Orthopoxvirus-specific antibodies wane to undetectable levels 1 year after MVA-BN vaccination of at-risk individuals, the Netherlands, 2022 to 2023.

In response to the mpox outbreak in 2022 and 2023, widespread vaccination with modified vaccinia Ankara-Bavarian Nordic (MVA-BN, also known as JYNNEOS or Imvanex) was initiated. Here, we demonstrate that orthopoxvirus-specific binding and MVA-neutralising antibodies waned to undetectable levels 1 year post vaccination in at-risk individuals who received two doses of MVA-BN administered subcutaneously with an interval of 4 weeks, without prior smallpox or mpox vaccination. Continuous surveillance is essential to understand the impact of declining antibody levels.

Fourth dose bivalent COVID-19 vaccines outperform monovalent boosters in eliciting cross-reactive memory B cells to Omicron subvariants.

Bivalent COVID-19 vaccines comprising ancestral Wuhan-Hu-1 (WH1) and the Omicron BA.1 or BA.5 subvariant elicit enhanced serum antibody responses to emerging Omicron subvariants. Here, we characterized the RBD-specific memory B cell (Bmem) response following a fourth dose with a BA.1 or BA.5 bivalent vaccine, in direct comparison with a WH1 monovalent fourth dose. Healthcare workers previously immunized with mRNA or adenoviral vector monovalent vaccines were sampled before and one month after a fourth dose with a monovalent or a BA.1 or BA.5 bivalent vaccine. Serum neutralizing antibodies (NAb) were quantified, as well as RBD-specific Bmem with an in-depth spectral flow cytometry panel including recombinant RBD proteins of the WH1, BA.1, BA.5, BQ.1.1, and XBB.1.5 variants. Both bivalent vaccines elicited higher NAb titers against Omicron subvariants compared to the monovalent vaccine. Following either vaccine type, recipients had slightly increased WH1 RBD-specific Bmem numbers. Both bivalent vaccines significantly increased WH1 RBD-specific Bmem binding of all Omicron subvariants tested by flow cytometry, while recognition of Omicron subvariants was not enhanced following monovalent vaccination. IgG1+ Bmem dominated the response, with substantial IgG4+ Bmem only detected in recipients of an mRNA vaccine for their primary dose. Thus, Omicron-based bivalent vaccines can significantly boost NAb and Bmem specific for ancestral WH1 and Omicron variants and improve recognition of descendent subvariants by pre-existing, WH1-specific Bmem beyond that of a monovalent vaccine. This provides new insights into the capacity of variant-based mRNA booster vaccines to improve immune memory against emerging SARS-CoV-2 variants and potentially protect against severe disease. ONE-SENTENCE SUMMARY: Omicron BA.1 and BA.5 bivalent COVID-19 boosters, used as a fourth dose, increase RBD-specific Bmem cross-recognition of Omicron subvariants, both those encoded by the vaccines and antigenically distinct subvariants, further than a monovalent booster.

Potential determinants of the decline in mpox cases in Belgium: A behavioral, epidemiological and seroprevalence study.

OBJECTIVES: The 2022 mpox epidemic reached a peak in Belgium and the rest of Europe in July 2022, after which it unexpectedly subsided. This study investigates epidemiological, behavioral, and immunological factors behind the waning of the epidemic in Belgium. METHODS: We investigated temporal evolutions in the characteristics and behavior of mpox patients using national surveillance data and data from a prospective registry of mpox patients in the Institute of Tropical Medicine (Antwerp). We studied behavioral changes in the population at risk using a survey among HIV-preexposure prophylaxis (PrEP) users. We determined the seroprevalence of anti-orthopoxvirus antibodies among HIV-PrEP users across four-time points in 2022. RESULTS: Mpox patients diagnosed at the end of the epidemic had less sexual risk behavior compared to those diagnosed earlier: they engaged less in sex at mass events, had fewer sexual partners, and were less likely to belong to the sexual network's central group. Among HIV-PrEP users there were no notable changes in sexual behavior. Anti-orthopoxvirus seroprevalence did not notably increase before the start of national vaccination campaigns. CONCLUSION: The observed changes in group immunity and behavior in the population at greater risk of exposure to mpox seem unable to explain the waning of the mpox epidemic. A change in the profile of mpox patients might have contributed to the decline in cases.

Original COVID-19 priming regimen impacts the immunogenicity of bivalent BA.1 and BA.5 boosters.

Waning antibody responses after COVID-19 vaccination combined with the emergence of the SARS-CoV-2 Omicron lineage led to reduced vaccine effectiveness. As a countermeasure, bivalent mRNA-based booster vaccines encoding the ancestral spike protein in combination with that of Omicron BA.1 or BA.5 were introduced. Since then, different BA.2-descendent lineages have become dominant, such as XBB.1.5, JN.1, or EG.5.1. Here, we report post-hoc analyses of data from the SWITCH-ON study, assessing how different COVID-19 priming regimens affect the immunogenicity of bivalent booster vaccinations and breakthrough infections (NCT05471440). BA.1 and BA.5 bivalent vaccines boosted neutralizing antibodies and T-cells up to 3 months after boost; however, cross-neutralization of XBB.1.5 was poor. Interestingly, different combinations of prime-boost regimens induced divergent responses: participants primed with Ad26.COV2.S developed lower binding antibody levels after bivalent boost while neutralization and T-cell responses were similar to mRNA-based primed participants. In contrast, the breadth of neutralization was higher in mRNA-primed and bivalent BA.5 boosted participants. Combined, our data further support the current use of monovalent vaccines based on circulating strains when vaccinating risk groups, as recently recommended by the WHO. We emphasize the importance of the continuous assessment of immune responses targeting circulating variants to guide future COVID-19 vaccination policies.

Scenarios of future mpox outbreaks among men who have sex with men: a modelling study based on cross-sectional seroprevalence data from the Netherlands, 2022.

BackgroundFollowing the 2022-2023 mpox outbreak, crucial knowledge gaps exist regarding orthopoxvirus-specific immunity in risk groups and its impact on future outbreaks.AimWe combined cross-sectional seroprevalence studies in two cities in the Netherlands with mathematical modelling to evaluate scenarios of future mpox outbreaks among men who have sex with men (MSM).MethodsSerum samples were obtained from 1,065 MSM attending Centres for Sexual Health (CSH) in Rotterdam or Amsterdam following the peak of the Dutch mpox outbreak and the introduction of vaccination. For MSM visiting the Rotterdam CSH, sera were linked to epidemiological and vaccination data. An in-house developed ELISA was used to detect vaccinia virus (VACV)-specific IgG. These observations were combined with published data on serial interval and vaccine effectiveness to inform a stochastic transmission model that estimates the risk of future mpox outbreaks.ResultsThe seroprevalence of VACV-specific antibodies was 45.4% and 47.1% in Rotterdam and Amsterdam, respectively. Transmission modelling showed that the impact of risk group vaccination on the original outbreak was likely small. However, assuming different scenarios, the number of mpox cases in a future outbreak would be markedly reduced because of vaccination. Simultaneously, the current level of immunity alone may not prevent future outbreaks. Maintaining a short time-to-diagnosis is a key component of any strategy to prevent new outbreaks.ConclusionOur findings indicate a reduced likelihood of large future mpox outbreaks among MSM in the Netherlands under current conditions, but emphasise the importance of maintaining population immunity, diagnostic capacities and disease awareness.

Analysis of Nipah Virus Replication and Host Proteome Response Patterns in Differentiated Porcine Airway Epithelial Cells Cultured at the Air-Liquid Interface.

Respiratory tract epithelium infection plays a primary role in Nipah virus (NiV) pathogenesis and transmission. Knowledge about infection dynamics and host responses to NiV infection in respiratory tract epithelia is scarce. Studies in non-differentiated primary respiratory tract cells or cell lines indicate insufficient interferon (IFN) responses. However, studies are lacking in the determination of complex host response patterns in differentiated respiratory tract epithelia for the understanding of NiV replication and spread in swine. Here we characterized infection and spread of NiV in differentiated primary porcine bronchial epithelial cells (PBEC) cultivated at the air-liquid interface (ALI). After the initial infection of only a few apical cells, lateral spread for 12 days with epithelium disruption was observed without releasing substantial amounts of infectious virus from the apical or basal sides. Deep time course proteomics revealed pronounced upregulation of genes related to type I/II IFN, immunoproteasomal subunits, transporter associated with antigen processing (TAP)-mediated peptide transport, and major histocompatibility complex (MHC) I antigen presentation. Spliceosomal factors were downregulated. We propose a model in which NiV replication in PBEC is slowed by a potent and broad type I/II IFN host response with conversion from 26S proteasomes to immunoproteasomal antigen processing and improved MHC I presentation for adaptive immunity priming. NiV induced cytopathic effects could reflect the focal release of cell-associated NiV, which may contribute to efficient airborne viral spread between pigs.

Immunogenicity of bivalent omicron (BA.1) booster vaccination after different priming regimens in health-care workers in the Netherlands (SWITCH ON): results from the direct boost group of an open-label, multicentre, randomised controlled trial.

BACKGROUND: Bivalent mRNA-based COVID-19 vaccines encoding the ancestral and omicron spike (S) protein were developed as a countermeasure against antigenically distinct SARS-CoV-2 variants. We aimed to assess the (variant-specific) immunogenicity and reactogenicity of mRNA-based bivalent omicron (BA.1) vaccines in individuals who were primed with adenovirus-based or mRNA-based vaccines encoding the ancestral spike protein. METHODS: We analysed results of the direct boost group of the SWITCH ON study, an open-label, multicentre, randomised controlled trial. Health-care workers from four academic hospitals in the Netherlands aged 18-65 years who had completed a primary COVID-19 vaccination regimen and received one booster of an mRNA-based vaccine, given no later than 3 months previously, were eligible. Participants were randomly assigned (1:1) using computer software in block sizes of 16 and 24 to receive an omicron BA.1 bivalent booster straight away (direct boost group) or a bivalent omicron BA.5 booster, postponed for 90 days (postponed boost group), stratified by priming regimen. The BNT162b2 OMI BA.1 boost was given to participants younger than 45 years, and the mRNA-1273.214 boost was given to participants 45 years or older, as per Dutch guidelines. The direct boost group, whose results are presented here, were divided into four subgroups for analysis: (1) Ad26.COV2.S (Johnson & Johnson) prime and BNT162b2 OMI BA.1 (BioNTech-Pfizer) boost (Ad/P), (2) mRNA-based prime and BNT162b2 OMI BA.1 boost (mRNA/P), (3) Ad26.COV2.S prime and mRNA-1273.214 (Moderna) boost (Ad/M), and (4) mRNA-based prime and mRNA-1273.214 boost (mRNA/M). The primary outcome was fold change in S protein S1 subunit-specific IgG antibodies before and 28 days after booster vaccination. The primary outcome and safety were assessed in all participants except those who withdrew, had a SARS-CoV-2 breakthrough infection, or had a missing blood sample at day 0 or day 28. This trial is registered with ClinicalTrials.gov, NCT05471440. FINDINGS: Between Sept 2 and Oct 4, 2022, 219 (50%) of 434 eligible participants were randomly assigned to the direct boost group; 187 participants were included in the primary analyses; exclusions were mainly due to SARS-CoV-2 infection between days 0 and 28. From the 187 included participants, 138 (74%) were female and 49 (26%) were male. 42 (22%) of 187 participants received Ad/P and 44 (24%) mRNA/P (those aged <45 years), and 45 (24%) had received Ad/M and 56 (30%) mRNA/M (those aged ≥45 years). S1-specific binding antibody concentrations increased 7 days after bivalent booster vaccination and remained stable over 28 days in all four subgroups (geometric mean ratio [GMR] between day 0 and day 28 was 1·15 [95% CI 1·12-1·19] for the Ad/P group, 1·17 [1·14-1·20] for the mRNA/P group, 1·20 [1·17-1·23] for the Ad/M group, and 1·16 [1·13-1·19] for the mRNA/M group). We observed no significant difference in the GMR between the Ad/P and mRNA/P groups (p=0·51). The GMR appeared to be higher in the Ad/M group than in the mRNA/M group, but was not significant (p=0·073). Most side-effects were mild to moderate in severity and resolved within 48 h in most individuals. INTERPRETATION: Booster vaccination with mRNA-1273.214 or BNT162b2 OMI BA.1 in adult healthcare workers resulted in a rapid recall of humoral and cellular immune responses independent of the priming regimen. Monitoring of SARS-CoV-2 immunity at the population level, and simultaneously antigenic drift at the virus level, remains crucial to assess the necessity and timing of COVID-19 variant-specific booster vaccinations. FUNDING: The Netherlands Organization for Health Research and Development (ZonMw).

Monkeypox Virus Cross-Neutralizing Antibodies in Clinical Trial Participants Vaccinated With Modified Vaccinia Virus Ankara Encoding Middle East Respiratory Syndrome-Coronavirus Spike Protein.

Modified vaccinia virus Ankara (MVA) is used as a vaccine against monkeypox virus and as a viral vaccine vector. MVA-MERS-S is a vaccine candidate against Middle East respiratory syndrome (MERS)-associated coronavirus. Here, we report that cross-reactive monkeypox virus neutralizing antibodies were detectable in only a single study participant after the first dose of MVA-MERS-S vaccine, in 3 of 10 after the second dose, and in 10 of 10 after the third dose.

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

TGF-ß/IFN-γ Antagonism in Subversion and Self-Defense of Phase II Coxiella burnetii-Infected Dendritic Cells.

Dendritic cells (DCs) belong to the first line of innate defense and come into early contact with invading pathogens, including the zoonotic bacterium Coxiella burnetii, the causative agent of Q fever. However, the pathogen-host cell interactions in C. burnetii-infected DCs, particularly the role of mechanisms of immune subversion beyond virulent phase I lipopolysaccharide (LPS), as well as the contribution of cellular self-defense strategies, are not understood. Using phase II Coxiella-infected DCs, we show that impairment of DC maturation and MHC I downregulation is caused by autocrine release and action of immunosuppressive transforming growth factor-ß (TGF-ß). Our study demonstrates that IFN-γ reverses TGF-ß impairment of maturation/MHC I presentation in infected DCs and activates bacterial elimination, predominantly by inducing iNOS/NO. Induced NO synthesis strongly affects bacterial growth and infectivity. Moreover, our studies hint that Coxiella-infected DCs might be able to protect themselves from mitotoxic NO by switching from oxidative phosphorylation to glycolysis, thus ensuring survival in self-defense against C. burnetii. Our results provide new insights into DC subversion by Coxiella and the IFN-γ-mediated targeting of C. burnetii during early steps in the innate immune response.

Low levels of monkeypox virus-neutralizing antibodies after MVA-BN vaccination in healthy individuals.

In July 2022, the ongoing monkeypox (MPX) outbreak was declared a public health emergency of international concern. Modified vaccinia Ankara-Bavarian Nordic (MVA-BN, also known as Imvamune, JYNNEOS or Imvanex) is a third-generation smallpox vaccine that is authorized and in use as a vaccine against MPX. To date, there are no data showing MPX virus (MPXV)-neutralizing antibodies in vaccinated individuals nor vaccine efficacy against MPX. Here we show that MPXV-neutralizing antibodies can be detected after MPXV infection and after historic smallpox vaccination. However, a two-shot MVA-BN immunization series in non-primed individuals yields relatively low levels of MPXV-neutralizing antibodies. Dose-sparing of an MVA-based influenza vaccine leads to lower MPXV-neutralizing antibody levels, whereas a third vaccination with the same MVA-based vaccine significantly boosts the antibody response. As the role of MPXV-neutralizing antibodies as a correlate of protection against disease and transmissibility is currently unclear, we conclude that cohort studies following vaccinated individuals are necessary to assess vaccine efficacy in at-risk populations.

Analyzing the immunogenicity of bivalent booster vaccinations in healthcare workers: The SWITCH ON trial protocol.

Vaccination against coronavirus disease 2019 (COVID-19) has contributed greatly to providing protection against severe disease, thereby reducing hospital admissions and deaths. Several studies have reported reduction in vaccine effectiveness over time against the Omicron sub-lineages. However, the willingness to receive regular booster doses in the general population is declining. To determine the need for repeated booster vaccinations in healthy individuals and to aid policymakers in future public health interventions for COVID-19, we aim to gain insight into the immunogenicity of the additional bivalent booster vaccination in a representative sample of the healthy Dutch population. The SWITCH ON study was initiated to investigate three main topics: i) immunogenicity of bivalent vaccines after priming with adenovirus- or mRNA-based vaccines, ii) immunological recall responses and reactivity with relevant variants after booster vaccination, and iii) the necessity of booster vaccinations for the healthy population in the future. Clinical trial registration: https://clinicaltrials.gov/, identifier NCT05471440.

Chlamydia trachomatis Cell-to-Cell Spread through Tunneling Nanotubes.

Tunneling nanotubes (TNTs) are transient cellular connections that consist of dynamic membrane protrusions. They play an important role in cell-to-cell communication and mediate the intercellular exchanges of molecules and organelles. TNTs can form between different cell types and may contribute to the spread of pathogens by serving as cytoplasmic corridors. We demonstrate that Chlamydia (C.) trachomatis-infected human embryonic kidney (HEK) 293 cells and other cells form TNT-like structures through which reticulate bodies (RBs) pass into uninfected cells. Observed TNTs have a life span of 1 to 5 h and contain microtubules, which are essential for chlamydial transfer. They can bridge distances of up to 50 µm between connecting neighboring cells. Consistent with the biological role for TNTs, we show that C. trachomatis spread also occurs under conditions in which the extracellular route of chlamydial entry into host cells is blocked. Based on our findings, we propose that TNTs play a critical role in the direct, cell-to-cell transmission of chlamydia. IMPORTANCE Intracellular bacterial pathogens often undergo a life cycle in which they parasitize infected host cells in membranous vacuoles. Two pathways have been described by which chlamydia can exit infected host cells: lytic cell destruction or exit via extrusion formation. Whether direct, cell-to-cell contact may also play a role in the spread of infection is unknown. Tunneling nanotubes (TNTs) interconnect the cytoplasm of adjacent cells to mediate efficient communication and the exchange of material between them. We used Chlamydia trachomatis and immortalized cells to analyze whether TNTs mediate bacterial transmission from an infected donor to uninfected acceptor cells. We show that chlamydia-infected cells build TNTs through which the intracellular reticulate bodies (RBs) of the chlamydia can pass into uninfected neighboring cells. Our study contributes to the understanding of the function of TNTs in the cell-to-cell transmission of intracellular pathogens and provides new insights into the strategies by which chlamydia spreads among multicellular tissues.

Comparative pathogenesis of different phylogroup I bat lyssaviruses in a standardized mouse model.

A plethora of bat-associated lyssaviruses potentially capable of causing the fatal disease rabies are known today. Transmitted via infectious saliva, occasionally-reported spillover infections from bats to other mammals demonstrate the permeability of the species-barrier and highlight the zoonotic potential of bat-related lyssaviruses. However, it is still unknown whether and, if so, to what extent, viruses from different lyssavirus species vary in their pathogenic potential. In order to characterize and systematically compare a broader group of lyssavirus isolates for their viral replication kinetics, pathogenicity, and virus release through saliva-associated virus shedding, we used a mouse infection model comprising a low (102 TCID50) and a high (105 TCID50) inoculation dose as well as three different inoculation routes (intramuscular, intranasal, intracranial). Clinical signs, incubation periods, and survival were investigated. Based on the latter two parameters, a novel pathogenicity matrix was introduced to classify lyssavirus isolates. Using a total of 13 isolates from ten different virus species, this pathogenicity index varied within and between virus species. Interestingly, Irkut virus (IRKV) and Bokeloh bat lyssavirus (BBLV) obtained higher pathogenicity scores (1.14 for IRKV and 1.06 for BBLV) compared to rabies virus (RABV) isolates ranging between 0.19 and 0.85. Also, clinical signs differed significantly between RABV and other bat lyssaviruses. Altogether, our findings suggest a high diversity among lyssavirus isolates concerning survival, incubation period, and clinical signs. Virus shedding significantly differed between RABVs and other lyssaviruses. Our results demonstrated that active shedding of infectious virus was exclusively associated with two RABV isolates (92% for RABV-DogA and 67% for RABV-Insectbat), thus providing a potential explanation as to why sustained spillovers are solely attributed to RABVs. Interestingly, 3D imaging of a selected panel of brain samples from bat-associated lyssaviruses demonstrated a significantly increased percentage of infected astrocytes in mice inoculated with IRKV (10.03%; SD±7.39) compared to RABV-Vampbat (2.23%; SD±2.4), and BBLV (0.78%; SD±1.51), while only individual infected cells were identified in mice infected with Duvenhage virus (DUVV). These results corroborate previous studies on RABV that suggest a role of astrocyte infection in the pathogenicity of lyssaviruses.

Innate Immune Signaling and Role of Glial Cells in Herpes Simplex Virus- and Rabies Virus-Induced Encephalitis.

The environment of the central nervous system (CNS) represents a double-edged sword in the context of viral infections. On the one hand, the infectious route for viral pathogens is restricted via neuroprotective barriers; on the other hand, viruses benefit from the immunologically quiescent neural environment after CNS entry. Both the herpes simplex virus (HSV) and the rabies virus (RABV) bypass the neuroprotective blood-brain barrier (BBB) and successfully enter the CNS parenchyma via nerve endings. Despite the differences in the molecular nature of both viruses, each virus uses retrograde transport along peripheral nerves to reach the human CNS. Once inside the CNS parenchyma, HSV infection results in severe acute inflammation, necrosis, and hemorrhaging, while RABV preserves the intact neuronal network by inhibiting apoptosis and limiting inflammation. During RABV neuroinvasion, surveilling glial cells fail to generate a sufficient type I interferon (IFN) response, enabling RABV to replicate undetected, ultimately leading to its fatal outcome. To date, we do not fully understand the molecular mechanisms underlying the activation or suppression of the host inflammatory responses of surveilling glial cells, which present important pathways shaping viral pathogenesis and clinical outcome in viral encephalitis. Here, we compare the innate immune responses of glial cells in RABV- and HSV-infected CNS, highlighting different viral strategies of neuroprotection or Neuroinflamm. in the context of viral encephalitis.

Point Mutations in the Glycoprotein Ectodomain of Field Rabies Viruses Mediate Cell Culture Adaptation through Improved Virus Release in a Host Cell Dependent and Independent Manner.

Molecular details of field rabies virus (RABV) adaptation to cell culture replication are insufficiently understood. A better understanding of adaptation may not only reveal requirements for efficient RABV replication in cell lines, but may also provide novel insights into RABV biology and adaptation-related loss of virulence and pathogenicity. Using two recombinant field rabies virus clones (rRABV Dog and rRABV Fox), we performed virus passages in three different cell lines to identify cell culture adaptive mutations. Ten passages were sufficient for the acquisition of adaptive mutations in the glycoprotein G and in the C-terminus of phosphoprotein P. Apart from the insertion of a glycosylation sequon via the mutation D247N in either virus, both acquired additional and cell line-specific mutations after passages on BHK (K425N) and MDCK-II (R346S or R350G) cells. As determined by virus replication kinetics, complementation, and immunofluorescence analysis, the major bottleneck in cell culture replication was the intracellular accumulation of field virus G protein, which was overcome after the acquisition of the adaptive mutations. Our data indicate that limited release of extracellular infectious virus at the plasma membrane is a defined characteristic of highly virulent field rabies viruses and we hypothesize that the observed suboptimal release of infectious virions is due to the inverse correlation of virus release and virulence in vivo.

Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus.

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.