TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.

Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus.

In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111-130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61-110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.

Molecular aspects of Rift Valley fever virus and the emergence of reassortants.

Rift Valley fever phlebovirus (RVFV) is a mosquito-transmitted pathogen endemic to sub-Saharan Africa and the Arabian Peninsula. RVFV is a threat to both animal and human health and has costly economic consequences mainly related to livestock production and trade. Competent hosts and vectors for RVFV are widespread, existing outside of endemic countries including the USA. Thus, the possibility of RVFV spreading to the USA or other countries worldwide is of significant concern. RVFV (genus Phlebovirus) is comprised of an enveloped virion containing a three-segmented, negative-stranded RNA genome that is able to undergo genetic reassortment. Reassortment has the potential to produce viruses that are more pathogenic, easily transmissible, and that have wider vector or host range. This is especially concerning because of the wide use of live attenuated vaccine strains throughout endemic countries. This review focuses on the molecular aspects of RVFV, genetic diversity of RVFV strains, and RVFV reassortment.

ACE2 and TMPRSS2 distribution in the respiratory tract of different animal species and its correlation with SARS-CoV-2 tissue tropism.

A wide range of animal species show variable susceptibility to SARS-CoV-2; however, host factors associated with varied susceptibility remain to be defined. Here, we examined whether susceptibility to SARS-CoV-2 and virus tropism in different animal species are dependent on the expression and distribution of the virus receptor angiotensin-converting enzyme 2 (ACE2) and the host cell factor transmembrane serine protease 2 (TMPRSS2). We cataloged the upper and lower respiratory tract of multiple animal species and humans in a tissue-specific manner and quantitatively evaluated the distribution and abundance of ACE2 and TMPRSS2 mRNA in situ. Our results show that: (i) ACE2 and TMPRSS2 mRNA are abundant in the conduction portion of the respiratory tract, (ii) ACE2 mRNA occurs at a lower abundance compared to TMPRSS2 mRNA, (iii) co-expression of ACE2-TMPRSS2 mRNAs is highest in those species with the highest susceptibility to SARS-CoV-2 infection (i.e., cats, Syrian hamsters, and white-tailed deer), and (iv) expression of ACE2 and TMPRSS2 mRNA was not altered following SARS-CoV-2 infection. Our results demonstrate that while specific regions of the respiratory tract are enriched in ACE2 and TMPRSS2 mRNAs in different animal species, this is only a partial determinant of susceptibility to SARS-CoV-2 infection.IMPORTANCESARS-CoV-2 infects a wide array of domestic and wild animals, raising concerns regarding its evolutionary dynamics in animals and potential for spillback transmission of emerging variants to humans. Hence, SARS-CoV-2 infection in animals has significant public health relevance. Host factors determining animal susceptibility to SARS-CoV-2 are vastly unknown, and their characterization is critical to further understand susceptibility and viral dynamics in animal populations and anticipate potential spillback transmission. Here, we quantitatively assessed the distribution and abundance of the two most important host factors, angiotensin-converting enzyme 2 and transmembrane serine protease 2, in the respiratory tract of various animal species and humans. Our results demonstrate that while specific regions of the respiratory tract are enriched in these two host factors, they are only partial determinants of susceptibility. Detailed analysis of additional host factors is critical for our understanding of the underlying mechanisms governing viral susceptibility and reservoir hosts.

Rift Valley Fever Phlebovirus Reassortment Study in Sheep.

Rift Valley fever (RVF) in ungulates and humans is caused by a mosquito-borne RVF phlebovirus (RVFV). Live attenuated vaccines are used in livestock (sheep and cattle) to control RVF in endemic regions during outbreaks. The ability of two or more different RVFV strains to reassort when co-infecting a host cell is a significant veterinary and public health concern due to the potential emergence of newly reassorted viruses, since reassortment of RVFVs has been documented in nature and in experimental infection studies. Due to the very limited information regarding the frequency and dynamics of RVFV reassortment, we evaluated the efficiency of RVFV reassortment in sheep, a natural host for this zoonotic pathogen. Co-infection experiments were performed, first in vitro in sheep-derived cells, and subsequently in vivo in sheep. Two RVFV co-infection groups were evaluated: group I consisted of co-infection with two wild-type (WT) RVFV strains, Kenya 128B-15 (Ken06) and Saudi Arabia SA01-1322 (SA01), while group II consisted of co-infection with the live attenuated virus (LAV) vaccine strain MP-12 and a WT strain, Ken06. In the in vitro experiments, the virus supernatants were collected 24 h post-infection. In the in vivo experiments, clinical signs were monitored, and blood and tissues were collected at various time points up to nine days post-challenge for analyses. Cell culture supernatants and samples from sheep were processed, and plaque-isolated viruses were genotyped to determine reassortment frequency. Our results show that RVFV reassortment is more efficient in co-infected sheep-derived cells compared to co-infected sheep. In vitro, the reassortment frequencies reached 37.9% for the group I co-infected cells and 25.4% for the group II co-infected cells. In contrast, we detected just 1.7% reassortant viruses from group I sheep co-infected with the two WT strains, while no reassortants were detected from group II sheep co-infected with the WT and LAV strains. The results indicate that RVFV reassortment occurs at a lower frequency in vivo in sheep when compared to in vitro conditions in sheep-derived cells. Further studies are needed to better understand the implications of RVFV reassortment in relation to virulence and transmission dynamics in the host and the vector. The knowledge learned from these studies on reassortment is important for understanding the dynamics of RVFV evolution.

Pigs are highly susceptible to but do not transmit mink-derived highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b.

ABSTRACTRapid evolution of highly pathogenic avian influenza viruses (HPAIVs) is driven by antigenic drift but also by reassortment, which might result in robust replication in and transmission to mammals. Recently, spillover of clade 2.3.4.4b HPAIV to mammals including humans, and their transmission between mammalian species has been reported. This study aimed to evaluate the pathogenicity and transmissibility of a mink-derived clade 2.3.4.4b H5N1 HPAIV isolate from Spain in pigs. Experimental infection caused interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract and 100% seroconversion. Infected pigs shed limited amount of virus, and importantly, there was no transmission to contact pigs. Notably, critical mammalian-like adaptations such as PB2-E627 K and HA-Q222L emerged at low frequencies in principal-infected pigs. It is concluded that pigs are highly susceptible to infection with the mink-derived clade 2.3.4.4b H5N1 HPAIV and provide a favorable environment for HPAIV to acquire mammalian-like adaptations.

Gene editing of pigs to control influenza A virus infections.

Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to WT pigs. Our findings could support the commercial use of GE pigs to minimize (i) the economic losses caused by IAV infection in pigs, and (ii) the emergence of novel IAVs with pandemic potential through genetic reassortment in the "mixing vessel", the pig.

Experimental inoculation of pigs with monkeypox virus results in productive infection and transmission to sentinels.

Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at 12 through 18 days post-challenge (DPC). No virus was isolated and no viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.

Experimental co-infection of calves with SARS-CoV-2 Delta and Omicron variants of concern.

Since emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.

Fomite Transmission of SARS-CoV-2 and Its Contributing Factors.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has drastically changed our lives, from our personal freedoms and habits to public health and socioeconomics [...].

Ancestral Lineage of SARS-CoV-2 Is More Stable in Human Biological Fluids than Alpha, Beta, and Omicron Variants of Concern.

SARS-CoV-2 is a zoonotic virus first identified in 2019, and has quickly spread worldwide. The virus is primarily transmitted through respiratory droplets from infected persons; however, the virus-laden excretions can contaminate surfaces which can serve as a potential source of infection. Since the beginning of the pandemic, SARS-CoV-2 has continued to evolve and accumulate mutations throughout its genome leading to the emergence of variants of concern (VOCs) which exhibit increased fitness, transmissibility, and/or virulence. However, the stability of SARS-CoV-2 VOCs in biological fluids has not been thoroughly investigated. The aim of this study was to determine and compare the stability of different SARS-CoV-2 strains in human biological fluids. Here, we demonstrate that the ancestral strain of the Wuhan-like lineage A was more stable than the Alpha VOC B.1.1.7, and the Beta VOC B.1.351 strains in human liquid nasal mucus and sputum. In contrast, there was no difference in stability among the three strains in dried biological fluids. Furthermore, we also show that the Omicron VOC B.1.1.529 strain was less stable than the ancestral Wuhan-like strain in liquid nasal mucus. These studies provide insight into the effect of the molecular evolution of SARS-CoV-2 on environmental virus stability, which is important information for the development of countermeasures against SARS-CoV-2. IMPORTANCE Genetic evolution of SARS-CoV-2 leads to the continuous emergence of novel virus variants, posing a significant concern to global public health. Five of these variants have been classified to date into variants of concern (VOCs); Alpha, Beta, Gamma, Delta, and Omicron. Previous studies investigated the stability of SARS-CoV-2 under various conditions, but there is a gap of knowledge on the survival of SARS-CoV-2 VOCs in human biological fluids which are clinically relevant. Here, we present evidence that Alpha, Beta, and Omicron VOCs were less stable than the ancestral Wuhan-like strain in human biological fluids. Our findings highlight the potential risk of contaminated human biological fluids in SARS-CoV-2 transmission and contribute to the development of countermeasures against SARS-CoV-2.

Stability of SARS-CoV-2 in Biological Fluids of Animals.

Since its first emergence in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to evolve genetically, jump species barriers, and expand its host range. There is growing evidence of interspecies transmission including infection of domestic animals and widespread circulation in wildlife. However, knowledge of SARS-CoV-2 stability in animal biological fluids and their role in transmission is still limited as previous studies focused on human biological fluids. Therefore, this study aimed to determine the SARS-CoV-2 stability in biological fluids from three animal species, cats, sheep and white-tailed deer (WTD). Saliva, feces, 10% fecal suspensions, and urine of cats, sheep, and WTD were mixed with a known concentration of virus and incubated under indoor and three different climatic conditions. Our results show that the virus was stable for up to 1 day in the saliva of cats, sheep, and WTD regardless of the environmental conditions. The virus remained infectious for up to 6 days in feces and 15 days in fecal suspension of WTD, whereas the virus was rather unstable in cat and sheep feces and fecal suspensions. We found the longest survival of SARS-CoV-2 in the urine of cats, sheep, and WTD. Furthermore, side-by-side comparison with different SARS-CoV-2 strains showed that the Alpha, Delta, and Omicron variants of concern were less stable than the ancestral Wuhan-like strain in WTD fecal suspension. The results of our study provide valuable information for assessing the potential role of various animal biological fluids in SARS-CoV-2 transmission.

Synthetic graphene-copper nanocomposites interact with the hACE-2 enzyme and inhibit its biochemical activity.

This study demonstrates the copper nanocomposite-induced enzymatic inhibition of human angiotensin I-converting enzyme-2 (hACE-2) by complex stabilization through the formation of the enzyme nanocomposite. The immediate application of this work is related to ACE-2 as a mechanism of SARS-CoV-2 entry into cells. Moreover, ACE-2 enzyme regulation is a potential therapeutic strategy in hypertension and cardiovascular disease, diabetes, lung injury, and fibrotic disorders. Thus, inhibition of ACE-2 with nanocomposite therapy, may have pharmacologic application with regard to infectious and non-infectious diseases. Synthesized copper nanocomposites described here alone with a commercially available compound, were tested for their potential to inhibit hACE-2 activities. Following wet chemical synthesis, Cu/CuO nanoparticles and graphene-copper (GO-Cu) complexes were synthesized and characterized for their chemical integrity. Cu/CuO formed well-dispersed clusters of 390 ± 100 nm, that when complexed with the hACE-2 enzyme exhibited larger clusters of 506 ± 56 nm. The formation of the Cu/CuO and hACE-2 enzyme complex was monitored by analyzing the zeta potential, which reflected the surface charge distribution of the complex. A negatively charged Cu/CuO nanocomposite nearly becomes neutral when complexed with hACE-2 further assuring the complex formation. Formation of this complex and its inactivation of hACE-2 was evaluated using a standardized protocal for enzymatic activity. Similarly, carboxylate-functionalized graphene was complexed with copper, and its inhibitory effect was studied. Each step in the GO-Cu composite formation was monitored by characterizing its surface electrical properties, resulting in a decrease in its zeta potential and conductivity when complexed with copper. The interaction of the nanocomposites with hACE-2 was confirmed by 2D-FDS and gel electrophoresis analysis. GO-Cu was a rapid and efficacious inhibitor compared to Cu-CuO, especially at lower concentrations (2 µg ml-1). Considering the environmental friendliness of copper and graphene and their use in industries as surface coating materials, we anticipate that use of these composites once proven effective, may have future antimicrobial application. Utility of nanocomposites as antimicrobials, either as a surface antimicrobial or as an in vivo therapeutic, could be invisioned for use against current unknown and/or emergent pathogens.

RT-qPCR genotyping assays for differentiating Rift Valley fever phlebovirus strains.

Rift Valley fever phlebovirus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen. Real time RT-qPCR genotyping (GT) assays were developed to differentiate between two RVFV wild-type strains (128B-15 and SA01-1322) and a vaccine strain (MP-12). The GT assay uses a one-step RT-qPCR mix, with two different RVFV strain-specific primers (either forward or reverse) with long or short G/C tags and a common primer (either forward or reverse) for each of the 3 genomic segments. The GT assay produces PCR amplicons with unique melting temperatures that are resolved in a post PCR melt curve analysis for strain identification. Furthermore, a strain specific RT-qPCR (SS-PCR) assay was developed to allow for specific detection of low titer RVFV strains in mixed RVFV samples. Our data shows that the GT assays are capable of differentiating L, M, and S segments of RVFV strains 128B-15 versus MP-12, and 128B-15 versus SA01-1322. The SS-PCR assay results revealed that it can specifically amplify and detect a low titer MP-12 strain in mixed RVFV samples. Overall, these two novel assays are useful as screening tools for determining reassortment of the segmented RVFV genome during co-infections, and could be adapted and applied for other segmented pathogens of interest.

De novo design of a stapled peptide targeting SARS-CoV-2 spike protein receptor-binding domain.

Although effective vaccines have been developed against SARS-CoV-2, many regions in the world still have low rates of vaccination and new variants with mutations in the viral spike protein have reduced the effectiveness of most available vaccines and treatments. There is an urgent need for a drug to cure this disease and prevent infection. The SARS-CoV-2 virus enters the host cell through protein-protein interaction between the virus's spike protein and the host's angiotensin converting enzyme (ACE2). Using protein design software and molecular dynamics simulations, we have designed a 17-residue peptide (pep39), that binds to the spike protein receptor-binding domain (RBD) and blocks interaction of spike protein with ACE2. We have confirmed the binding activity of the designed peptide for the original spike protein and the delta variant spike protein using micro-cantilever and bio-layer interferometry (BLI) based methods. We also confirmed that pep39 strongly inhibits SARS-CoV-2 virus replication in Vero E6 cells. Taken together these data suggest that a newly designed spike protein RBD blocking peptide pep39 has a potential as a SARS-CoV-2 virus inhibitor.

Rapid Identification of ASFV, CSFV and FMDV from Mongolian Outbreaks with MinION Short Amplicon Sequencing.

African swine fever virus (ASFV), classical swine fever virus (CSFV), and foot-and-mouth disease virus (FMDV) cause important transboundary animal diseases (TADs) that have a significant economic impact. The rapid and unequivocal identification of these pathogens and distinction from other animal diseases based on clinical symptoms in the field is difficult. Nevertheless, early pathogen detection is critical in limiting their spread and impact as is the availability of a reliable, rapid, and cost-effective diagnostic test. The purpose of this study was to evaluate the feasibility to identify ASFV, CSFV, and FMDV in field samples using next generation sequencing of short PCR products as a point-of-care diagnostic. We isolated nucleic acids from tissue samples of animals in Mongolia that were infected with ASFV (2019), CSFV (2015), or FMDV (2018), and performed conventional (RT-) PCR using primers recommended by the Terrestrial Animal Health Code of the World Organization for Animal Health (WOAH). The (RT-) PCR products were then sequenced in Mongolia using the MinION nanopore portable sequencer. The resulting sequencing reads successfully identified the respective pathogens that exhibited 91-100% nucleic acid similarity to the reference strains. Phylogenetic analyses suggest that the Mongolian virus isolates are closely related to other isolates circulating in the same geographic region. Based on our results, sequencing short fragments derived by conventional (RT-) PCR is a reliable approach for rapid point-of-care diagnostics for ASFV, CSFV, and FMDV even in low-resource countries.

Identification of Host Factors for Rift Valley Fever Phlebovirus.

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.

Identification of host factors for Rift Valley Fever Phlebovirus.

Background: Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. Methodology: To identify the host factors or genes essential for RVFV replication, we conducted a CRISPR-Cas9 knock-out screen in human A549 cells. We then validated the putative genes using siRNA-mediated knockdowns and CRISPR-Cas9-mediated knockout studies, respectively. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers by plaque assay or TCID50 assay. Findings: We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knockdowns found that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of LRP1 in RVFV replication was previously described in detail. Knock-out A549 cell lines were generated and used to dissect the effect of WRD7 on RVFV and another bunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knock-out cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24h) when compared to LACV which was affected an earlier replication phase (12h). Conclusion: In summary, we have identified WDR7 as an essential host factor for the replication of two relevant bunyaviruses, RVFV and LACV. Future studies will investigate the mechanistic role by which WDR7 facilitates Phlebovirus replication.

Preliminary Study on the Efficacy of a Recombinant, Subunit SARS-CoV-2 Animal Vaccine against Virulent SARS-CoV-2 Challenge in Cats.

The objective of this work was to evaluate the safety and efficacy of a recombinant, subunit SARS-CoV-2 animal vaccine in cats against virulent SARS-CoV-2 challenge. Two groups of cats were immunized with two doses of either a recombinant SARS-CoV-2 spike protein vaccine or a placebo, administered three weeks apart. Seven weeks after the second vaccination, both groups of cats were challenged with SARS-CoV-2 via the intranasal and oral routes simultaneously. Animals were monitored for 14 days post-infection for clinical signs and viral shedding before being humanely euthanized and evaluated for macroscopic and microscopic lesions. The recombinant SARS-CoV-2 spike protein subunit vaccine induced strong serologic responses post-vaccination and significantly increased neutralizing antibody responses post-challenge. A significant difference in nasal and oral viral shedding, with significantly reduced virus load (detected using RT-qPCR) was observed in vaccinates compared to mock-vaccinated controls. Duration of nasal, oral, and rectal viral shedding was also significantly reduced in vaccinates compared to controls. No differences in histopathological lesion scores were noted between the two groups. Our findings support the safety and efficacy of the recombinant spike protein-based SARS-CoV-2 vaccine which induced high levels of neutralizing antibodies and reduced nasal, oral, and rectal viral shedding, indicating that this vaccine will be efficacious as a COVID-19 vaccine for domestic cats.

Development of an Indirect ELISA for the Detection of SARS-CoV-2 Antibodies in Cats.

Companion animals are susceptible to a variety of coronaviruses, and recent studies show that felines are highly susceptible to SARS-CoV-2 infection. RT-PCR diagnostic is currently the method of choice to detect the presence of SARS-CoV-2-specific viral nucleic acids in animal samples during an active infection; however, serological assays are critical to determine whether animals were exposed to the virus and to determine the seroprevalence of SARS-CoV-2-specific antibodies in a defined population. In this study, we utilized recombinant nucleocapsid (N) protein and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 expressed in E. coli (N) and mammalian cells (N, RBD) to develop indirect ELISA (iELISA) tests using well-characterized SARS-CoV-2-positive and -negative cat serum panels from previous experimental cat challenge studies. The optimal conditions for the iELISA tests were established based on checkerboard dilutions of antigens and antibodies. The diagnostic sensitivity for the detection of feline antibodies specific for the N or RBD proteins of the iELISA tests was between 93.3 and 97.8%, respectively, and the diagnostic specificity 95.5%. The iELISAs developed here can be used for high-throughput screening of cat sera for both antigens. The presence of SARS-CoV-2-specific antibodies in a BSL-2 biocontainment environment, unlike virus neutralization tests with live virus which have to be performed in BSL-3 laboratories.