Gang Pei

Highly pathogenic Hendra and Nipah viruses encode accessory P gene products (C, V and W) that antagonize innate immunity and contribute to pathogenicity. Cedar virus (CedV), an apathogenic bat-borne henipavirus, is presumed to lack P gene mRNA editing and therefore is unable to express V and W proteins. Here, we identify CedV peptides originating from a frameshifted P gene open reading frame and demonstrate a previously unrecognized, noncanonical editing event at a homopolymeric adenine tract that introduces single-nucleotide A or G insertion. This mRNA editing produces a V-like protein whose C-terminal domain shares sequence and predicted structural features with those of the henipavirus V protein and displays cytoplasmic and nuclear localization. Recombinant CedV mutants defective in mRNA editing or with a truncated C-terminus were only recoverable by trans-complementation and showed markedly reduced release of infectious virus in cell culture and attenuated replication in mice lacking type I interferon receptor. Our data revise the CedV gene expression models and reveal a noncanonical editing mechanism that supports the production of a V-like protein critical for efficient infectious virus release. These results expand the fundamental concepts of paramyxovirus gene expression and reveal an unexpected, pivotal role for mRNA editing in virus egress, with direct implications for the evaluation of potentially high-consequence paramyxoviruses.

Cedar virus (CedV), closely related to the Hendra and Nipah viruses, is a novel Henipavirus that was originally isolated from flying foxes in Australia in 2012. Although its glycoprotein G exhibits relatively low sequence similarity with its counterparts of the Hendra and Nipah viruses, CedV also uses ephrin receptors, i.e., ephrins B1, B2, A2 and A5, to enters human cells. Nevertheless, the entry mechanism of CedV into bat cells remains unexplored. Considering that Rousettus aegyptiacus (Egyptian Rousette bat, ERB) is postulated to be a reservoir host for henipaviruses, we aim to reveal the receptors utilized by CedV to enable its entry into ERB cells. To this end, we cloned the class A and B ephrins of ERB and generated CHO-K1 cells stably expressing individual ephrins. We also developed a lentivirus-based pseudovirus system containing the firefly luciferase reporter. Assessment of the luciferase activity in cells expressing single ephrins demonstrated that the ERB ephrin B1 and B2 mediated CedV pseudovirus entry. Further, we generated a recombinant CedV expressing the fluorescent protein TurboFP635 (rCedV-nTurbo635). By performing high-content microscopy and flow cytometry, we unveiled that, in addition to ephrin B1 and B2, ephrin A5 was also able to mediate rCedV-nTurbo635 entry, although to a much lesser extent. In contrast to human ephrin A2, ERB ephrin A2 failed to mediate rCedV-nTurbo635 entry. Finally, we generated ERB epithelial cells with ephrin B1 and/or ephrin B2 knockdown (KD). The entry of rCedV-nTurbo635 into ERB epithelial cells was drastically impaired by ephrin B1/B2 KD, validating the importance of ephrin B1 and B2 in its entry. Altogether, we conclude that CedV primarily employs ERB ephrin B1, B2 and, possibly, A5 for its entry into ERB cells.

African swine fever virus (ASFV) is a large DNA virus of the Asfarviridae family that causes a fatal hemorrhagic disease in domestic swine and wild boar. Infections with moderately virulent strains predominantly result in a milder clinical course and lower lethality. As target cells of ASFV, monocytes play a crucial role in triggering T-cell-mediated immune defense and ASF pathogenesis. We compared the effect of the highly virulent “Armenia2008” (ASFV-A) virus strain with that of the naturally attenuated “Estonia2014” (ASFV-E) on cellular immune activation in vivo and on primary monocytes ex vivo. Specifically, we asked whether antigen presentation of porcine monocytes is impaired upon ASFV-A infection. ASFV-A-infected monocytes are characterized by lower levels of swine leukocyte antigen (SLA) class I on the cell surface than ASFV-E-infected and uninfected monocytes. Despite stable steady-state SLA I mRNA/protein levels and expression of critical components of the antigen processing machinery, a marked decrease in maturation and reduced surface transport of SLA I were observed in ASFV-A-infected monocytes. The intracellular maturation block of SLA I was accompanied by a loss of functional rough ER structures and a pronounced formation of ER-associated aggresomes. This unsolved cellular stress resulted in a shutdown of overall host cell protein translation, mitochondrial dysfunction, and caspase-3-mediated apoptosis. In contrast, no such cellular subversion phenomenon was found in ASFV-E-infected monocytes. Our findings suggest that in domestic pigs infected with highly virulent ASFV-A, sequential subversion events occur in infected monocytes, likely leading to compromised T-cell activation and impaired downstream responses against ASFV.

Increasing evidence suggests bats are the ancestral hosts of the majority of coronaviruses. In general, coronaviruses primarily target the gastrointestinal system, while some strains, especially Betacoronaviruses with the most relevant representatives SARS-CoV, MERS-CoV, and SARS-CoV-2, also cause severe respiratory disease in humans and other mammals. We previously reported the susceptibility of Rousettus aegyptiacus (Egyptian fruit bats) to intranasal SARS-CoV-2 infection. Here, we compared their permissiveness to an oral infection versus respiratory challenge (intranasal or orotracheal) by assessing virus shedding, host immune responses, tissue-specific pathology, and physiological parameters. While respiratory challenge with a moderate infection dose of 1 × 10⁴ TCID₅₀ caused a systemic infection with oral and nasal shedding of replication-competent virus, the oral challenge only induced nasal shedding of low levels of viral RNA. Even after a challenge with a higher infection dose of 1 × 10⁶ TCID₅₀, no replication-competent virus was detectable in any of the samples of the orally challenged bats. We postulate that SARS-CoV-2 is inactivated by HCl and digested by pepsin in the stomach of R. aegyptiacus, thereby decreasing the efficiency of an oral infection. Therefore, fecal shedding of RNA seems to depend on systemic dissemination upon respiratory infection. These findings may influence our general understanding of the pathophysiology of coronavirus infections in bats.

Bats are natural reservoirs for zoonotic pathogens, yet the determinants of microbial persistence as well as the specific functionality of their immune system remain largely enigmatic. Their propensity to harbor viruses lethal to humans and/or livestock, mostly in absence of clinical disease, makes bats stand out among mammals. Defending against pathogens relies on avoidance, resistance, and/or tolerance strategies. In bats, disease tolerance has recently gained increasing attention as a prevailing host defense paradigm. We here summarize the current knowledge on immune responses in bats in the context of infection with zoonotic agents and discuss concepts related to disease tolerance. Acknowledging the wide diversity of bats, the broad spectrum of bat-associated microbial species, and immune-related knowledge gaps, we identify research priorities necessary to provide evidence-based proofs for disease tolerance in bats. Since disease tolerance relies on networks of biological processes, we emphasize that investigations beyond the immune system, using novel technologies and computational biology, could jointly advance our knowledge about mechanisms conferring bats reservoir abilities. Although disease tolerance may not be the “one fit all” defense strategy, deciphering disease tolerance in bats could translate into novel therapies and inform prevention of spillover infections to humans and livestock.

Viral replication fully relies on the host cell machinery, and physical interactions between viral and host proteins mediate key steps of the viral life cycle. Therefore, identifying virus–host protein–protein interactions (PPIs) provides insights into the molecular mechanisms governing virus infection and is crucial for designing novel antiviral strategies. In the case of the African swine fever virus (ASFV), a large DNA virus that causes a deadly panzootic disease in pigs, the limited understanding of host and viral targets hinders the development of effective vaccines and treatments. This review summarizes the current knowledge of virus–host and virus–virus PPIs by collecting and analyzing studies of individual viral proteins. We have compiled a dataset of experimentally determined host and virus protein targets, the molecular mechanisms involved, and the biological functions of the identified virus–host and virus–virus protein interactions during infection. Ultimately, this work provides a comprehensive and systematic overview of ASFV interactome, identifies knowledge gaps, and proposes future research directions.

Mouse guanylate-binding proteins (mGBPs) are recruited to various invasive pathogens, thereby conferring cell-autonomous immunity against these pathogens. However, whether and how human GBPs (hGBPs) target M. tuberculosis (Mtb) and L. monocytogenes (Lm) remains unclear. Here, we describe hGBPs association with intracellular Mtb and Lm, which was dependent on the ability of bacteria to induce disruption of phagosomal membranes. hGBP1 formed puncta structures which were recruited to ruptured endolysosomes. Furthermore, both GTP-binding and isoprenylation of hGBP1 were required for its puncta formation. hGBP1 was required for the recovery of endolysosomal integrity. In vitro lipid-binding assays demonstrated direct binding of hGBP1 to PI4P. Upon endolysosomal damage, hGBP1 was targeted to PI4P and PI(3,4)P2-positive endolysosomes in cells. Finally, live-cell imaging demonstrated that hGBP1 was recruited to damaged endolysosomes, and consequently mediated endolysosomal repair. In summary, we uncover a novel interferon-inducible mechanism in which hGBP1 contributes to the repair of damaged phagosomes/endolysosomes.

Nucleotide binding oligomerization domain-containing protein 1 (NOD1) and NOD2 have been identified as intracellular receptors for bacterial peptidoglycan for almost two decades; however, the direct binding with their respective ligands has only been recently demonstrated due to the difficulty of achieving large quantity of proteins with high purity. Here we describe a strategy combining immunoprecipitation of GFP-tagged proteins and microscale thermophoresis (MST) for efficient one-step purification of NOD1-GFP and NOD2-GFP and easy measurement of the binding affinities of NOD1 or NOD2 with sphingosine-1-phosphate (S1P) using small amount of proteins (nM range). This method will allow the identification of novel agonists/antagonists for NOD1/2. Key words Nucleotide binding oligomerization domain (NOD)-like receptors NOD1 NOD2 Dissociation constant Sphingosine-1-phosphate (S1P) Microscale thermophoresis

Bats are the only mammals capable of powered flight and their body temperature can reach up to 42 °C during flight. Additionally, bats display robust type I IFN interferon (IFN-I) responses and some species constitutively express IFN-α. Reference genes with stable expression under temperature oscillations and IFN-I release are therefore critical for normalization of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) data in bats. The expression stability of reference genes in Rousettus aegyptiacus remains elusive, although this species is frequently used in the infection research. We selected ACTB, EEF1A1, GAPDH and PGK1 as candidate reference genes and evaluated their expression stability in various tissues and cells from this model bat species upon IFN-I treatment at 35 °C, 37 °C and 40 °C by qRT-PCR. We employed two statistical algorithms, BestKeeper and NormFinder, and found that EEF1A1 exhibited the highest expression stability under all tested conditions. ACTB and GAPDH displayed unstable expression upon temperature change and IFN-I treatment, respectively. By normalizing to EEF1A1, we uncovered that GAPDH expression was significantly induced by IFN-I in R. aegyptiacus. Our study identifies EEF1A1 as the most suitable reference gene for qRT-PCR studies upon temperature changes and IFN-I treatment and unveils the induction of GAPDH expression by IFN-I in R. aegyptiacus. These findings are pertinent to other bat species and may be relevant for non-volant mammals that show physiological fluctuations of core body temperature.