Biology subjects

Rockx, B.

Publications and source records attributed to Rockx, B..

14 recordsLinked to original sources

bioRxiv2025

Effect of mosquito saliva from distinct species on human dermal endothelial cell function in vitro and West Nile virus pathogenesis in vivo

During probing and feeding, an infected mosquito injects both virus and saliva into the host skin. The presence of mosquito saliva in the skin increases arbovirus pathogenesis in the bitten host, however the exact mechanism behind this remains to be determined. It is hypothesized that disease enhancement is dependent on the function of the dermal endothelium, where an increased permeability aids in the influx of virus-susceptible cells to the bite site and therefore more cells for the virus to replicate in. Here, we investigate the effects of saliva from Culex and Aedes species on the human dermal endothelial cell function in vitro. Furthermore, we investigate the effect of Culex saliva on West Nile virus (WNV) pathogenesis in a mouse model. We found that salivary gland extract from anthropophilic mosquito species (Aedes and Cx. pipiens molestus) induce permeability of the human dermal endothelium, while an ornithophilic mosquito species (Cx. pip. pipiens) does not. We identified that this effect is likely due to the presence of protease(s) in Cx. pipiens molestus saliva that are absent in Cx. pipiens pipiens saliva. In addition, we show that the presence of Culex saliva at the WNV inoculation site in vivo leads to more consistent weight loss, increased permeability in the inoculation site, and increased mortality compared to inoculation of WNV alone. Identification and characterization of novel salivary proteins from similar but genetically distinct mosquito species will advance the development of intervention methods to combat potential transmission risks and disease severity of emerging mosquito-borne pathogens.

microbiology↗

bioRxiv2025

The effect of increased CpG and UpA dinucleotides in the West Nile virus genome on virus transmission by Culex mosquitoes and pathogenesis in a vertebrate host

Vertebrate animals and many small DNA and single-stranded RNA viruses that infect vertebrates have evolved to suppress genomic CpG dinucleotides. All organisms and most viruses additionally suppress UpA dinucleotides in protein coding RNA. Synonymously recoding viral genomes to introduce CpG or UpA dinucleotides has emerged as an approach for viral attenuation and vaccine development. However, studies that investigate the effects of this recoding strategy on viral replication and pathogenesis in vivo are still limited. Flaviviruses including West Nile virus (WNV) are transmitted between vertebrate hosts by invertebrate vectors. In humans, WNV infection can cause flu-like symptoms and neuroinvasive disease. We investigated how alterations in WNV dinucleotide frequencies impact virus replication, transmission by vector mosquitoes, as well as pathogenesis and neuroinvasiveness in vertebrates. In Culex pipiens vector mosquitoes and Culex cell lines only WNV with elevated UpA frequencies displayed attenuated replication. In vertebrate cell lines and primary human neuro-astrocyte co-cultures both UpA and CpG enrichment reduced viral replication. In mice, the CpG-high WNV mutant demonstrated partial attenuation with delayed weight loss compared to wild-type WNV, though infection still resulted in 100% mortality. In contrast, 75% of animals survived inoculation with the UpA-high WNV mutant and were protected against wild-type WNV challenge. Notably, all animals that succumbed to infection had similar levels of virus in the brain, irrespective of the WNV mutant. Our results underscore the complex interplay between viral genome composition and host immune responses, highlighting potential safety concerns for dinucleotide manipulation as a strategy for live-attenuated vaccine development in flaviviruses. ImportanceFlaviviruses such as West Nile virus (WNV) pose significant public health concerns due to their potential to cause severe neurological disease. Synonymously recoding flavivirus genomes to introduce CpG or UpA dinucleotides has emerged as an approach for viral attenuation and vaccine development. However, the in vivo effects of manipulating these frequencies across the complete transmission cycle remained unexplored. Our study provides comprehensive insights of how CpG and UpA recoding affects WNV replication in both the mosquito vector and vertebrate hosts. We demonstrate that elevated UpA content attenuates virus replication throughout the transmission cycle, while CpG enrichment only impacts replication in the vertebrate host. Although UpA-high WNV shows significant attenuation and provides protection against wild-type infection, animals that succumb exhibit similar brain viral loads as wild-type infections. These findings have critical implications for live-attenuated vaccine development based on dinucleotide manipulation, specifically highlighting the importance of carefully evaluating the risk of neuroinvasion.

microbiology↗

bioRxiv2024

Experimental monkeypox virus infection in rats

The global spread of Monkeypox virus (MPXV) clade IIb in 2022/2023 raised concerns about spillback into new animal reservoirs. Experimental inoculation of rats with MPXV resulted in skin lesions and viral shedding in the respiratory tract and skin. These findings suggest a potential role for rats in MPXV transmission.

microbiology↗

bioRxiv2024

Usutu virus and West Nile virus use a transcellular route of neuroinvasion across an in vitro model of the human blood-brain barrier

West Nile virus (WNV) leads to thousands of cases of severe neurological disease in humans each year. Usutu virus (USUV) is closely related to WNV, but rarely induces disease in humans. We hypothesised that USUV is less able to cross the blood-brain barrier, and is therefore less likely to infect the brain. Therefore, we developed an in vitro BBB model consisting of primary human brain microvascular endothelial cells (BMECs), pericytes and astrocytes. Both USUV and WNV invaded across the in vitro BBB via a transcellular mechanism in absence of barrier disruption. USUV replicated to lower titres than WNV but induced a comparable cytokine and chemokine response, with modulation of key factors associated with barrier function and immune-cell migration. In conclusion, USUV appears attenuated in its ability to replicate at this interface compared with WNV, but further work must to done to identify key determinants underlying the differing clinical presentations.

microbiology↗

bioRxiv2022

Differences in neuroinflammation in the olfactory bulb between D614G, Delta and Omicron BA.1 SARS-CoV-2 variants in the hamster model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with various neurological complications. SARS-CoV-2 infection induces neuroinflammation in the central nervous system (CNS), whereat the olfactory bulb seems to be involved most frequently. Here we show differences in the neuroinvasiveness and neurovirulence among SARS-CoV-2 variants in the hamster model five days post inoculation. Replication in the olfactory mucosa was observed in all hamsters, but most prominent in D614 inoculated hamsters. We observed neuroinvasion into the CNS via the olfactory nerve in D614G-, but not Delta (B.1.617.2)- or Omicron BA.1 (B.1.1.529) inoculated hamsters. Neuroinvasion was associated with neuroinflammation in the olfactory bulb of hamsters inoculated with D614G but hardly in Delta or Omicron BA.1. Altogether, this indicates that there are differences in the neuroinvasive and neurovirulent potential among SARS-CoV-2 variants in the acute phase of the infection in the hamster model.

microbiology↗

bioRxiv2022

Pulmonary lesions following inoculation with the SARS-CoV-2 Omicron BA.1 (B.1.1.529) variant in Syrian golden hamsters

The Omicron BA.1 (B.1.1.529) SARS-CoV-2 variant is characterized by a high number of mutations in the viral genome, associated with immune-escape and increased viral spread. It remains unclear whether milder COVID-19 disease progression observed after infection with Omicron BA.1 in humans is due to reduced pathogenicity of the virus or due to pre-existing immunity from vaccination or previous infection. Here, we inoculated hamsters with Omicron BA.1 to evaluate pathogenicity and kinetics of viral shedding, compared to Delta (B.1.617.2) and to animals re-challenged with Omicron BA.1 after previous SARS-CoV-2 614G infection. Omicron BA.1 infected animals showed reduced clinical signs, pathological changes, and viral shedding, compared to Delta-infected animals, but still showed gross- and histopathological evidence of pneumonia. Pre-existing immunity reduced viral shedding and protected against pneumonia. Our data indicate that the observed decrease of disease severity is in part due to intrinsic properties of the Omicron BA.1 variant.

microbiology↗

bioRxiv2022

Omicron BA.1 and BA.2 are antigenically distinct SARS-CoV-2 variants

The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly1. The Omicron variant termed BA.2, which differs genetically substantially from BA.1, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed2,3. Here, we used antigenic cartography to quantify and visualize antigenic differences between SARS-CoV-2 variants using hamster sera obtained after primary infection. Whereas early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape (vaccine-induced) antibody responses as a result of different antigenic characteristics. Close monitoring of the antigenic changes of SARS-CoV-2 using antigenic cartography can be helpful in the selection of future vaccine strains.

microbiology↗

bioRxiv2021

3D visualization of SARS-CoV-2 infection and receptor distribution in Syrian hamster lung lobes display distinct spatial arrangements

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRRS2) to facilitate viral-host membrane fusion. ACE2 and TMPRRS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq level, however, biologically relevant protein receptor organization in whole tissues is still poorly understood. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRRS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals were stained using antibodies against ACE2 and TMPRRS2, combined with fluorescent spike protein and SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize expression patterns. The data demonstrates that infection is restricted to sites with both ACE2 and TMPRRS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the terminal bronchioles and alveoli. Conversely, infection completely overlaps at these sites where ACE2 and TMPRSS2 co-localize. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/435771v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@150fc65org.highwire.dtl.DTLVardef@1ea6a56org.highwire.dtl.DTLVardef@eb3c43org.highwire.dtl.DTLVardef@1c1877e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Replication kinetic, cell tropism and associated immune responses in SARS-CoV-2 and H5N1 virus infected human iPSC derived neural models

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite ACE2, TMPRSS2 and NPR1 expression in all cultures. However, this sparse infection did result in the production of type-III-interferons and IL-8. In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS.

microbiology↗

bioRxiv2020

Intranasal fusion inhibitory lipopeptide prevents direct contact SARS-CoV-2 transmission in ferrets

Containment of the COVID-19 pandemic requires reducing viral transmission. SARS-CoV-2 infection is initiated by membrane fusion between the viral and host cell membranes, mediated by the viral spike protein. We have designed a dimeric lipopeptide fusion inhibitor that blocks this critical first step of infection for emerging coronaviruses and document that it completely prevents SARS-CoV-2 infection in ferrets. Daily intranasal administration to ferrets completely prevented SARS-CoV-2 direct-contact transmission during 24-hour co-housing with infected animals, under stringent conditions that resulted in infection of 100% of untreated animals. These lipopeptides are highly stable and non-toxic and thus readily translate into a safe and effective intranasal prophylactic approach to reduce transmission of SARS-CoV-2. One-sentence summaryA dimeric form of a SARS-CoV-2-derived lipopeptide is a potent inhibitor of fusion and infection in vitro and transmission in vivo.

microbiology↗

bioRxiv2020

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies using recombinant SARS-CoV proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric RBD proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that fully glycosylated trimeric RBD proteins are attractive to analyze receptor binding and explore ACE2 expression profiles in tissues.

molecular biology↗

bioRxiv2020

SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.

microbiology↗

bioRxiv2020

SARS-CoV-2 is transmitted via contact and via the air between ferrets.

SARS-CoV-2, a coronavirus that newly emerged in China in late 2019 1,2 and spread rapidly worldwide, caused the first witnessed pandemic sparked by a coronavirus. As the pandemic progresses, information about the modes of transmission of SARS-CoV-2 among humans is critical to apply appropriate infection control measures and to slow its spread. Here we show that SARS-CoV-2 is transmitted efficiently via direct contact and via the air (via respiratory droplets and/or aerosols) between ferrets. Intranasal inoculation of donor ferrets resulted in a productive upper respiratory tract infection and long-term shedding, up to 11 to 19 days post-inoculation. SARS-CoV-2 transmitted to four out of four direct contact ferrets between 1 and 3 days after exposure and via the air to three out of four independent indirect recipient ferrets between 3 and 7 days after exposure. The pattern of virus shedding in the direct contact and indirect recipient ferrets was similar to that of the inoculated ferrets and infectious virus was isolated from all positive animals, showing that ferrets were productively infected via either route. This study provides experimental evidence of robust transmission of SARS-CoV-2 via the air, supporting the implementation of community-level social distancing measures currently applied in many countries in the world and informing decisions on infection control measures in healthcare settings 3.

microbiology↗

bioRxiv2020

Comparative Pathogenesis Of COVID-19, MERS And SARS In A Non-Human Primate Model

A novel coronavirus, SARS-CoV-2, was recently identified in patients with an acute respiratory syndrome, COVID-19. To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or MERS-CoV and compared with historical SARS-CoV infections. In SARS-CoV-2-infected macaques, virus was excreted from nose and throat in absence of clinical signs, and detected in type I and II pneumocytes in foci of diffuse alveolar damage and mucous glands of the nasal cavity. In SARS-CoV-infection, lung lesions were typically more severe, while they were milder in MERS-CoV infection, where virus was detected mainly in type II pneumocytes. These data show that SARS-CoV-2 can cause a COVID-19-like disease, and suggest that the severity of SARS-CoV-2 infection is intermediate between that of SARS-CoV and MERS-CoV. One Sentence SummarySARS-CoV-2 infection in macaques results in COVID-19-like disease with prolonged virus excretion from nose and throat in absence of clinical signs.

microbiology↗