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Mettenleiter, T. C.

Publications and source records attributed to Mettenleiter, T. C..

15 recordsLinked to original sources

The core herpes simplex-1 fusion complex drives cell-to-cell spreading of pathological Tau

Neurodegenerative diseases such as Alzheimers disease (AD) are characterized by the pathological aggregation of the host-encoded protein Tau into amyloid fibrils. Pathologic protein aggregates composed of Tau are able to spread from cell-to-cell, thereby contributing to disease progression. The exact mechanisms of intercellular dissemination remain ill-defined. Mounting evidence links the herpes simplex virus-1 (HSV-1) to the aetiology of AD. HSV-1 is a prevalent neurotropic virus that replicates in epithelial cells at the site of infection, and subsequently establishes lifelong latency in the peripheral and central nervous systems. In the quiescent latent state, AD brains show elevated expression of structural viral proteins in the absence of virus production. We hypothesized that HSV-1 glycoproteins involved in viral attachment and fusion facilitate the intercellular spreading of proteopathic seeds. Using cellular models, we demonstrate that expression of the HSV-1 core fusion complex, essential for viral entry and direct cell-to-cell transmission, is sufficient to promote aggregate dissemination between cells. Moreover, anti-HSV-1 antibodies present in the cerebrospinal fluid of patients with HSV-1 encephalitis efficiently neutralize viral infection, block viral cell-to-cell transmission, and inhibit propagation of pathological Tau. Thus, latent HSV-1 infection of the brain could contribute to neurodegenerative disease progression, and antiviral antibodies may represent a potential therapeutic strategy.

neuroscience↗

Spatiotemporal dynamics of alphaherpesviral latency and reactivation in the murine central nervous system

Alphaherpesviruses, including Herpes Simplex Virus 1 (HSV-1) and Pseudorabies Virus (PrV), establish lifelong latency in the nervous system and can cause recurrent disease. While latency has classically been attributed to peripheral sensory ganglia, accumulating evidence indicates that the central nervous system (CNS) also act as a relevant reservoir for viral latency and reactivation. Here, we investigated the CNS as a site of latency using the attenuated mutant PrV-{Delta}UL21/US3{Delta}kin which reproduces key features of herpes simplex encephalitis (HSE) in female CD1 mice. We mapped brain regions permissive to alphaherpesviral latency and analyzed the temporal dynamics of viral transcription, histopathology, and host clinical and immune responses. Following intranasal inoculation, mice were analyzed at 11 to 14, 21, 28, 42, 105, and 190 days post infection (dpi). To assess the potential of reactivation, a subset received cyclophosphamide/dexamethasone at 170 dpi. Viral transcripts were detected by RNAscope in situ hybridization and RT-qPCR targeting the lytic gene UL19 and the latency-associated transcript (LAT). Histopathological analyses included hematoxylin and eosin (H&E) staining and immunohistochemistry for CD3, Iba1, GFAP, and cleaved caspase-3. Major capsid protein (UL19) expression displayed marked regional and temporal heterogeneity, with prominent signals in mesiotemporal structures (piriform cortex, hippocampus, entorhinal cortex), coinciding with pronounced T-cell infiltration. LAT expression remained overall low, with a transient peak during the acute infection (11-14 dpi). RT-qPCR confirmed high viral transcript levels for both UL19 and LAT in mesiotemporal regions during early infection, while LAT expression returned to baseline levels thereafter. Histopathology demonstrated a transition from acute necrotizing meningoencephalitis to prolonged or recurrent low-grade inflammation, accompanied by glial activation and localized apoptosis. Notably, UL19 expression strongly correlated with CD3+ T-cell infiltration, particularly at 42 dpi. These findings define the spatiotemporal interplay between viral transcriptional activity and neuroinflammation and identify selected CNS regions as reservoirs for latent or recurrent alphaherpesvirus infection. Author SummaryAlphaherpesviruses are pathogens that not only cause acute disease but also establish lifelong latency in the nervous system. Under certain conditions, they can reactivate and trigger recurrent disease. Using a genetically modified pseudorabies virus in mice, we mapped how latent and lytic phases occur within the brain over several months. By combining molecular, histological, and clinical analyses, we show that specific brain regions act as long-term viral reservoirs, where signs of inflammation and viral activity can reappear long after the initial infection. These findings provide new insights into how alphaherpesviruses persist in the central nervous system and suggest that recurrent or subclinical reactivation may contribute to long-term neurological complications, including those resembling human herpes simplex encephalitis.

neuroscience↗

Different genetic determinants for high virulence, transmission and replication of high pathogenicity H7N7 avian influenza virus in turkeys and chickens

High pathogenicity (HP) avian influenza viruses (AIV) generally evolve from low pathogenicity (LP) precursors after transmission from wild birds to chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo), causing severe economic losses worldwide. Turkeys are more susceptible to AIV infection than chickens and are considered potential bridging hosts that facilitate the emergence of HPAIV. Beyond the polybasic cleavage site (pCS) in hemagglutinin (HA), little is known about other virulence determinants of HPAIV in these species. In 2015, HPAIV H7N7 and its LP ancestor were isolated from the same chicken farm, which differed by 16 nonsynonymous mutations across all eight gene segments, in addition to the pCS. Here we identify the genetic determinants, including the pCS, that contributed to the HPAIV H7N7 virulence, transmission, replication, and tissue distribution in chickens and turkeys. Notably, the non-structural (NS1) or matrix (M) proteins encoding segments in turkeys, or NS segment in chickens, rendered viruses as virulent and transmissible as the original HPAIV. Endotheliotropism, observed exclusively in chickens, was driven by the pCS and, to a lesser extent, the neuraminidase (NA). In vitro, the M2-V68L mutation influenced NS1 expression and virus morphology in chicken and turkey cells. Additionally, HPAIV NS1 enhanced polymerase activity and effectively suppressed interferon induction, a process further modulated by M2-V68L. These findings underscore the critical role of turkeys as a "hub" in the evolution of HPAIV from LP precursors, offering crucial insights into the genotypic and phenotypic factors that facilitate viral adaptation in different poultry species. ImportanceHigh pathogenicity avian influenza viruses (HPAIV) cause severe economic losses for the poultry industry worldwide. HPAIV generally evolve from low pathogenicity (LP) ancestors in galliform birds, with turkeys being more susceptible to severe disease and death than chickens. The mechanisms underlying HPAIV emergence in these species remain unclear. This study reveals two distinct evolutionary pathways for HPAIV. In turkeys, both the polybasic hemagglutinin cleavage site (pCS) and mutations in the NS or M segments contributed to high virulence and transmission. In chickens, only the NS segment was critical, in addition to the pCS. These segments increased virus replication in both chicken and turkey cells. However, unlike chicken cells, the M and NS segments did not play a role in blocking the innate immune response. Understanding these species-specific mechanisms highlights the role of turkeys as a bridging host and provides insights into the molecular evolution of HPAIV from LP precursors.

microbiology↗

Oral immunization of goats and foxes with a recombinant NDV vectored rabies vaccine

Vaccination of the reservoir species is a key component in the global fight against rabies. For wildlife reservoir species and hard to reach spillover species (e. g. ruminant farm animals), oral vaccination is the only solution. In search for a novel potent and safe oral rabies vaccine, we generated a recombinant vector virus based on lentogenic Newcastle disease virus (NDV) strain Clone 30 that expresses the glycoprotein G of rabies virus (RABV) vaccine strain SAD L16 (rNDV_GRABV). Transgene expression and virus replication was verified in avian and mammalian cells. To test immunogenicity and viral shedding, in a proof-of-concept study six goats and foxes, representing herbivore and carnivore species susceptible to rabies, each received a single dose of rNDV_GRABV (108.5 TCID50/animal) by direct oral application. For comparison, three animals received the similar dose of the empty viral vector (rNDV). All animals remained clinically inconspicuous during the trial. Viral RNA could be isolated from oral and nasal swabs until four (goats) or seven days (foxes) post vaccination, while infectious NDV could not be re-isolated. After four weeks, three out of six rNDV_GRABV vaccinated foxes developed RABV binding and virus neutralizing antibodies. Five out of six rNDV_GRABV vaccinated goats displayed RABV G specific antibodies either detected by ELISA or RFFIT. Additionally, NDV and RABV specific T cell activity was demonstrated in some of the vaccinated animals by detecting antigen specific interferon {gamma} secretion in lymphocytes isolated from pharyngeal lymph nodes. In conclusion, the NDV vectored rabies vaccine rNDV_GRABV was safe and immunogenic after a single oral application in goats and foxes, and highlight the potential of NDV as vector for oral vaccines in mammals. Author SummaryOral vaccination of rabies reservoir and spill-over species is the key to control the disease and prevent human rabies. In the past, baits containing live-attenuated rabies vaccines decreased significantly carnivore-mediated rabies in Central and Western Europe as well as North America. However, certain susceptible species are refractory to the oral immunization using so far licensed vaccines. Our vector vaccine based on avian Newcastle disease virus (NDV) has the potential to contribute to the targeted rabies eradication as it was safe and immunogenic after oral administration in goats and foxes. A single vaccine application elicited a rabies virus (RABV) specific systemic humoral immune response in the majority of the vaccinated animals as well as RABV specific T cells in some of the vaccinated animals. NDV can be manufactured at low-cost using already existing infrastructure of influenza vaccines, opening new possibilities especially for middle- and low-income countries that suffer under the economically burden of rabies.

microbiology↗

NECing goes: flexibility of the herpesvirus nuclear egress complex

The nuclear egress complex (NEC) allows herpesvirus capsids to escape from the nucleus without breaking the nuclear envelope barrier. It assembles into a lattice on the inner nuclear membrane enveloping newly assembled nucleocapsids, which bud into the perinuclear space. The primary virion envelope subsequently fuses with the outer nuclear membrane, releasing the capsid into the cytosol. Here we interrogated the NEC in the context of intact cells infected with pseudorabies or herpes simplex virus using focused-ion beam milling and electron cryo- tomography. We determined the structure of NEC in different conformations and show that it consists of a flexible hexameric lattice that generates curvature through a combination of ordered and disordered domains. After interrogating the intermediate stages of capsid formation, we show that capsid vertex binding may initiate envelopment but does not directly induce curvature formation. These data and many examples of the intermediate stages of nuclear egress paint a detailed holistic view of a versatile transport system.

microbiology↗

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs

Factors of the innate immune response to SARS-CoV-2 in the lungs are pivotal for the ability of the host to deal with the infection. In humans, excessive macrophage infiltration is associated with disease severity. Using 3D spatiotemporal analysis of optically cleared hamster lung slices in combination with virological, immunohistochemical and RNA sequence analyses, we visualized the spread of SARS-CoV-2 through the lungs and the rapid anti-viral response in infected lung epithelial cells, followed by a wave of monocyte-derived macrophage (MDM) infiltration and virus elimination from the tissue. These SARS-CoV-2 induced innate immune processes are closely related to the onset of necrotizing inflammatory and consecutive remodelling responses in the lungs, which manifests as extensive cell death, vascular damage, thrombosis, and cell proliferation. Here we show that MDM are directly linked to virus clearance, and appear in connection with tissue injury and blood vessel damage. Rapid initiation of prothrombotic factor upregulation, tissue repair and alveolar cell proliferation results in tissue remodelling, which is followed by fibrosis development despite a decrease in inflammatory and anti-viral activities. Thus, although the hamsters are able to resolve the infection following the MDM influx and repair lung tissue integrity, longer-term alterations of the lung tissues arise as a result of concurrent tissue damage and regeneration processes.

immunology↗

Compellingly high SARS-CoV-2 susceptibility of Golden Syrian hamsters suggests multiple zoonotic infections of pet hamsters during the COVID-19 pandemic

Golden Syrian hamsters (Mesocricetus auratus) are used as a research model for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Millions of Golden Syrian hamsters are also kept as pets in close contact to humans. To determine the minimum infective dose (MID) for assessing the zoonotic transmission risk, and to define the optimal infection dose for experimental studies, we orotracheally inoculated hamsters with SARS-CoV-2 doses from 1*105 to 1*10-4 tissue culture infectious dose 50 (TCID50). Body weight and virus shedding were monitored daily. 1*10-3 TCID50 was defined as the MID, and this was still sufficient to induce virus shedding at levels up to 102.75 TCID50/ml, equaling the estimated MID for humans. Virological and histological data revealed 1*102 TCID50 as the optimal dose for experimental infections. This compellingly high susceptibility resulting in productive infections in Golden Syrian hamsters needs to be considered also as a source of SARS-CoV-2 infections in humans.

microbiology↗

Low-dose bivalent mRNA vaccine is highly effective against different SARS-CoV-2 variants in a transgenic mouse model

Combining optimized spike (S) protein-encoding mRNA vaccines to target multiple SARS-CoV-2 variants could improve COVID-19 control. We compared monovalent and bivalent mRNA vaccines encoding B.1.351 (Beta) and/or B.1.617.2 (Delta) SARS-CoV-2 S-protein, primarily in a transgenic mouse model and a Wistar rat model. The low-dose bivalent mRNA vaccine contained half the mRNA of each respective monovalent vaccine, but induced comparable neutralizing antibody titres, enrichment of lung-resident memory CD8+ T cells, specific CD4+ and CD8+ responses, and fully protected transgenic mice from SARS-CoV-2 lethality. The bivalent mRNA vaccine significantly reduced viral replication in both Beta- and Delta-challenged mice. Sera from bivalent mRNA vaccine immunized Wistar rats also contained neutralizing antibodies against the B.1.1.529 (Omicron BA.1) variant. These data suggest that low-dose and fit-for-purpose multivalent mRNA vaccines encoding distinct S-proteins is a feasible approach for increasing the potency of vaccines against emerging and co-circulating SARS-CoV-2 variants.

immunology↗

Serological screening in wild ruminants in Germany, 2021/22: No evidence of SARS-CoV-2, bluetongue virus or pestivirus spread but high seroprevalences against Schmallenberg virus

Wildlife animals may be susceptible for multiple infectious agents of public health or veterinary relevance, thereby potentially forming a reservoir that bears the constant risk of re-introduction into the human or livestock population. Here, we serologically investigated 493 wild ruminant samples collected in the 2021/22 hunting season in Germany for the presence of antibodies against the severe acute respiratory coronavirus 2 (SARS-CoV-2) and four viruses pathogenic for domestic ruminants, namely the orthobunyavirus Schmallenberg virus (SBV), the reovirus bluetongue virus (BTV) and ruminant pestiviruses like bovine viral diarrhoea virus or border disease virus. The animal species comprised fallow deer, red deer, roe deer, mouflon and wisent. For coronavirus serology, additional 307 fallow, roe and red deer samples collected between 2017 and 2020 at three military training areas were included. While antibodies against SBV could be detected in about 13.6% of the samples collected in 2021/22, only one fallow deer of unknown age tested positive for anti-BTV antibodies and all samples reacted negative for antibodies against ruminant pestiviruses. In an ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2, 25 out of 493 (5.1%) samples collected in autumn and winter 2021/22 scored positive. This sero-reactivity could not be confirmed by the highly specific virus neutralization test, occurred also in 2017, 2018 and 2019, i.e. prior to the human SARS-CoV-2 pandemic, and was likewise observed against the RBD of the related SARS-CoV-1. Therefore, the SARS-CoV-2-seroreactivity was most likely induced by another, hitherto unknown deer virus belonging to the subgenus Sarbecovirus of betacoronaviruses.

microbiology↗

CVnCoV protects human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic necessitates the fast development of vaccines as the primary control option. Recently, viral mutants termed "variants of concern" (VOC) have emerged with the potential to escape host immunity. VOC B.1.351 was first discovered in South Africa in late 2020, and causes global concern due to poor neutralization with propensity to evade preexisting immunity from ancestral strains. We tested the efficacy of a spike encoding mRNA vaccine (CVnCoV) against the ancestral strain BavPat1 and the novel VOC B.1.351 in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice developed elevated SARS-CoV-2 RBD-specific antibody as well as neutralization titers against the ancestral strain BavPat1. Neutralization titers against VOC B.1.351 were readily detectable but significantly reduced compared to BavPat1. VOC B.1.351-infected control animals experienced a delayed course of disease, yet nearly all SARS-CoV-2 challenged naive mice succumbed with virus dissemination and high viral loads. CVnCoV vaccine completely protected the animals from disease and mortality caused by either viral strain. Moreover, SARS-CoV-2 was not detected in oral swabs, lung, or brain in these groups. Only partial protection was observed in mice receiving the formalin-inactivated virus preparation. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV shows complete disease protection against the novel VOC B.1.351 in our studies.

microbiology↗

Preferential selection and contribution of non-structural protein 1 (NS1) to the efficient transmission of the panzootic avian influenza H5N8 2.3.4.4 clades A and B viruses in chickens and ducks

Highly pathogenic avian influenza viruses H5N8 clade 2.3.4.4 caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade-2.3.4.4A in 2014/2015 and clade-2.3.4.4B since 2016 up to today. Both clades were highly virulent in chickens, but only clade-B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 2.3.4.4 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and in-vitro experiments indicated that clade-2.3.4.4A and clade-2.3.4.4B viruses have a preference for NS1 of 237-aa and 217-aa, respectively over NS1 of 230-aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C-terminus of clade-B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade-A virus was observed. Remarkably, the NS1 of clade-A and clade-B, regardless of length, was efficient to block interferon induction in infected chickens and changes in the NS1 C-terminus reduced the efficiency for interferon antagonism. Together, the NS1 C-terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks. ImportanceThe panzootic H5N8 highly pathogenic avian influenza viruses of clade 2.3.4.4A and 2.3.4.4B devastated poultry industry globally. Clade 2.3.4.4A was predominant in 2014/2015 while clade 2.3.4.4B was widely spread in 2016/2017. Both clades exhibited different pathotypes in ducks. Virus factors contributing to virulence and transmission are largely unknown. The NS1 protein is typically composed of 230 amino-acids (aa) and is an essential interferon (IFN) antagonist. Here, we found that the NS1 protein of clade 2.3.4.4A preferentially evolved toward long NS1 with 237-aa, while clade 2.3.4.4B evolved toward shorter NS1 with 217-aa (exclusively found in Europe) due stop-codons in the C-terminus (CTE). We showed that the NS1 CTE of H5N8 is required for efficient virus replication, transmission and endotheliotropism in ducks. In chickens, H5N8 NS1 evolved toward higher efficiency to block IFN-response. These findings may explain the preferential pattern for short NS1 and high fitness of the panzootic H5N8 in birds. Subject categoryAnimal, RNA Viruses

microbiology↗

3D reconstruction of SARS-CoV-2 infection in ferrets emphasizes focal infection pattern in the upper respiratory tract

The visualization of viral pathogens in infected tissues is an invaluable tool to understand spatial virus distribution, localization, and cell tropism in vivo. Commonly, virus-infected tissues are analyzed using conventional immunohistochemistry in paraffin-embedded thin sections. Here, we demonstrate the utility of volumetric three-dimensional (3D) immunofluorescence imaging using tissue optical clearing and light sheet microscopy to investigate host-pathogen interactions of pandemic SARS-CoV-2 in ferrets at a mesoscopic scale. The superior spatial context of large, intact samples (> 150 mm3) allowed detailed quantification of interrelated parameters like focus-to-focus distance or SARS-CoV-2-infected area, facilitating an in-depth description of SARS-CoV-2 infection foci. Accordingly, we could confirm a preferential infection of the ferret upper respiratory tract by SARS-CoV-2 and emphasize a distinct focal infection pattern in nasal turbinates. Conclusively, we present a proof-of-concept study for investigating critically important respiratory pathogens in their spatial tissue morphology and demonstrate the first specific 3D visualization of SARS-CoV-2 infection.

microbiology↗

Experimental infection of cattle with SARS-CoV-2

Six cattle (Bos taurus) were intranasally inoculated with SARS-CoV-2 and kept together with three naive in-contact animals. Low-level virus replication and a specific sero-reactivity were observed in two inoculated animals, despite the presence of high antibody titers against a bovine betacoronavirus. The in-contact animals did not become infected.

microbiology↗

Susceptibility of raccoon dogs for experimental SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019, and became pandemic. The zoonotic virus most likely originated from bats, but definite intermediate hosts have not yet been identified. Raccoon dogs (Nyctereutes procyonoides) are kept for fur production, in particular in China, and were suspected as potential intermediate host for both SARS-CoV6 and SARS-CoV2. Here we demonstrate susceptibility of raccoon dogs for SARS-CoV-2 infection after intranasal inoculation and transmission to direct contact animals. Rapid, high level virus shedding, in combination with minor clinical signs and pathohistological changes, seroconversion and absence of viral adaptation highlight the role of raccoon dogs as a potential intermediate host. The results are highly relevant for control strategies and emphasize the risk that raccoon dogs may represent a potential SARS-CoV-2 reservoir. Our results support the establishment of adequate surveillance and risk mitigation strategies for kept and wild raccoon dogs. Article Summary LineRaccoon dogs are susceptible to and efficiently transmit SARS-CoV2 and may serve as intermediate host

microbiology↗

Pitfalls in SARS-CoV-2 PCR diagnostics

To combat the COVID-19 pandemic, millions of PCR tests are performed worldwide. Any deviation of the diagnostic sensitivity and specificity will reduce the predictive values of the test. Here, we report the occurrence of contaminations of commercial primers/probe sets with the SARS-CoV-2 target sequence of the RT-qPCR as an example for pitfalls during PCR diagnostics affecting diagnostic specificity. In several purchased in-house primers/probe sets, quantification cycle values as low as 17 were measured for negative control samples. However, there were also primers/probe sets that displayed very low-level contaminations, which were detected only during thorough internal validation. Hence, it appears imperative to pre-test each batch of reagents extensively before use in routine diagnosis, to avoid false-positive results and low positive predictive value in low-prevalence situations. As such, contaminations may have happened more widely, COVID-19 diagnostic results should be re-assessed retrospectively to validate the epidemiological basis for control measures.

microbiology↗