A new and threatened species of Bondarzewia from the Brazilian cloud forests.

A new and threatened polypore species, Bondarzewia loguerciae, is described from the cloud forests of southern Brazil. It is characterized by single-pileate basidiomata that grow on dead branches and along living stems of standing trunks and present a context with dark lines and resinous tubes. When growing in axenic culture, this species also develops chlamydospores. We provide an illustrated morphological description and molecular analysis. Our specimens from Brazil form a monophyletic group among other species of the Southern Hemisphere. The conservation status of B. loguerciae is assessed and published as "Critically Endangered" based on the International Union for Conservation of Nature  (IUCN) criteria. Additionally, a key to the species is provided.

ACE2-using merbecoviruses: Further evidence of convergent evolution of ACE2 recognition by NeoCoV and other MERS-CoV related viruses.

Angiotensin-converting enzyme 2 (ACE2) was recognized as an entry receptor shared by coronaviruses from Sarbecovirus and Setracovirus subgenera, including three human coronaviruses: SARS-CoV, SARS-CoV-2, and NL63. We recently disclosed that NeoCoV and three other merbecoviruses (PDF-2180, MOW15-22, PnNL 2018B), which are MERS-CoV relatives found in African and European bats, also utilize ACE2 as their functional receptors through unique receptor binding mechanisms. This unexpected receptor usage assumes significance, particularly in light of the prior recognition of Dipeptidyl peptidase-4 (DPP4) as the only known protein receptor for merbecoviruses. In contrast to other ACE2-using coronaviruses, NeoCoV and PDF-2180 engage a distinct and relatively compact binding surface on ACE2, facilitated by protein-glycan interactions, which is demonstrated by the Cryo-EM structures of the receptor binding domains (RBDs) of these viruses in complex with a bat ACE2 orthologue. These findings further support the hypothesis that phylogenetically distant coronaviruses, characterized by distinct RBD structures, can independently evolve to acquire ACE2 affinity during inter-species transmission and adaptive evolution. To date, these viruses have exhibited limited efficiency in entering human cells, although single mutations like T510F in NeoCoV can overcome the incompatibility with human ACE2. In this review, we present a comprehensive overview of ACE2-using merbecoviruses, summarize our current knowledge regarding receptor usage and host tropism determination, and deliberate on potential strategies for prevention and intervention, with the goal of mitigating potential future outbreaks caused by spillover of these viruses.

The avian influenza A virus receptor SA-α2,3-Gal is expressed in the porcine nasal mucosa sustaining the pig as a mixing vessel for new influenza viruses.

Influenza A viruses (IAVs) originate from wild birds but have on several occasions jumped host barriers and are now also circulating in humans and mammals. The IAV host receptors (glycans with galactose linked to a sialic acid (SA) in an α2,3 or α2,6 linkage) are crucial host factors restricting inter-species transmission. In general, avian-origin IAVs show a preference for SA-α2,3 (avian receptor), whereas IAVs isolated from humans and pigs prefer SA-α2,6 (human receptor). N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two major SAs. Neu5Ac is expressed in all species, whereas Neu5Gc is only expressed in a limited number of domestic species such as pigs and horses, but not in humans. Despite that previous studies have shown that the IAV host receptor distribution appears to be similar in pigs and humans, none of these studies have investigated the expression of Neu5Gc-α2,6 in situ in porcine tissues. Thus, the aim of this study was to elucidate the distribution of IAV host receptors expressed in the porcine respiratory tract and relate the expression to the viral tropism of diverse host-adapted IAVs. The IAV receptor (SA-α2,3 and SA-α2,6) distribution and the presence of specifically Neu5Gc-α2,6 in the porcine nasal, tracheal, and lung tissues was investigated by lectin histochemistry. Furthermore, IAV immunohistochemistry was performed on tissues from pigs experimentally infected with IAVs, either adapted to pigs or humans, to investigate the significance of the IAV host receptors and the tropism of the diverse host-adapted IAVs. We document for the first time the expression of the avian receptor on the surface of the porcine nasal mucosa and an equal expression of Neu5Ac-α2,6 and Neu5Gc-α2,6 on the surface of the tracheal epithelium and alveoli. In all IAV-infected pigs, we found a low amount of IAV-positive cells in the trachea despite a high expression of the human receptor. Cumulatively, these findings suggest that optimal IAV replication involves a complex interplay between the viruses and their host receptors and that there might be other less clearly defined host factors that determine the site of replication.

Structural evolution of an immune evasion determinant shapes pathogen host tropism.

Modern infectious disease outbreaks often involve changes in host tropism, the preferential adaptation of pathogens to specific hosts. The Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the molecular mechanisms of host tropism, because different variants of these tick-transmitted bacteria are distinctly maintained in rodents or bird reservoir hosts. To survive in hosts and escape complement-mediated immune clearance, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants differ in human FH-binding ability. Together with the FH polymorphisms between vertebrate hosts, these findings suggest that minor sequence variation in this bacterial outer surface protein may confer dramatic differences in host-specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, and identifying minor variation localized in the FH-binding interface yielding bird and rodent FH-specific binding activity that impacts infectivity. Swapping the divergent region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. We further linked these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ phylogenetic lineages, elucidating evolutionary mechanisms driving host tropism emergence. Our multidisciplinary work provides a novel molecular basis for how a single, short protein motif could greatly modulate pathogen host tropism.

Altered hACE2 binding affinity and S1/S2 cleavage efficiency of SARS-CoV-2 spike protein mutants affect viral cell entry.

SARS-CoV-2 variants are constantly emerging, hampering public health measures in controlling the number of infections. While it is well established that mutations in spike proteins observed for the different variants directly affect virus entry into host cells, there remains a need for further expansion of systematic and multifaceted comparisons. Here, we comprehensively studied the effect of spike protein mutations on spike expression and proteolytic activation, binding affinity, viral entry efficiency and host cell tropism of eight variants of concern (VOC) and variants of interest (VOI). We found that both the full-length spike and its receptor-binding domain (RBD) of Omicron bind to hACE2 with an affinity similar to that of the wild-type. In addition, Alpha, Beta, Delta and Lambda pseudoviruses gained significantly enhanced cell entry ability compared to the wild-type, while the Omicron pseudoviruses showed a slightly increased cell entry, suggesting the vastly increased rate of transmission observed for Omicron variant is not associated with its affinity to hACE2. We also found that the spikes of Omicron and Mu showed lower S1/S2 cleavage efficiency and inefficiently utilized TMPRSS2 to enter host cells than others, suggesting that they prefer the endocytosis pathway to enter host cells. Furthermore, all variants' pseudoviruses we tested gained the ability to enter the animal ACE2-expressing cells. Especially the infection potential of rats and mice showed significantly increased, strongly suggesting that rodents possibly become a reservoir for viral evolution. The insights gained from this study provide valuable guidance for a targeted approach to epidemic control, and contribute to a better understanding of SARS-CoV-2 evolution.

Genome organization and genomics in Chlamydia: whole genome sequencing increases understanding of chlamydial virulence, evolution, and phylogeny.

The genus Chlamydia contains important obligate intracellular bacterial pathogens to humans and animals, including C. trachomatis and C. pneumoniae. Since 1998, when the first Chlamydia genome was published, our understanding of how these microbes interact, evolved and adapted to different intracellular host environments has been transformed due to the expansion of chlamydial genomes. This review explores the current state of knowledge in Chlamydia genomics and how whole genome sequencing has revolutionised our understanding of Chlamydia virulence, evolution, and phylogeny over the past two and a half decades. This review will also highlight developments in multi-omics and other approaches that have complemented whole genome sequencing to advance knowledge of Chlamydia pathogenesis and future directions for chlamydial genomics.

Broad Host Tropism of Flaviviruses during the Entry Stage.

The genus Flavivirus consists of viruses with various hosts, including insect-specific flaviviruses (ISFs), mosquito-borne flaviviruses (MBFs), tick-borne flaviviruses (TBFs), and no-known vector (NKV) flaviviruses. Using the reporter viral particle (RVP) system, we found the efficient entry of ISFs into vertebrate cells, MBFs into tick cells, as well as NKVs and TBFs into mosquito cells with similar entry characteristics. By construction of reverse genetics, we found that Yokose virus (YOKV), an NKV, could enter and replicate in mosquito cells but failed to produce infectious particles. The complete removal of the glycosylation modification on the envelope proteins of flaviviruses had no obvious effect on the entry of all MBFs and TBFs. Our results demonstrate an entry-independent host-tropism mechanism and provide a new insight into the evolution of flaviviruses. IMPORTANCE Vector-borne flaviviruses, such as Zika virus, have extremely broad host and cell tropism, even though no critical entry receptors have yet been identified. Using an RVP system, we found the efficient entry of ISFs, MBFs, TBFs, and NKVs into their nonhost cells with similar characteristics. However, glycan-binding proteins cannot serve as universal entry receptors. Our results demonstrate an entry-independent host-tropism mechanism and give a new insight into the cross-species evolution of flaviviruses.

Hepatitis B Virus Capsid: The Core in Productive Entry and Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) relies on the core protein (HBc) to establish productive infection, as defined by the formation of the covalently closed circularized DNA (cccDNA), as well as to carry out almost every step of the lifecycle following cccDNA formation. Multiple copies of HBc form an icosahedral capsid shell that encapsidates the viral pregenomic RNA (pgRNA) and facilitates the reverse transcription of pgRNA to a relaxed circular DNA (rcDNA) within the capsid. During infection, the complete HBV virion, which contains an outer envelope layer in addition to the internal nucleocapsid containing rcDNA, enters human hepatocytes via endocytosis and traffics through the endosomal compartments and the cytosol to deliver its rcDNA to the nucleus to produce cccDNA. In addition, progeny rcDNA, newly formed in cytoplasmic nucleocapsids, is also delivered to the nucleus in the same cell to form more cccDNA in a process called intracellular cccDNA amplification or recycling. Here, we focus on recent evidence demonstrating differential effects of HBc in affecting cccDNA formation during de novo infection vs. recycling, obtained using HBc mutations and small molecule inhibitors. These results implicate a critical role of HBc in determining HBV trafficking during infection, as well as in nucleocapsid disassembly (uncoating) to release rcDNA, events essential for cccDNA formation. HBc likely functions in these processes via interactions with host factors, which contributes critically to HBV host tropism. A better understanding of the roles of HBc in HBV entry, cccDNA formation, and host species tropism should accelerate ongoing efforts to target HBc and cccDNA for the development of an HBV cure and facilitate the establishment of convenient animal models for both basic research and drug development.

The Sialyl Lewis X Glycan Receptor Facilitates Infection of Subtype H7 Avian Influenza A Viruses.

Subtype H7 avian influenza A viruses (IAVs) are enzootic in wild aquatic birds and have caused sporadic spillovers into domestic poultry and humans. Here, we determined the distribution of fucosylated α2,3 sialoglycan (i.e., sialyl Lewis X [SLeX]) in chickens and five common dabbling duck species and the association between SLeX and cell/tissue/host tropisms of H7 IAVs. Receptor binding analyses showed that H7 IAVs bind to both α2,3-linked (SA2,3Gal) and α2,6-linked sialic acids (SA2,6Gal), but with a higher preference for SLeX; H7 IAVs replicated more efficiently in SLeX-overexpressed than SLeX-deficient MDCK cells. While chickens and all tested dabbling ducks expressed abundant SA2,3Gal and SA2,6Gal, SLeX was detected in both respiratory and gastrointestinal tissues of chickens and mallard ducks and in only the respiratory tissues of gadwall, green-wing teal, and northern shoveler but not in wood ducks. Viral-tissue binding assays showed that H7 IAVs bind to chicken colon crypt cells that express SLeX but fewer bind to mallard colon crypt cells, which do not express SLeX; H7 IAVs bind efficiently to epithelial cells of all tissues expressing SA2,3Gal. High viral replication was identified in both chickens and mallards infected with an H7 virus, regardless of SLeX expression, and viruses were detected in all cells to the same degree as viruses detected in the viral-tissue binding assays. In summary, this study suggests that SLeX facilitates infection of H7 viruses, but other types of SA2,3Gal glycan receptors shape the tissue/host tropisms of H7 IAVs. IMPORTANCE In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLeX expression varies among wild dabbling ducks. Although it facilitated virus binding and affected infection of H7 IAV in cells, SLeX expression is not the only determinant of viral replication at either the tissue or host level. This study suggested that access to heterologous SA2,3Gal glycan receptors, including fucosylated α2,3-linked sialoglycans, shape tissue and host tropism of H7 IAVs in aquatic wild birds.

Domestic poultry are not susceptible to avian-origin H3N2 subtype canine influenza A virus.

At present, avian-origin H3N2 subtype canine influenza virus (H3N2 CIV) is prevalent in East Asian and North American countries. The host tropism of H3N2 CIV to mammals, including mice, guinea pigs, ferrets, and pigs, has been evaluated. However, it has not previously been determined whether avian-origin H3N2 CIV can be transmitted back to birds. In China, ducks, chickens, geese, pigeons, and quails are economically important domestic poultry that are susceptible to numerous subtypes of influenza A virus. These poultry occasionally or frequently come into contact with dogs. In this study, the infectivity of the first- and last-isolated Chinese H3N2 CIV strains in these poultry species was evaluated, and oropharyngeal and cloacal swabs of these animals were negative for virus, as determined by specific pathogen-free (SPF) chicken egg inoculation and real-time RT-qPCR assays. Clinical signs and gross lesions were not observed in any of these species, and seroconversion also did not occur. The results showed that all these avian species were unsusceptible to the first- and last-isolated H3N2 CIVs, indicating unidirectional evolution of the mammalian host tropism of H3N2 CIV.

Wild and domestic animals variably display Neu5Ac and Neu5Gc sialic acids.

Sialic acids are used as a receptor by several viruses and variations in the linkage type or C-5 modifications affect the binding properties. A species barrier for multiple viruses is present due to α2,3- or α2,6-linked sialic acids. The C-5 position of the sialic acid can be modified to form N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc), which acts as a determinant for host susceptibility for pathogens such as influenza A virus, rotavirus, and transmissible gastroenteritis coronavirus. Neu5Gc is present in most mammals such as pigs and horses but is absent in humans, ferrets, and dogs. However, little is known about C-5 content in wildlife species or how many C-5 modified sialic acids are present on N-linked glycans or glycolipids. Using our previously developed tissue microarray system, we investigated how 2 different lectins specific for Neu5Gc can result in varying detection levels of Neu5Gc glycans. We used these lectins to map Neu5Gc content in wild Suidae, Cervidae, tigers, and European hedgehogs. We show that Neu5Gc content is highly variable among different species. Furthermore, the removal of N-linked glycans reduces the binding of both Neu5Gc lectins while retention of glycolipids by omitting methanol treatment of tissues increases lectin binding. These findings highlight the importance of using multiple Neu5Gc lectins as the rich variety in which Neu5Gc is displayed can hardly be detected by a single lectin.

Zoonotic Origins of Human Metapneumovirus: A Journey from Birds to Humans.

Metapneumoviruses, members of the family Pneumoviridae, have been identified in birds (avian metapneumoviruses; AMPV's) and humans (human metapneumoviruses; HMPV's). AMPV and HMPV are closely related viruses with a similar genomic organization and cause respiratory tract illnesses in birds and humans, respectively. AMPV can be classified into four subgroups, A-D, and is the etiological agent of turkey rhinotracheitis and swollen head syndrome in chickens. Epidemiological studies have indicated that AMPV also circulates in wild bird species which may act as reservoir hosts for novel subtypes. HMPV was first discovered in 2001, but retrospective studies have shown that HMPV has been circulating in humans for at least 50 years. AMPV subgroup C is more closely related to HMPV than to any other AMPV subgroup, suggesting that HMPV has evolved from AMPV-C following zoonotic transfer. In this review, we present a historical perspective on the discovery of metapneumoviruses and discuss the host tropism, pathogenicity, and molecular characteristics of the different AMPV and HMPV subgroups to provide increased focus on the necessity to better understand the evolutionary pathways through which HMPV emerged as a seasonal endemic human respiratory virus.

A host non-coding RNA, nc886, plays a pro-viral role by promoting virus trafficking to the nucleus.

Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.

Influenza D binding properties vary amongst the two major virus clades and wildlife species.

The influenza D virus (IDV) uses a trimeric hemagglutinin-esterase fusion protein (HEF) for attachment to 9-O-acetylated sialic acid receptors on the cell surface of host species. So far research has revealed that farm animals such as cattle, domestic pigs, goats, sheep and horses contain the necessary receptors on the epithelial surface of the respiratory tract to accommodate binding of the IDV HEF protein of both worldwide clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660). More recently, seroprevalence studies have identified IDV-seropositive wildlife such as wild boar, deer, dromedaries, and small ruminants. However, no research has thus far been conducted in wildlife to reveal the distribution of acetylated sialic acid receptors that accommodate binding of IDV. Using our previously developed tissue microarray (TMA) system, we developed TMAs containing respiratory tissues of various wild and domestic species including wild boar, deer, dromedary, springbok, water buffalo, tiger, hedgehog, and Asian elephant. Protein histochemical staining of these TMAs with HEF proteins showed no receptor binding for wild Suidae, Cervidae and tiger. However, receptors were present in dromedary, springbok, water buffalo, Asian elephant, and hedgehog. In contrast to previously tested farm animals, a difference in host tropism was observed between the D/OK and D/660 clade HEF proteins in Asian elephant, and water buffalo. These results show that IDV can attach to the respiratory tract of wildlife which might facilitate transmission of IDV between wildlife and domestic animals.

Generation and characterization of genetically and antigenically diverse infectious clones of dengue virus serotypes 1-4.

Dengue is caused by four genetically distinct viral serotypes, dengue virus (DENV) 1-4. Following transmission by Aedes mosquitoes, DENV can cause a broad spectrum of clinically apparent disease ranging from febrile illness to dengue hemorrhagic fever and dengue shock syndrome. Progress in the understanding of different dengue serotypes and their impacts on specific host-virus interactions has been hampered by the scarcity of tools that adequately reflect their antigenic and genetic diversity. To bridge this gap, we created and characterized infectious clones of DENV1-4 originating from South America, Africa, and Southeast Asia. Analysis of whole viral genome sequences of five DENV isolates from each of the four serotypes confirmed their broad genetic and antigenic diversity. Using a modified circular polymerase extension reaction (CPER), we generated de novo viruses from these isolates. The resultant clones replicated robustly in human and insect cells at levels similar to those of the parental strains. To investigate in vivo properties of these genetically diverse isolates, representative viruses from each DENV serotype were administered to NOD Rag1-/-, IL2rgnull Flk2-/- (NRGF) mice, engrafted with components of a human immune system. All DENV strains tested resulted in viremia in humanized mice and induced cellular and IgM immune responses. Collectively, we describe here a workflow for rapidly generating de novo infectious clones of DENV - and conceivably other RNA viruses. The infectious clones described here are a valuable resource for reverse genetic studies and for characterizing host responses to DENV in vitro and in vivo.

Bactericidal activity of avian complement: a contribution to understand avian-host tropism of Lyme borreliae.

Complement has been considered as an important factor impacting the host-pathogen association of spirochetes belonging to the Borrelia burgdorferi sensu lato complex, and may play a role in the spirochete's ecology. Birds are known to be important hosts for ticks and in the maintenance of borreliae. Recent field surveys and laboratory transmission studies indicated that certain avian species act as reservoir hosts for different Borrelia species. Nevertheless, our current understanding of the molecular mechanisms determining host tropism of Borrelia is still in its fledgling stage. Concerning the role of complement in avian-host tropism, only a few bird species and Borrelia species have been analysed so far. Here, we performed in vitro serum bactericidal assays with serum samples collected from four bird species including the European robin Erithacus rubecula, the great tit Parus major, the Eurasian blackbird Turdus merula, and the racing pigeon Columba livia, as well as four Borrelia species (B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi sensu stricto). From July to September 2019, juvenile wild birds were caught using mist nets in Portugal. Racing pigeons were sampled in a loft in October 2019. Independent of the bird species analysed, all Borrelia species displayed an intermediate serum-resistant or serum-resistant phenotype except for B. afzelii challenged with serum from blackbirds. This genospecies was efficiently killed by avian complement, suggesting that blackbirds served as dead-end hosts for B. afzelii. In summary, these findings suggest that complement contributes in the avian-spirochete-tick infection cycle and in Borrelia-host tropism.

Tissue Microarrays to Visualize Influenza D Attachment to Host Receptors in the Respiratory Tract of Farm Animals.

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids.

Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus.

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as a cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture-adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig-expressing cells and areas of nonexpressing cells, and one single cell line revealed not to express any CD46pig The CD46pig-deficient porcine lymphoma cell line, known to facilitate CSFV replication, was the only porcine cell line nonpermissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig-independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized.IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and is highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in the absence of CD46pig Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

Human Noroviruses Attach to Intestinal Tissues of a Broad Range of Animal Species.

Human noroviruses are the most common nonbacterial cause of gastroenteritis outbreaks, with new variants and genotypes frequently emerging. The origin of these new viruses is unknown; however, animals have been proposed as a potential source, as human noroviruses have been detected in animal species. Here, we investigated the potential of animals to serve as a reservoir of human noroviruses by testing norovirus attachment to formalin-fixed intestinal tissues of a range of potential reservoir animals. We set up a novel method to study norovirus binding using fluorescein isothiocyanate (FITC)-labeled virus-like particles (VLPs). In humans, noroviruses interact with histo-blood group antigens (HBGAs), carbohydrates that are expressed, among others, on the epithelial lining of the gastrointestinal tract. In animals, this interaction is not well understood. To test if virus binding depends on HBGAs, we characterized the HBGA phenotype in animal tissues by immunohistochemistry. With the exception of the black-headed gull and the straw-colored fruitbat, we observed the attachment of several human norovirus genotypes to the intestinal epithelium of all tested animal species. However, we did not find an association between the expression of a specific HBGA phenotype and virus-like particle (VLP) attachment. We show that selected human noroviruses can attach to small-intestinal tissues across species, supporting the hypothesis that human noroviruses can reside in an animal reservoir. However, whether this attachment can subsequently lead to infection needs to be further assessed.IMPORTANCE Noroviruses are a major cause of acute gastroenteritis in humans. New norovirus variants and recombinants (re)emerge regularly in the human population. From animal experiments and surveillance studies, it has become clear that at least seven animal models are susceptible to infection with human strains and that domesticated and wild animals shed human noroviruses in their feces. As virus attachment is an important first step for infection, we used a novel method utilizing FITC-labeled VLPs to test for norovirus attachment to intestinal tissues of potential animal hosts. We further characterized these tissues with regard to their HBGA expression, a well-studied norovirus susceptibility factor in humans. We found attachment of several human strains to a variety of animal species independent of their HBGA phenotype. This supports the hypothesis that human strains could reside in an animal reservoir.

Animal coronaviruses and SARS-CoV-2.

COVID-19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has rapidly spread to 216 countries and territories since first outbreak in December of 2019, posing a substantial economic losses and extraordinary threats to the public health worldwide. Although bats have been suggested as the natural host of SARS-CoV-2, transmission chains of this virus, role of animals during cross-species transmission, and future concerns remain unclear. Diverse animal coronaviruses have extensively been studied since the discovery of avian coronavirus in 1930s. The current article comprehensively reviews and discusses the current understanding about animal coronaviruses and SARS-CoV-2 for their emergence, transmission, zoonotic potential, alteration of tissue/host tropism, evolution, status of vaccines and surveillance. This study aims at providing guidance for control of COVID-19 and preventative strategies for possible future outbreaks of zoonotic coronavirus via cross-species transmission.