Bolivian river dolphin (Inia boliviensis), and a lingual sample from an Atlantic spotted dolphin (Stenella frontalis) were histologically evaluated. Additional tissue samples from these animals were also PCR-positive for HV, including a novel sequence obtained from the dwarf sperm whale's stomach and mesenteric lymph node. Four novel HV species were detected in the Guiana dolphin (one), the dwarf sperm whale (two) and the Bolivian river dolphin (one). The cutaneous lesions (marked, focally extensive, chronic proliferative dermatitis) of the Guiana dolphin and the Bolivian river dolphin were similar to previous HV reports in cetaceans, despite the absence of intranuclear inclusion bodies. This is the largest HV survey in South American cetaceans and the first detection of HV infection in riverine dolphins worldwide.

Highlights • A quantitative LAMP method for identification of EEDV has been developed. • Analytical sensitivity of the qLAMP is as low as 78 pg extracted DNA from tissue. • The method is highly specific for EEDV. • The EEDV qLAMP method was evaluated against the qPCR method., Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl2, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus-1, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R2 = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.

The synthesis of constrained nucleosides has become an important tool to understand the SAR in the interaction between biological and synthetic nucleotides in the context of antisense oligonucleotide therapy. The incorporation of a cyclopropane into a furanose ring of a nucleoside induces some degree of constrain without affecting significantly the steric environment of a nucleoside. Here, we report a new, short and stereocontrolled synthesis of two constrained nucleosides analogues, 1′,2′- methano-2′,3′-dideoxyuridine 9, and the corresponding cytidine analog 12. X-ray crystallography revealed that the furanose ring in the constrained uridine and cytidine analogues was flattened with virtual loss of pseudorotation. The phosphoramidate esters of the novel constrained uridine and cytidine nucleosides, intended as prodrugs, were tested in cell-based assays for viral replication across the herpes virus family and HIV inhibition courtesy of Merck laboratories, Rahway. They were also tested in antiproliferative assays against colorectal and melanoma cell lines. Unfortunately, none of the compounds showed activity in these assays.

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus is a global pathogen causing mass mortality in koi and common carp, against which improved vaccines are urgently needed. In this study we investigated the role of four nonessential, but immunogenic envelope glycoproteins encoded by members of the ORF25 gene family (ORF25, ORF65, ORF148 and ORF149) during CyHV-3 replication. Single deletion of ORF65 did not affect in vitro replication, and deletion of ORF148 even slightly enhanced virus growth on common carp brain (CCB) cells. Deletions of ORF25 or ORF149 led to reduced plaque sizes and virus titers, which was due to delayed entry into host cells. An ORF148/ORF149 double deletion mutant exhibited wild-type like growth indicating opposing functions of the two proteins. Electron microscopy of CCB cells infected with either mutant did not indicate any effects on virion formation and maturation in nucleus or cytoplasm, nor on release of enveloped particles. The ORF148, ORF149 and double deletion mutants were also tested in animal experiments using juvenile carp, and proved to be insufficiently attenuated for use as live virus vaccines. However, surviving fish were protected against challenge with wild-type CyHV-3, demonstrating that these antibody inducing proteins are dispensable for an efficient immune response in vivo.

Herpesvirus (HVs) infection has already been reported in cetaceans, but available information on its epidemiology is scarce. In this study we surveyed a total of 179 cetaceans belonging to 6 different species. Samples were obtained from cetaceans stranded along the Portuguese coastline, belonging to populations that roam the north-east region of the Atlantic Ocean. Detection of HVs was performed by conventional nested PCR. Amplicons were sequenced by Sanger's method and sequences used to construct phylogenetic trees by Maximum Likelihood method. Our results show that prevalence of positive samples, among fresh carcasses, reached 14.3% (10/70) and both alpha and gammaherpesvirus were detected. Histopathology showed that herpesvirus infection varied from absence of signs compatible with disease, localized genital lesions and systemic disease. Phylogenetic analysis revealed three clusters within the alphaherpesvirus family; within the gammaherpesvirus no subdivision was detected. All clusters included animals from different species and geographic origins. In seven of the positive HVs samples, co-infections with other agents such as morbillivirus and toxoplasma gondii were detected. The viral nucleotide sequences were not assigned to a specific animal species, nor presented a given geographic distribution, which may imply a wider distribution of herpesvirus in these animal populations. Our results are also the first report of herpesvirus infection in common dolphins (Delphinus delphis), with both alpha and gammaherpesvirus detected.

A key step in the replication of human cytomegalovirus (HCMV) in the host cell is the generation and packaging of unit-length genomes into preformed capsids. Enzymes required for this process are so-called terminases, first described for double-stranded DNA bacteriophages. The HCMV terminase consists of the two subunits, the ATPase pUL56 and the nuclease pUL89, and a potential third component pUL51. The terminase subunits are essential for virus replication and are highly conserved throughout the Herpesviridae family. Together with the portal protein pUL104 they form a powerful biological nanomotor. It has been shown for tailed dsDNA bacteriophages that DNA translocation into preformed capsid needs an extraordinary amount of energy. The HCMV terminase subunit pUL56 provides the required ATP hydrolyzing activity. The necessary nuclease activity to cleave the concatemers into unit-length genomes is mediated by the terminase subunit pUL89. Whether this cleavage is mediated by site-specific duplex nicking has not been demonstrated, however, it is required for packaging. Binding to the portal is a prerequisite for DNA translocation. To date, it is a common view that during translocation the terminase moves along some domains of the DNA by a binding and release mechanism. These critical structures have proven to be outstanding targets for drugs to treat HCMV infections because corresponding structures do not exist in mammalian cells. Herein we examine the HCMV terminase as a target for drugs and review several inhibitors discovered by both lead-directed medicinal chemistry and by target-specific design. In addition to producing clinically active compounds the research also has furthered the understanding of the role and function of the terminase itself.

Motor symptoms of Parkinson's disease (PD) are apparent after a high proportion of dopamine neurons in the substantia nigra have degenerated. The vast majority of PD cases are sporadic, and the underlying pathobiological causes are poorly understood. Adults exhibit great variability in the numbers of nigral dopamine neurons, suggesting that factors during embryonic or early life regulate the development and physiology of dopaminergic neurons. Furthermore, exposure to infections and inflammation in utero has been shown to affect fetal brain development in models of schizophrenia and autism. Here, we utilize a mouse maternal infection model to examine how maternal herpesvirus infection impacts dopaminergic neuron-related gene and protein expression in the adult offspring.Pregnant mice were injected with murine cytomegalovirus (MCMV), murine gamma herpes virus-68 (MHV68) or phosphate buffered saline (PBS) at embryonic day 8.5. Offspring were sacrificed at eight weeks of age and midbrains were processed for whole genome RNA sequencing, DNA methylation analysis, targeted protein expression and high-performance liquid chromatography for quantification of dopamine and its metabolites.The midbrain of adult offspring from MHV68 infected dams had significantly decreased expression of genes linked to dopamine neurons (Th, Lmx1b, and Foxa1) and increased Lrrk2, a gene involved in familial PD and PD risk that associates with neuroinflammation. Deconvolution analysis revealed that the proportion of dopamine neuron genes in the midbrain was reduced. There was an overall increase in DNA methylation in the midbrain of animals from MHV68-infected dams and pathway analyses indicated mitochondrial dysfunction, with reductions in genes associated with ATP synthesis, mitochondrial respiratory chain, and mitochondrial translation in the offspring of dams infected with MHV68. TIGAR (a negative regulator of mitophagy) and SDHA (mitochondrial complex II subunit) protein levels were increased, and the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum were increased in these offspring compared to offspring from uninfected control dams. No such changes were observed in the offspring of dams infected with MCMV.Our data suggest that maternal infection with Herpesviridae, specifically MHV68, can trigger changes in the development of the midbrain that impact dopamine neuron physiology in adulthood. Our work is of importance for the understanding of neuronal susceptibility underlying neurodegenerative disease, with particular relevance for PD.

Summary Amyloid-β peptide (Aβ) fibrilization and deposition as β-amyloid are hallmarks of Alzheimer's disease (AD) pathology. We recently reported Aβ is an innate immune protein that protects against fungal and bacterial infections. Fibrilization pathways mediate Aβ antimicrobial activities. Thus, infection can seed and dramatically accelerate β-amyloid deposition. Here, we show Aβ oligomers bind herpesvirus surface glycoproteins, accelerating β-amyloid deposition and leading to protective viral entrapment activity in 5XFAD mouse and 3D human neural cell culture infection models against neurotropic herpes simplex virus 1 (HSV1) and human herpesvirus 6A and B. Herpesviridae are linked to AD, but it has been unclear how viruses may induce β-amyloidosis in brain. These data support the notion that Aβ might play a protective role in CNS innate immunity, and suggest an AD etiological mechanism in which herpesviridae infection may directly promote Aβ amyloidosis.

In the past decades, global freshwater fish production has been rapidly growing, while cyprinid takes the largest portion. Along with the rapid rise of novel forms of intensive aquaculture, increased global aquatic animal movement and various anthropogenic stress to aquatic ecosystems during the past century, freshwater fish farming industry encounter the emergence and breakout of many diseases, especially viral diseases. Because of the ability to safely and effectively prevent aquaculture diseases, vaccines have become the mainstream technology for prevention and control of aquatic diseases in the world. In this review, authors summarized six major cyprinid viral diseases, including koi herpesvirus disease (KHVD), spring viraemia of carp (SVC), grass carp hemorrhagic disease (GCHD), koi sleepy disease (KSD), carp pox disease (CPD) and herpesviral haematopoietic necrosis (HPHN). The present review described the characteristics of these diseases from epidemiology, pathology, etiology and diagnostics. Furthermore, the development of specific vaccines respective to these diseases is stated according to preparation methods and immunization approaches. It is hoped that the review could contribute to aquaculture in prevention and controlling of cyprinid viral diseases, and serve the healthy and sustainable development of aquaculture industry., Highlights • Reviewed the six most popular cyprinid viral diseases. • Summarized the development of aquaculture vaccine. • Characterized the vaccines specifically against KHV, GCRV and SVCV in detail.

Since emerged in the late 1990s, cyprinid herpesvirus 3 (CyHV-3) has caused huge economic losses in common and koi carp culture worldwide. Accumulating evidences suggest that teleost fish microRNA (miRNA), a class of non-coding RNA of ∼22 nucleotides, can participate in many cellular processes, especially in host antiviral defenses. However, the roles of miRNAs in CyHV-3 infection are still unclear. Here, using high-throughput miRNA sequencing and quantitative real-time PCR (qRT-PCR) verification, we found that miR-155 was significantly upregulated in common carp brain (CCB) cells upon CyHV-3 infection. Overexpression of miR-155 effectively inhibited CyHV-3 replication in CCB cells and promoted type I interferon (IFN-I) expression. Further study revealed that miR-155 targeted the 3' untranslated region (UTR) of the mRNA of 5'AMP-activated protein kinase (AMPK), and that AMPK could interact with and degrade the mitochondrial antiviral signaling protein (MAVS), resulting in the reduction of interferon (IFN) expression. Collectively, our results show that miR-155, induced by CyHV-3 infection, exhibits anti-CyHV-3 activity via regulating AMPK-MAVS-IFN axis, which will help design anti-CyHV-3 drugs.

Natural products represent valuable chemical scaffolds for drug development. A recent success story in this context was artemisinin, which is not only active against malaria but also to other diseases. This raised the interest of artemisinin's potential for drug repurposing. On the present review, we give an overview on artemisinin's antiviral activity. There is good in vitro and in vivo evidence for the activity of artemisinin and its derivatives against DNA viruses of the Herpesviridae and Hepadnaviridae families such as cytomegaloviruses, human herpesvirus 6, herpes simplex viruses 1 and 2, Epstein-Barr virus and Hepatitis B virus. The evidence is weaker for Polyomaviruses and papilloma viruses. Weaker or no inhibitory activity in vitro has been reported for RNA viruses such as human immunodeficiency viruses 1 and 2, hepatitis C virus, influenza virus and others. Interestingly, the artemisinin derivative artesunate did not exert cross-resistance to ganciclovir-resistant HCMV and exerted synergistic inhibition in combination with several clinically established antiviral standard drugs. The antiviral activity of first generation artemisinin derivatives (e.g. artesunate, artemether, etc.) was enhanced by novel derivatives, including dimer and trimer molecules. First results on patients indicating activity in a subset of HCMV patients. Novel developments in the field of nanotechnology and synthetic biology to bioengineer microorganisms for artemisinin production may pave the way for novel drugs to fight viral infections with artemisinin-based drugs.

The establishment and in-depth characterization of a novel continuous cell line derived from fin tissue of common carp ( Cyprinus carpio) , CCApin, is reported. The cells of the cell line could be propagated in Leibovitz's L-15 medium containing 15% foetal calf serum and 0.5% carp serum for >150 passages during the last 24 months, with a stable fast growth. Furthermore, antibody staining indicated that cell types obtained in primary cultures, containing the epithelial stem-cell marker tumorprotein 63, were different from cells in long-term cell cultures, containing tight junction protein zona occludens 1 and cytokeratin 7. These observations suggest a switch of dominant cell types. Molecular analysis of gene expression profiles of caudal fin tissue and CCApin cells showed that genes relevant in epithelial cells but also in mesenchymal cells were expressed. However, during cultivation of CCApin a set of very steadily expressed, primarily mesenchymal genes like collagen 1 alpha 1, fibronectin or cadherin 2 was found. In summary, the long-term cell culture could be described as a stably growing epithelial population with some mesenchymal features. There are several application possibilities, especially for virus susceptibility studies, e.g. cyprinid herpesvirus-3 (CyHV-3). The study leads to a better understanding of molecular and physiological mechanisms of in vitro fish cell cultures.

The search for new compounds with a broad spectrum of antiviral activity is important and requires the evaluation of many compounds against several distinct viruses. Researchers attempting to develop new antiviral therapies for DNA virus infections currently use a variety of cell lines, assay conditions and measurement methods to determine in vitro drug efficacy, making it difficult to compare results from within the same laboratory as well as between laboratories. In this paper, we describe the assessment of antiviral activity of a set of nucleoside analogs against BK polyomavirus, JC polyomavirus, Epstein-Barr virus, human herpesvirus 6B, and human herpesvirus 8 in an automated 384-well format and utilize qPCR assays to measure the accumulation of viral DNA. In an accompanying paper, we present a standardized approach to evaluating antivirals against additional herpesviruses, orthopoxviruses, and adenovirus. Together, they reveal new activities for reference compounds and help to define the spectrum of antiviral activity for a set of nucleoside analogs against a set of 12 DNA viruses that infect humans including representative human herpesviruses, orthopoxviruses, adenoviruses, and polyomaviruses. This analysis helps provide perspective on combinations of agents that would help provide broad coverage of significant pathogens in immunocompromised patients as well as against emerging infections.

Human cytomegalovirus (HCMV) is a major human pathogen with seropositivity rates in the adult population ranging between 40% and 95%. HCMV infection is associated with severe pathology, such as life-threatening courses of infection in immunocompromised individuals and neonates. Current standard therapy with valganciclovir has the disadvantage of adverse side effects and viral drug resistance. A novel anti-HCMV drug, letermovir, has been approved recently, so that improved therapy options are available. Nevertheless, even more so far unexploited classes of compounds and molecular modes of action will be required for a next generation of antiherpesviral treatment strategies. In this study, we focused on the analysis of the antiviral potency of a novel class of compounds, i.e. pyrrolopyridine analogs, and identified both hit compounds and their target protein candidates. In essence, we provide novel evidence as follows: (i) screening hit SC88941 is highly active in inhibiting HCMV replication in primary human fibroblasts with an EC50 value of 0.20 ± 0.01 μM in the absence of cytotoxicity, (ii) inhibition occurs at the early-late stage of viral protein production and shows reinforcing effects upon LMV cotreatment, (iii) among the viruses analyzed, antiviral activity was most pronounced against β-herpesviruses (HCMV, HHV-6A) and intermediate against adenovirus (HAdV-2), (iv) induction of SC88941 resistance was not detectable, thus differed from the induction of ganciclovir resistance, (v) a linker-coupled model compound was used for mass spectrometry-based target identification, thus yielding several drug-binding target proteins and (vi) a first confocal imaging approach used for addressing intracellular effects of SC88941 indicated qualitative and quantitative alteration of viral protein expression and localization. Thus, our findings suggest a multifaceted pattern of compound-target binding in connection with an unusual mode of action, opening up further opportunities of antiviral drug development.

Cyprinid herpesvirus 2 (CyHV-2) infection is a recently discovered viral disease of farmed silver crucian carp (Carassius auratus gibelio) in mainland China that has resulted in massive fish mortality and appears to be spreading cross the world. Various detection methods based on nucleic acids have been developed, but effective immunological diagnostic method has not been reported for CyHV-2. In this study, recombinant ORF92 protein was used as a capture antigen to identify cells and tissues infected with CyHV-2 by immunological methods. Firstly, the open reading frame of CyHV-2 ORF92 was cloned into the PGEX-4T-3 vector and expressed in Escherichia coli. Purified recombinant ORF92 protein was then used as an antigen to prepare monoclonal antibodies, and an efficient hybridoma cell line was selected by dot-blot assay. The resulting mAb-1B7 was applied to detect CyHV-2 in infected Ryukin goldfish fin (RyuF-2) cells and fish tissues by western blotting and immunofluorescence assays. The monoclonal antibody could specifically identify CyHV-2 in infected RyuF-2 cells and gill, kidney, and spleen tissues, suggesting it could be applied for CyHV-2 detection in crucian carp.

Cyprinid herpesvirus 3 (CyHV-3) infection in carp causes a fatal and highly contagious disease that results in huge economic losses in common and koi carp aquaculture worldwide. Thus the development of an effective vaccine to protect carp stocks against the CyHV3 virus is imperative. In this study, we immunized common carps with a DNA vaccine consisting of a plasmid that co-expresses the CyHV-3 envelope protein ORF25 and the carp IL-1β gene in order to evaluate the adjuvant potential of IL-1β. Our result shows that antibodies specific to ORF25 can be detected as early as one week after intramuscular injection of the DNA vaccine at low dosage. Moreover, the co-expression of IL-1β can enhance the potency of the vaccine, as demonstrated by a higher antibody level after the third immunizations. Importantly, the DNA vaccine reduced mortality in carps when they were immunized prior to a CyHV-3 challenge, as compared to negative control groups. However, despite being able to induce higher neutralizing antibody titres, the co-expression of IL-1β in the DNA vaccine did not significantly improve the overall survival of immunized fish following virus challenge. Furthermore, the DNA vaccine can protect carps from tissue damage and histopathological alteration caused by viral infection. These strongly suggests that the vaccine can efficiently elicit protective immunity against CyHV-3 infection. In conclusion, the DNA vaccine formulated with the pIRES-ORF25-IL-1β DNA construct can protect carp against CyHV-3 infection and has potential applicability in the aquaculture industry.

Diverse immunoglobulin (Ig) domain-containing protein (DICP) family is a novel bony fish-specific multi-gene family encoding diversified immune receptors. However, their function and the implication of binding partners remain unknown. In this study, we first identified 28 DICPs from three gibel carp gynogenetic clones and revealed their high variability and clone-specific feature. After crucian carp herpesvirus (CaHV) infection, these DICPs were significantly upregulated in head kidney, kidney and spleen. The up-regulation folds in clone A+, F and H were related to the susceptibility to CaHV, progressively increasing from resistant clone to susceptible clone. Overexpression of gibel carp DICPs inhibited interferon (IFN) and viperin promoter-driven luciferase activity. The additions of E. coli extracts and lipid A significantly enhanced the inhibition effect. In addition, gibel carp DICPs can interact with SHP-1 and SHP-2. These findings suggest that gible carp DICPs, as inhibitory receptors, might specifically recognize lipid A, and then interact with SHP-1 and SHP-2 to inhibit the induction of IFN and ISGs.

The herpesviridae family are important causes of central nervous system disease in children. We review the spectrum of disease caused by the viruses in the context of a brief description of their epidemiology and transmission. Herpesviridae establish lifelong latency following primary infection and we clearly categorize those diseases associated with primary infection and those associated with reactivation with a particular emphasis on the immunocompromised child. Though a causal association with neurological disease in children is established for HSV and VZV syndromes, we identify the ongoing challenge of establishing causation for other viruses because of the rarity of presentations and viral latency. We review the diagnosis, management and outcome issues with recognition that more research is required.

Crucian carp (Carassius auratus gibelio) is a popular food fish in Asia, and cyprinid herpesvirus 2 (CyHV-2) is the only known viral pathogen for crucian carp. Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms. In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge, a partial interferon transcript was identified to be significantly up-regulated in the kidney of infected fish, which was named as crucian carp IFNc (ccIFNc). The complete ORF of ccIFNc was further determined by RACE technique, which spanned over 546 bp and encoded a polypeptide containing 182 amino acids. Phylogenetic analysis revealed that ccIFNc clustered with known type I IFN genes from other aquatic organisms. Quantitative RT-PCR analysis demonstrated that ccIFNc was constitutively expressed in all investigated tissues with a comparably higher expression level in spleen, gill, kidney, and muscle. Following challenge with CyHV-2, the transcriptional levels of ccIFNc were dramatically up-regulated in all of the tested tissues, especially in the spleen and gill with increased folds of 436 and 158, respectively. The intramuscular (i.m.) injection of a eukaryotic expression plasmid encoding ccIFNc (pEGFP-cIFNc) resulted in increased ccIFNc expression and reduced the mortality after the CyHV-2 challenge significantly. In summary, our data suggested that the ccIFNc belongs to the type I interferon family with a potential role in countering CyHV-2 infection in crucian carp.

Blanding’s turtles (Emydoidea blandingii), an endangered species in Illinois, have experienced range-wide declines because of habitat degradation and fragmentation, predation, and road mortality. While ongoing studies are crucial to a thorough understanding of the natural history and demographics in these disjointed Illinois populations, infectious disease threats have been largely unevaluated. Herpesvirus outbreaks have been associated with high morbidity and mortality in populations of captive tortoises and turtles worldwide, including the family Emydidae (pond and box turtles). However, novel herpesviruses including Terrapene herpesvirus 1, Emydid herpesvirus 1 and 2, and Glyptemys herpesvirus 1 and 2, have recently been identified in apparently healthy free-ranging freshwater turtles. In 2015, 20 free-ranging Blanding’s turtles in DuPage County, Illinois were screened for a herpesvirus using consensus PCR. A novel herpesvirus species (Emydoidea herpesvirus 1; EBHV1) was identified in two animals and shared a high degree of sequence homology to other freshwater turtle herpesviruses. Two quantitative real-time PCR assays, using EBHV1 primer-1 and primer-2, were developed to target an EBHV1-specific segment of the DNA-dependent DNA polymerase gene and validated. Both assays performed with high efficiency (slope = −3.2; R2 = 1), low intra-assay variability, and low inter-assay variability (coefficient of variation

Herpesviruses thymidine kinase (TK) and protein kinase (PK) allow the activation of nucleoside analogues used in anti-herpesvirus treatments. Mutations emerging in these two genes often lead to emergence of drug-resistant strains responsible for life-threatening diseases in immunocompromised populations. In this review, we analyze the binding of different nucleoside analogues to the TK active site of the three α-herpesviruses [Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2) and Varicella-Zoster Virus (VZV)] and present the impact of known mutations on the structure of the viral TKs. Furthermore, models of β-herpesviruses [Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6)] PKs allow to link amino acid changes with resistance to ganciclovir and/or maribavir, an investigational chemotherapeutic used in patients with multidrug-resistant HCMV. Finally, we set the basis for the understanding of drug-resistance in γ-herpesviruses [Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV)] TK and PK through the use of animal surrogate models.

Artificial lipid bilayers in the form of planar supported or vesicular bilayers are commonly used as models for studying interaction of biological membranes with different substances such as proteins and small molecule pharmaceutical compounds. Lipid membranes are typically regarded as inert and passive scaffolds for membrane proteins, but both non-specific and specific interactions between biomolecules and lipid membranes are indeed ubiquitous; dynamic exchange of proteins from the environment at the membrane interface can strongly influence the function of biological membranes. Such exchanges would either be of a superficial (peripheral) or integrative (penetrating) nature. In the context of viral membranes (termed envelopes), this could contribute to the emergence of zoonotic infections as well as change the virulence and/or pathogenicity of viral diseases. In this study, we analyze adsorption/desorption patterns upon challenging tethered liposomes and enveloped virus particles with proteins - or protein mixtures - such as bovine serum albumin, glycosylphosphatidylinositol anchored proteins and serum, chosen for their different lipid-interaction capabilities. We employed quartz crystal microbalance and dual polarization interferometry measurements to measure protein/membrane interaction in real time. We identified differences in mass uptake between the challenges, as well as differences between variants of lipid bilayers. Tethered viral particles showed a similar adsorption/desorption behavior to liposomes, underlining their value as model system. We believe that this methodology may be developed into a new approach in virology and membrane research by enabling the combination of biophysical and biochemical information.