Identification of TMEM53 as a novel SADS-CoV restriction factor that targets viral RNA-dependent RNA polymerase.

ABSTRACTZoonotic transmission of coronaviruses (CoVs) poses a serious public health threat. Swine acute diarrhea syndrome coronavirus (SADS-CoV), originating from a bat HKU2-related CoV, causes devastating swine diseases and poses a high risk of spillover to humans. Currently, licensed therapeutics that can prevent potential human outbreaks are unavailable. Identifying the cellular proteins that restrict viral infection is imperative for developing effective interventions and therapeutics. We utilized a large-scale human cDNA screening and identified transmembrane protein 53 (TMEM53) as a novel cell-intrinsic SADS-CoV restriction factor. The inhibitory effect of TMEM53 on SADS-CoV infection was found to be independent of canonical type I interferon responses. Instead, TMEM53 interacts with non-structural protein 12 (NSP12) and disrupts viral RNA-dependent RNA polymerase (RdRp) complex assembly by interrupting NSP8-NSP12 interaction, thus suppressing viral RdRp activity and RNA synthesis. Deleting the transmembrane domain of TMEM53 resulted in the abrogation of TMEM53-NSP12 interaction and TMEM53 antiviral activity. Importantly, TMEM53 exhibited broad antiviral activity against multiple HKU2-related CoVs. Our findings reveal a novel role of TMEM53 in SADS-CoV restriction and pave the way to host-directed therapeutics against HKU2-related CoV infection.

Characterization of vB_ValM_PVA8, a broad-host-range bacteriophage infecting Vibrio alginolyticus and Vibrio parahaemolyticus.

Phage therapy was taken as an alternative strategy to antibiotics in shrimp farming for the control of Vibrio species of Vibrio parahaemolyticus and Vibrio alginolyticus, which cause substantial mortality and significant economic losses. In this study, a new Vibrio phage vB_ValM_PVA8 (PVA8), which could efficiently infect pathogenic isolates of V. alginolyticus and V. parahaemolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. The phage was characterized to be a member of the Straboviridae family with elongated head and contractile tail by transmission electron microscopy. Genome sequencing showed that PVA8 had a 246,348-bp double-stranded DNA genome with a G + C content of 42.6%. It harbored totally 388 putative open reading frames (ORFs), among them 92 (23.71%) assigned to functional genes. Up to 27 transfer RNA (tRNA) genes were found in the genome, and the genes for virulence, antibiotic resistance, and lysogeny were not detected. NCBI genomic blasting results and the phylogenetic analysis based on the sequences of the large terminase subunits and the DNA polymerase indicated that PVA8 shared considerable similarity with Vibrio phage V09 and bacteriophage KVP40. The phage had a latent period of 20 min and a burst size of 309 PFUs/infected cell with the host V. alginolyticus, and it was stable over a broad pH range (4.0-11.0) and a wide temperature span (-80°C to 60°C), respectively, which may benefit its feasibility for phage therapy. In addition, it had the minimum multiplicity of infection (MOI) of 0.0000001, which revealed its strong multiplication capacity. The shrimp cultivation lab trials demonstrated that PVA8 could be applied in treating pathogenic V. parahaemolyticus infection disease of shrimp with a survival rate of 88.89% comparing to that of 34.43% in the infected group, and the pond application trails confirmed that the implementation of PVA8 could rapidly yet effectively reduce the level of the Vibrio. Taken together, PVA8 may be potential to be explored as a promising biological agent for Vibrio control in aquaculture farming industry.

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

Characterization and Genomic Analysis of a Bacteriophage with Potential in Lysing Vibrio alginolyticus

Vibrio alginolyticus is one of the major pathogens causing vibriosis to a variety of aquatic animals as well as bringing about severe food safety concerns. Nowadays, phage therapy has received increasing attention as an alternative to the antibiotics that have being limited for use in aquaculture industries. In this work, a potent bacteriophage, vB_ValM_PVA23 (PVA23), which efficiently infects pathogenic strains of V. alginolyticus, was isolated from sewage water and characterized by microbiological and genomic analyses. Based on the transmission electronic observation, the phage was characterized to be the Myoviridae family. It has a latent period of 10 min and a burst size of 203 PFUs/infected bacterium, and was stable over a broad pH range (5.0−11.0) and a wide temperature span (−80 °C to 60 °C), respectively. Genome sequencing results show that PVA23 has a 246,962-bp double-stranded DNA with a G + C content of 41.25%. The lab and plant shrimp farming trials demonstrated that phage preparation derived from PVA23 out-performed the chemical disinfectant iodine treatment in the prevention of V. alginolyticus propagation, and the phage application could rapidly yet significantly reduce the level of V. alginolyticus in the pond within 12 h, with negligible rebound observed. These results suggests that phage PVA23 has the potential to be used as an anti-V. alginolyticus agent in aquaculture industries.