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.

A field test of the dilution effect hypothesis in four avian multi-host pathogens.

The Dilution Effect Hypothesis (DEH) argues that greater biodiversity lowers the risk of disease and reduces the rates of pathogen transmission since more diverse communities harbour fewer competent hosts for any given pathogen, thereby reducing host exposure to the pathogen. DEH is expected to operate most intensely in vector-borne pathogens and when species-rich communities are not associated with increased host density. Overall, dilution will occur if greater species diversity leads to a lower contact rate between infected vectors and susceptible hosts, and between infected hosts and susceptible vectors. Field-based tests simultaneously analysing the prevalence of several multi-host pathogens in relation to host and vector diversity are required to validate DEH. We tested the relationship between the prevalence in house sparrows (Passer domesticus) of four vector-borne pathogens-three avian haemosporidians (including the avian malaria parasite Plasmodium and the malaria-like parasites Haemoproteus and Leucocytozoon) and West Nile virus (WNV)-and vertebrate diversity. Birds were sampled at 45 localities in SW Spain for which extensive data on vector (mosquitoes) and vertebrate communities exist. Vertebrate censuses were conducted to quantify avian and mammal density, species richness and evenness. Contrary to the predictions of DEH, WNV seroprevalence and haemosporidian prevalence were not negatively associated with either vertebrate species richness or evenness. Indeed, the opposite pattern was found, with positive relationships between avian species richness and WNV seroprevalence, and Leucocytozoon prevalence being detected. When vector (mosquito) richness and evenness were incorporated into the models, all the previous associations between WNV prevalence and the vertebrate community variables remained unchanged. No significant association was found for Plasmodium prevalence and vertebrate community variables in any of the models tested. Despite the studied system having several characteristics that should favour the dilution effect (i.e., vector-borne pathogens, an area where vector and host densities are unrelated, and where host richness is not associated with an increase in host density), none of the relationships between host species diversity and species richness, and pathogen prevalence supported DEH and, in fact, amplification was found for three of the four pathogens tested. Consequently, the range of pathogens and communities studied needs to be broadened if we are to understand the ecological factors that favour dilution and how often these conditions occur in nature.

Niche dynamics along two centuries of multiple crayfish invasions.

The realised ecological niches of species may change in response to dynamic abiotic and biotic environments, particularly under fast global change. To fully understand the dynamics of niche features and their drivers, it is essential to have a long-term view of species distributions and the factors that may have influenced them. Here, we analysed the distribution and niche dynamics of the Italian crayfish (Austropotamobius fulcisianus) in the Iberian Peninsula over the past 200 years. The Italian crayfish was introduced to Spain in the 16th century, and spread due to multiple stocking events until the 1970s, when two North American crayfish (red swamp crayfish Procambarus clarkii, and signal crayfish Pacifastacus leniusculus) were introduced. Both North American species are carriers of a pathogen (Aphanomyces astaci, the causal agent of crayfish plague) lethal to the Italian crayfish. We hypothesised that the realised niche of the Italian crayfish, both in breadth and in position, has changed over time following changes in its range. The distribution of the Italian crayfish expanded from the mid-19th century until the mid-20th century, in association with an enlargement of its realised niched, mostly towards less abrupt and more coastal-influenced areas. After the introduction of the North American crayfishes, the collapse of the Italian crayfish involved a niche shift towards rough terrains in mountain areas. North American crayfish have eventually occupied most of the Italian crayfish's niche space, with the few no-coexistence areas being relegated to the most abrupt and high-elevation territories. Our historical approach allowed us to document and understand the highly dynamic distribution and niche of the Italian crayfish in the presence of invader counterparts, and to explore the environmental conditions under which their coexistence is minimised.

Retrospective Identification of Early Autochthonous Case of Crimean-Congo Hemorrhagic Fever, Spain, 2013.

Before this report, 7 autochthonous human cases of Crimean-Congo hemorrhagic fever had been reported in Spain, all occurring since 2016. We describe the retrospective identification of an eighth case dating back to 2013. This study highlights that the earliest cases of an emerging disease are often difficult to recognize.

Sequencing of SARS-CoV-2 genome using different nanopore chemistries.

Nanopore sequencing has emerged as a rapid and cost-efficient tool for diagnostic and epidemiological surveillance of SARS-CoV-2 during the COVID-19 pandemic. This study compared the results from sequencing the SARS-CoV-2 genome using R9 vs R10 flow cells and a Rapid Barcoding Kit (RBK) vs a Ligation Sequencing Kit (LSK). The R9 chemistry provided a lower error rate (3.5%) than R10 chemistry (7%). The SARS-CoV-2 genome includes few homopolymeric regions. Longest homopolymers were composed of 7 (TTTTTTT) and 6 (AAAAAA) nucleotides. The R10 chemistry resulted in a lower rate of deletions in thymine and adenine homopolymeric regions than the R9, at the expenses of a larger rate (~10%) of mismatches in these regions. The LSK had a larger yield than the RBK, and provided longer reads than the RBK. It also resulted in a larger percentage of aligned reads (99 vs 93%) and also in a complete consensus genome. The results from this study suggest that the LSK preparation library provided longer DNA fragments which contributed to a better assembly of the SARS-CoV-2, despite an impaired detection of variants in a R10 flow cell. Nanopore sequencing could be used in epidemiological surveillance of SARS-CoV-2. KEY POINTS: • Sequencing SARS-CoV-2 genome is of great importance for the pandemic surveillance. • Nanopore offers a low cost and accurate method to sequence SARS-CoV-2 genome. • Ligation sequencing is preferred rather than the rapid kit using transposases.

Experimental infection of grey partridges with Bagaza virus: pathogenicity evaluation and potential role as a competent host.

Bagaza virus (BAGV; synonymous to Israel turkey meningoencephalomyelitis virus, ITV) is a relevant arthropod-borne epornitic flavivirus. In its first emergence in Europe (southern Spain, 2010) BAGV caused an outbreak, severely affecting red-legged partridges and common pheasants. The effects (pathogenicity, role as reservoir host) of BAGV in other European phasianids are unknown. To fill this gap, grey partridges were experimentally infected with BAGV. The clinical course of the disease was severe, with neurological signs, significant weight loss and 40% mortality. Low viral loads in the blood and the absence of contact transmission suggest a limited-if any-role on BAGV transmission for this European phasianid.

Characterization of PTV-12, a newly described porcine teschovirus serotype: in vivo infection and cross-protection studies.

Porcine teschoviruses (PTVs) constitute 1 of the 31 genera within the Picornaviridae family, comprising at least 13 genetic types (PTV-1 to PTV-13), of which only 11 (PTV-1 to PTV-11) have been recognized as serotypes to date. Specific for swine and wild boars, most PTVs are usually non-pathogenic, but some viral variants cause severe disorders in the central nervous system (Teschen disease) or milder signs (Talfan disease), as well as reproductive, digestive and respiratory disorders and skin lesions. Previous studies revealed a high diversity of teschoviruses circulating in Spanish pig populations. Phylogenetic analysis performed with these sequences and others available in GenBank disclosed 13 clusters, 11 of which corresponded to the known PTV serotypes, and 1 of 2 additional groups is represented by isolate CC25, whose full-length genomic sequence has been obtained. This group is new to science, and was putatively named PTV-12. Here, a complete characterization of this isolate is presented, including the experimental infection of minipigs to assess tissue tropism and possible pathogenicity in vivo in the host species. In addition, using this experimental animal model, we investigated whether a pre-existing infection with this PTV-12 isolate could confer cross-protection against infection with a heterotypic PTV-1 virulent strain. Based on phylogenetic analysis and serological data, we propose CC25 as the prototype strain of a new teschovirus serotype, PTV-12.

Pathogenicity evaluation of twelve West Nile virus strains belonging to four lineages from five continents in a mouse model: discrimination between three pathogenicity categories.

Rodent models have been used extensively to study West Nile virus (WNV) infection because they develop severe neurological symptoms similar to those observed in human WNV neuroinvasive disease. Most of this research has focused on old lineage (L) 1 strains, while information about pathogenicity is lacking for the most recent L1 and L2 strains, as well as for newly defined lineages. In this study, 4-week-old Swiss mice were inoculated with a collection of 12 WNV isolates, comprising 10 old and recent L1 and L2 strains, the putative L6 strain from Malaysia and the proposed L7 strain Koutango (KOU). The intraperitoneal inoculation of 10-fold dilutions of each strain allowed the characterization of the isolates in terms of LD50, median survival times, ID50, replication in neural and extraneural tissues and antibody production. Based on these results, we classified the isolates in three groups: high virulence (all L1a strains, recent L2 strains and KOU), moderate virulence (B956 strain) and low virulence (Kunjin and Malaysian isolates). We determined that the inoculation of a single dose of 1000 p.f.u. would be sufficient to classify WNV strains by pathotype. We confirmed the enhanced virulence of the KOU strain with a high capacity to cause rapid systemic infection. We also corroborated that differences in pathogenicity among strains do not correlate with phylogenetic lineage or geographic origin, and confirmed that recent European and African WNV strains belonging to L1 and L2 are highly virulent and do not differ in their pathotype profile compared to the prototype NY99 strain.

Historical citizen science to understand and predict climate-driven trout decline.

Historical species records offer an excellent opportunity to test the predictive ability of range forecasts under climate change, but researchers often consider that historical records are scarce and unreliable, besides the datasets collected by renowned naturalists. Here, we demonstrate the relevance of biodiversity records developed through citizen-science initiatives generated outside the natural sciences academia. We used a Spanish geographical dictionary from the mid-nineteenth century to compile over 10 000 freshwater fish records, including almost 4 000 brown trout (Salmo trutta) citations, and constructed a historical presence-absence dataset covering over 2 000 10 × 10 km cells, which is comparable to present-day data. There has been a clear reduction in trout range in the past 150 years, coinciding with a generalized warming. We show that current trout distribution can be accurately predicted based on historical records and past and present values of three air temperature variables. The models indicate a consistent decline of average suitability of around 25% between 1850s and 2000s, which is expected to surpass 40% by the 2050s. We stress the largely unexplored potential of historical species records from non-academic sources to open new pathways for long-term global change science.