Immune Responses and Pathogenesis following Experimental SARS-CoV-2 Infection in Domestic Cats.

Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats (n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations.

Efficient Direct and Limited Environmental Transmission of SARS-CoV-2 Lineage B.1.22 in Domestic Cats.

The susceptibility of domestic cats to infection with SARS-CoV-2 has been demonstrated by several experimental studies and field observations. We performed an extensive study to further characterize the transmission of SARS-CoV-2 between cats, through both direct and indirect contact. To that end, we estimated the transmission rate parameter and the decay parameter for infectivity in the environment. Using four groups of pair-transmission experiment, all donor (inoculated) cats became infected, shed virus, and seroconverted, while three out of four direct contact cats got infected, shed virus, and two of those seroconverted. One out of eight cats exposed to a SARS-CoV-2-contaminated environment became infected but did not seroconvert. Statistical analysis of the transmission data gives a reproduction number R0 of 2.18 (95% CI = 0.92 to 4.08), a transmission rate parameter ß of 0.23 day-1 (95% CI = 0.06 to 0.54), and a virus decay rate parameter µ of 2.73 day-1 (95% CI = 0.77 to 15.82). These data indicate that transmission between cats is efficient and can be sustained (R0 > 1), however, the infectiousness of a contaminated environment decays rapidly (mean duration of infectiousness 1/2.73 days). Despite this, infections of cats via exposure to a SARS-CoV-2-contaminated environment cannot be discounted if cats are exposed shortly after contamination. IMPORTANCE This article provides additional insight into the risk of infection that could arise from cats infected with SARS-CoV-2 by using epidemiological models to determine transmission parameters. Considering that transmission parameters are not always provided in the literature describing transmission experiments in animals, we demonstrate that mathematical analysis of experimental data is crucial to estimate the likelihood of transmission. This article is also relevant to animal health professionals and authorities involved in risk assessments for zoonotic spill-overs of SARS-CoV-2. Last but not least, the mathematical models to calculate transmission parameters are applicable to analyze the experimental transmission of other pathogens between animals.

Engineering potent live attenuated coronavirus vaccines by targeted inactivation of the immune evasive viral deubiquitinase.

Coronaviruses express a papain-like protease (PLpro) that is required for replicase polyprotein maturation and also serves as a deubiquitinating enzyme (DUB). In this study, using a Middle East respiratory syndrome virus (MERS-CoV) PLpro modified virus in which the DUB is selectively inactivated, we show that the PLpro DUB is an important MERS-CoV interferon antagonist and virulence factor. Although the DUB-negative rMERS-CoVMA replicates robustly in the lungs of human dipeptidyl peptidase 4 knock-in (hDPP4 KI) mice, it does not cause clinical symptoms. Interestingly, a single intranasal vaccination with DUB-negative rMERS-CoVMA induces strong and sustained neutralizing antibody responses and sterilizing immunity after a lethal wt virus challenge. The survival of naïve animals also significantly increases when sera from animals vaccinated with the DUB-negative rMERS-CoVMA are passively transferred, prior to receiving a lethal virus dose. These data demonstrate that DUB-negative coronaviruses could be the basis of effective modified live attenuated vaccines.

Intranasal administration of a live-attenuated recombinant newcastle disease virus expressing the SARS-CoV-2 Spike protein induces high neutralizing antibody levels and protects from experimental challenge infection in hamsters.

The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.

Experimental and field investigations of exposure, replication and transmission of SARS-CoV-2 in pigs in the Netherlands.

In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely.

A Yellow Fever 17D Virus Replicon-Based Vaccine Platform for Emerging Coronaviruses.

The tremendous global impact of the current SARS-CoV-2 pandemic, as well as other current and recent outbreaks of (re)emerging viruses, emphasize the need for fast-track development of effective vaccines. Yellow fever virus 17D (YF17D) is a live-attenuated virus vaccine with an impressive efficacy record in humans, and therefore, it is a very attractive platform for the development of novel chimeric vaccines against various pathogens. In the present study, we generated a YF17D-based replicon vaccine platform by replacing the prM and E surface proteins of YF17D with antigenic subdomains from the spike (S) proteins of three different betacoronaviruses: MERS-CoV, SARS-CoV and MHV. The prM and E proteins were provided in trans for the packaging of these RNA replicons into single-round infectious particles capable of expressing coronavirus antigens in infected cells. YF17D replicon particles expressing the S1 regions of the MERS-CoV and SARS-CoV spike proteins were immunogenic in mice and elicited (neutralizing) antibody responses against both the YF17D vector and the coronavirus inserts. Thus, YF17D replicon-based vaccines, and their potential DNA- or mRNA-based derivatives, may constitute a promising and particularly safe vaccine platform for current and future emerging coronaviruses.

Predictive Value of Precision-Cut Lung Slices for the Susceptibility of Three Animal Species for SARS-CoV-2 and Validation in a Refined Hamster Model.

In assessing species susceptibility for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in the search for an appropriate animal model, multiple research groups around the world inoculated a broad range of animal species using various SARS-CoV-2 strains, doses and administration routes. Although in silico analyses based on receptor binding and diverse in vitro cell cultures were valuable, exact prediction of species susceptibility based on these tools proved challenging. Here, we assessed whether precision-cut lung slices (PCLS) could facilitate the selection of animal models, thereby reducing animal experimentation. Pig, hamster and cat PCLS were incubated with SARS-CoV-2 and virus replication was followed over time. Virus replicated efficiently in PCLS from hamsters and cats, while no evidence of replication was obtained for pig PCLS. These data corroborate the findings of many research groups that have investigated the susceptibility of hamsters, pigs and cats towards infection with SARS-CoV-2. Our findings suggest that PCLS can be used as convenient tool for the screening of different animal species for sensitivity to newly emerged viruses. To validate our results obtained in PCLS, we employed the hamster model. Hamsters were inoculated with SARS-CoV-2 via the intranasal route. Susceptibility to infection was evaluated by body weight loss, viral loads in oropharyngeal swabs and respiratory tissues and lung pathology. The broadly used hamster model was further refined by including activity tracking of the hamsters by an activity wheel as a very robust and sensitive parameter for clinical health. In addition, to facilitate the quantification of pathology in the lungs, we devised a semi-quantitative scoring system for evaluating the degree of histological changes in the lungs. The inclusion of these additional parameters refined and enriched the hamster model, allowing for the generation of more data from a single experiment.

Animal models for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by the introduction of a novel coronavirus into humans late in 2019 (first detected in Hubei province, China). As of 18 September 2020, SARS-CoV-2 has spread to 215 countries, has infected more than 30 million people and has caused more than 950,000 deaths. As humans do not have pre-existing immunity to SARS-CoV-2, there is an urgent need to develop therapeutic agents and vaccines to mitigate the current pandemic and to prevent the re-emergence of COVID-19. In February 2020, the World Health Organization (WHO) assembled an international panel to develop animal models for COVID-19 to accelerate the testing of vaccines and therapeutic agents. Here we summarize the findings to date and provides relevant information for preclinical testing of vaccine candidates and therapeutic agents for COVID-19.

Single-Round Infectious Particle Production by DNA-Launched Infectious Clones of Bungowannah Pestivirus.

Reverse genetics systems are powerful tools for functional studies of viral genes or for vaccine development. Here, we established DNA-launched reverse genetics for the pestivirus Bungowannah virus (BuPV), where cDNA flanked by a hammerhead ribozyme sequence at the 5' end and the hepatitis delta ribozyme at the 3' end was placed under the control of the CMV RNA polymerase II promoter. Infectious recombinant BuPV could be rescued from pBuPV-DNA-transfected SK-6 cells and it had very similar growth characteristics to BuPV generated by conventional RNA-based reverse genetics and wild type BuPV. Subsequently, DNA-based ERNS deleted BuPV split genomes (pBuPV∆ERNS/ERNS)-co-expressing the ERNS protein from a separate synthetic CAG promoter-were constructed and characterized in vitro. Overall, DNA-launched BuPV genomes enable a rapid and cost-effective generation of recombinant BuPV and virus mutants, however, the protein expression efficiency of the DNA-launched systems after transfection is very low and needs further optimization in the future to allow the use e.g., as vaccine platform.

Safety and efficacy of four-segmented Rift Valley fever virus in young sheep, goats and cattle.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. RVFV induces severe disease in newborns and abortion in pregnant ruminants. The viral genome consists of a small (S), medium (M) and large (L) RNA segment of negative polarity. The M segment encodes a glycoprotein precursor protein that is co-translationally cleaved into the two structural glycoproteins Gn and Gc, which are involved in receptor attachment and cell entry. We previously constructed a four-segmented RVFV (RVFV-4s) by splitting the M genome segment into two M-type segments encoding either Gn or Gc. RVFV-4s replicates efficiently in cell culture but was shown to be completely avirulent in mice, lambs and pregnant ewes. Here, we show that a RVFV-4s candidate vaccine for veterinary use (vRVFV-4s) does not disseminate in vaccinated animals, is not shed or spread to the environment and does not revert to virulence. Furthermore, a single vaccination of lambs, goat kids and calves was shown to induce protective immunity against a homologous challenge. Finally, the vaccine was shown to provide full protection against a genetically distinct RVFV strain. Altogether, we demonstrate that vRVFV-4s optimally combines efficacy with safety, holding great promise as a next-generation RVF vaccine.

SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020.

Respiratory disease and increased mortality occurred in minks on two farms in the Netherlands, with interstitial pneumonia and SARS-CoV-2 RNA in organ and swab samples. On both farms, at least one worker had coronavirus disease-associated symptoms before the outbreak. Variations in mink-derived viral genomes showed between-mink transmission and no infection link between the farms. Inhalable dust contained viral RNA, indicating possible exposure of workers. One worker is assumed to have attracted the virus from mink.

Novel Toscana Virus Reverse Genetics System Establishes NSs as an Antagonist of Type I Interferon Responses.

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.

Co-housing of Rift Valley Fever Virus Infected Lambs with Immunocompetent or Immunosuppressed Lambs Does Not Result in Virus Transmission.

Rift Valley fever virus (RVFV) is transmitted among susceptible animals by mosquito vectors. Although the virus can be isolated from nasal and oral swabs of infected animals and is known to be highly infectious when administered experimentally via oral or respiratory route, horizontal transmission of the virus is only sporadically reported in literature. We considered that immunosuppression resulting from stressful conditions in the field may increase the susceptibility to horizontally transmitted RVFV. Additionally, we reasoned that horizontal transmission may induce immune responses that could affect the susceptibility of contact-exposed animals to subsequent infection via mosquito vectors. To address these two hypotheses, viremic lambs were brought into contact with sentinel lambs. One group of sentinel lambs was treated with the immunosuppressive synthetic glucocorticosteroid dexamethasone and monitored for signs of disease and presence of virus in the blood and target organs. Another group of contact-exposed sentinel lambs remained untreated for three weeks and was subsequently challenged with RVFV. We found that none of the dexamethasone-treated contact-exposed lambs developed detectable viremia, antibody responses or significant increases in cytokine mRNA levels. Susceptibility of immunocompetent lambs to RVFV infection was not influenced by previous contact-exposure. Our results are discussed in light of previous findings.

Nonspreading Rift Valley Fever Virus Infection of Human Dendritic Cells Results in Downregulation of CD83 and Full Maturation of Bystander Cells.

Vaccines based on nonspreading Rift Valley fever virus (NSR) induce strong humoral and robust cellular immune responses with pronounced Th1 polarisation. The present work was aimed to gain insight into the molecular basis of NSR-mediated immunity. Recent studies have demonstrated that wild-type Rift Valley fever virus efficiently targets and replicates in dendritic cells (DCs). We found that NSR infection of cultured human DCs results in maturation of DCs, characterized by surface upregulation of CD40, CD80, CD86, MHC-I and MHC-II and secretion of the proinflammatory cytokines IFN-ß, IL-6 and TNF. Interestingly, expression of the most prominent marker of DC maturation, CD83, was consistently downregulated at 24 hours post infection. Remarkably, NSR infection also completely abrogated CD83 upregulation by LPS. Downregulation of CD83 was not associated with reduced mRNA levels or impaired CD83 mRNA transport from the nucleus and could not be prevented by inhibition of the proteasome or endocytic degradation pathways, suggesting that suppression occurs at the translational level. In contrast to infected cells, bystander DCs displayed full maturation as evidenced by upregulation of CD83. Our results indicate that bystander DCs play an important role in NSR-mediated immunity.

Paramyxovirus-based production of Rift Valley fever virus replicon particles.

Replicon-particle-based vaccines combine the efficacy of live-attenuated vaccines with the safety of inactivated or subunit vaccines. Recently, we developed Rift Valley fever virus (RVFV) replicon particles, also known as nonspreading RVFV (NSR), and demonstrated that a single vaccination with these particles can confer sterile immunity in target animals. NSR particles can be produced by transfection of replicon cells, which stably maintain replicating RVFV S and L genome segments, with an expression plasmid encoding the RVFV glycoproteins, Gn and Gc, normally encoded by the M-genome segment. Here, we explored the possibility to produce NSR with the use of a helper virus. We show that replicon cells infected with a Newcastle disease virus expressing Gn and Gc (NDV-GnGc) were able to produce high levels of NSR particles. In addition, using reverse genetics and site-directed mutagenesis, we were able to create an NDV-GnGc variant that lacks the NDV fusion protein and contains two amino acid substitutions in, respectively, Gn and HN. The resulting virus uses a unique entry pathway that facilitates the efficient production of NSR in a one-component system. The novel system provides a promising alternative for transfection-based NSR production.

Creation of Rift Valley fever viruses with four-segmented genomes reveals flexibility in bunyavirus genome packaging.

UNLABELLED: Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines. IMPORTANCE: Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines.

A single vaccination with an improved nonspreading Rift Valley fever virus vaccine provides sterile immunity in lambs.

Rift Valley fever virus (RVFV) is an important pathogen that affects ruminants and humans. Recently we developed a vaccine based on nonspreading RVFV (NSR) and showed that a single vaccination with this vaccine protects lambs from viremia and clinical signs. However, low levels of viral RNA were detected in the blood of vaccinated lambs shortly after challenge infection. These low levels of virus, when present in a pregnant ewe, could potentially infect the highly susceptible fetus. We therefore aimed to further improve the efficacy of the NSR vaccine. Here we report the expression of Gn, the major immunogenic protein of the virus, from the NSR genome. The resulting NSR-Gn vaccine was shown to elicit superior CD8 and CD4-restricted memory responses and improved virus neutralization titers in mice. A dose titration study in lambs revealed that the highest vaccination dose of 10(6.3) TCID50/ml protected all lambs from clinical signs and viremia. The lambs developed neutralizing antibodies within three weeks after vaccination and no anamnestic responses were observed following challenge. The combined results suggest that sterile immunity was achieved by a single vaccination with the NSR-Gn vaccine.

Creation of a nonspreading Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.

Autotransporter ß-domains have a specific function in protein secretion beyond outer-membrane targeting.

Autotransporters (ATs) of Gram-negative bacteria contain an N-proximal passenger domain that is transported to the extracellular milieu and a C-terminal ß-domain that inserts into the outer membrane (OM) in a ß-barrel conformation. This ß-domain facilitates translocation of the passenger domain across the OM and has long been considered to be the translocation pore. However, available crystal structures of ß-domains show that the ß-barrel pore is too narrow for the observed transport of folded elements within the passenger domains. ATs have recently been shown to interact with the ß-barrel assembly machinery. These findings questioned a direct involvement of the ß-domain in passenger translocation and suggested that it may only target the passenger to the ß-barrel assembly machinery pore. To address the function of the ß-domain in more detail, we have replaced the ß-domain of the Escherichia coli AT hemoglobin protease by ß-domains originating from other OM proteins. Furthermore, we have modified the diameter of the ß-domain pore. The mutant proteins were analyzed for their capacity to insert into the OM and for surface display of the passenger. Our results show that efficient passenger secretion requires a specific ß-domain that not only functions as a targeting device but also is directly involved in the translocation of the passenger to the cell surface.