Autophagy-linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS-CoV-2 entry into human cells.

Better understanding on interactions between SARS-CoV-2 and host cells should help to identify host factors that may be targetable to combat infection and COVID-19 pathology. To this end, we have conducted a genome-wide CRISPR/Cas9-based loss-of-function screen in human lung cancer cells infected with SARS-CoV-2-pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS-CoV-2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS-CoV-2 viruses confirm that loss-of-function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single-cell RNA-Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS-CoV-2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests.

BACKGROUND: The fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms. METHODS: We expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test. RESULTS: The performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05-98.44) and diagnostic specificity of 96.37 (95% CI: 93.05-98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892-0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA). CONCLUSION: This study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.

Two serological approaches for detection of antibodies to SARS-CoV-2 in different scenarios: a screening tool and a point-of-care test.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 8 million people worldwide, becoming a pandemic. Detecting antibodies against SARS-CoV-2 is of utmost importance and a good indicator of exposure and circulation of the virus within the general population. Two serological tools based on a double recognition assay [enzyme-linked immunosorbent assay (DR-ELISA) and lateral flow assay (DR-LFA)] to detect total antibodies to SARS-CoV-2 have been developed based on the recombinant nucleocapsid protein. A total of 1065 serum samples, including positive for COVID-19 and negative samples from healthy donors or infected with other respiratory pathogens, were analyzed. The results showed values of sensitivity between 91.2% and 100%, and specificity of 100% and 98.2% for DR-LFA and DR-ELISA, respectively. No cross-reactivity against seasonal coronavirus (HCoV-NL63, HCoV-229E, HCoV-HKU1, HCoV-OC43) was found. These results demonstrate the importance of serology as a complementary tool to polymerase chain reaction for follow-up of recovered patients and identification of asymptomatic individuals.

A monoclonal antibody to DIII E protein allowing the differentiation of West Nile virus from other flaviviruses by a lateral flow assay.

West Nile Virus (WNV) belongs to the Flaviviridae family, genus Flavivirus, which includes other emerging arthropod-borne viruses (arboviruses) pathogenic for animals and/or humans. West Nile Virus is a genetically diverse RNA virus with at least 7 different recognized lineages. Following its recent introduction and subsequent expansion to the Americas, WNV is currently one of the most widely spread arboviruses in the world having recently re-emerged in the Mediterranean basin, Central and Eastern Europe. Laboratory tests are essential to confirm WNV infection and monoclonal antibodies represent useful tools for the development of diagnostic assays. A monoclonal antibody, 1D11, recognizing an epitope in the domain III of the envelope glycoprotein of WNV, was selected for this study. Its suitability to detect a range of WNV variants representative of its whole genetic range, and to differentiate it from other flaviviruses and arboviruses, was assessed by means of an immunochromatographic assay in an LFA format. A panel of cell culture supernatants infected with 9 different WNV isolates representing a wide range of genetic lineages, and 16 non-WNV arboviruses, including flaviviruses closely related to WNV, were tested. The mAb correctly detected all WNV strains, and did not react with any of the non-WNV arboviruses.

Diagnostic aptitude of West Nile virus-like particles expressed in insect cells.

West Nile virus is a globally spread zoonotic arbovirus. The laboratory diagnosis of WNV infection relies on virus identification by RT-PCR or on specific antibody detection by serological tests, such as ELISA or virus-neutralization. These methods usually require a preparation of the whole virus as antigen, entailing biosafety issues and therefore requiring BSL-3 facilities. For this reason, recombinant antigenic structures enabling effective antibody recognition comparable to that of the native virions, would be advantageous as diagnostic reagents. WNV virions are enveloped spherical particles made up of 3 structural proteins (C, capsid; M, membrane and E, envelope) enclosing the viral RNA. This study describes the co-expression of these 3 proteins yielding non-infectious virus-like particles (VLPs) and the results of the initial assessment of these VLPs, used instead of the whole virus, that were shown to perform correctly in two different ELISAs for WNV diagnosis.

Bagaza virus and Israel turkey meningoencephalomyelitis virus are a single virus species.

Bagaza virus (BAGV) and Israel turkey meningoencephalomyelitis virus (ITV) are classified in the genus Flavivirus of the family Flaviviridae. Serologically, they are closely related, belonging to the Ntaya serocomplex. Nucleotide sequences available to date consist of several complete sequences of BAGV isolates, but only partial sequences of ITV isolates. Sequence comparisons of partial envelope (E) and NS5 regions reveal a close genetic relationship between these viruses. Despite this, BAGV and ITV are considered as separate virus species in the database of the International Committee on Taxonomy of Viruses. In this work, complete nucleotide sequences for five ITV isolates are provided, thereby permitting a phylogenetic comparison with other complete sequences of flaviviruses in the Ntaya serogroup. We conclude that BAGV and ITV are the same virus species and propose that both viruses be designated by a new unified name: Avian meningoencephalomyelitis virus.

Global gene expression analysis in skin biopsies of European red deer experimentally infected with bluetongue virus serotypes 1 and 8.

Bluetongue virus (BTV) is a double-stranded RNA virus transmitted by blood-feeding biting midges of the genus Culicoides to wild and domestic ruminants, causing high morbidity and variable mortality. The aim of this study was to characterize differential gene expression in skin biopsies of red deer (Cervus elaphus) hinds experimentally infected with BTV serotypes 1 and 8. Skin biopsies were collected from BTV-1 and BTV-8 experimentally infected and control hinds at 14 and 98 days post-infection (dpi). Global gene expression profile in response to BTV infection was characterized at 14 dpi using a bovine microarray together with real-time RT-PCR analysis of differentially expressed genes at 14 and 98 dpi. Eighteen genes were upregulated and three were downregulated in response to virus infection, with no significant differences between BTV-1 and BTV-8 infected hinds. Seven unique genes, six upregulated (ISG15, PSMB8, PSMB9, BOLA, C1qA, C4) and one downregulated (FOS) were over-represented after conditional test for biological process gene ontology, which affected five molecular pathways (RIG-1, proteasome, MHC-1, complement, TLR) implicated in host immune response. BTV infection had a minor and transient effect on gene expression in hinds, as shown by the very few genes that were differentially expressed in response to infection at 14 dpi, most of which had similar expression levels between infected and uninfected animals at 98 dpi. These results suggested that red deer could control BTV infection with little effect on host molecular pathways.

Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus.

West Nile virus (WNV) is an emerging zoonotic pathogen with a wide range of hosts, including birds, horses and humans. The development and evaluation of the performance of a new enzyme-linked immunosorbent assay (ELISA) are described for rapid detection of WNV-specific antibodies in samples originating from an extensive range of vertebrates susceptible to WNV infection. The assay uses a monoclonal antibody (MAb) which binds whole virus particles and neutralizes infection in vitro by recognizing a neutralizing epitope within the envelope (E) glycoprotein of the virus. This MAb, labelled with horseradish peroxidase, was used to compete with WNV-specific serum antibodies for virus-binding in vitro. The epitope-blocking ELISA was optimized in a manner that enabled its validation with a number of experimental and field sera, from a wide range of wild bird species, and susceptible mammals. The new ELISA exhibited high specificity (79.5-96.5%) and sensitivity (100%), using the virus-neutralization test as reference standard. It also required a much lower volume of sample (10 µl per analysis) compared to other ELISAs available commercially. This new method may be helpful for diagnosis and disease surveillance, particularly when testing samples from small birds, which are available in limited amounts.

A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain.

In the course of an epidemiologic surveillance program for swine diseases carried out in Spain, 206 cytopathic viruses were isolated from 600 porcine fecal samples between 2004 and 2005. The virus isolates were examined using reverse transcription polymerase chain reaction (RT-PCR) methods specific for different types of porcine picornaviruses, including members of the Teschovirus, Enterovirus, and Sapelovirus genera, and PCR for porcine adenoviruses. Of the 206 isolates, 97 (47%) were identified as teschoviruses, 18 (9%) as sapeloviruses, and 7 (3%) as porcine adenoviruses. Neither Porcine enterovirus B nor Swine vesicular disease virus was found among the isolates. The present study confirms that teschoviruses are highly prevalent in porcine fecal samples, at least in Spain. It also reveals that these viruses commonly circulate among apparently healthy pigs.

Heparan sulphate mediates swine vesicular disease virus attachment to the host cell.

Heparan sulphate (HS) has been found to serve as receptor for initial cell binding of numerous viruses. Different glycosaminoglycans (GAGs), including heparin and HS, were analysed for their ability to bind swine vesicular disease virus (SVDV), a picornavirus with close homology to human coxsackie B5 virus. Binding of SVDV was established by heparin-affinity chromatography. In addition, infection of IB-RS-2 epithelial porcine cells was inhibited by treating the virus with soluble HS, heparin, and chondroitin sulphate B (CS-B), as well as by enzymic digestion of cell surface GAGs. Analysis of the infection course showed that SVDV uses cellular HS for its binding to the cell surface and that this interaction occurs during attachment of the virus, prior to its internalization into the cell. Sequence analysis of SVDV variants selected for their lack of sensitivity to heparin inhibition in vitro led to the identification of two residues (A2135V and I1266K) potentially involved in heparin/HS interaction. The location of these residues in a three-dimensional model shows that they are clustered in a well-exposed region of the capsid, providing a physical mechanism that could account for the heparin-binding phenotype.