Major Envelope Protein Research Articles

First report of molecular epidemiology and phylogenetic characteristics of feline herpesvirus (FHV-1) from naturally infected cats in Kunshan, China

BackgroundFeline herpesvirus type 1 (FHV-1) is a life threatening highly contagious virus in cats and typically causes upper respiratory tract infections as well as conjunctival and corneal ulcers. Genetic variability could alter the severity of diseases and clinical signs. Despite regular vaccine practices against FHV-1 in China, new FHV-1 cases still commonly occur. The genetic and phylogenetic characteristics of FHV-1 in Kunshan city of China has not been studied yet. Therefore, this study was planned to investigate the prevalence, molecular characteristics of circulating strains, and phylogenetic analyses of FHV-1. This is the first report of molecular epidemiology and phylogenetic characteristics of FHV-1 from naturally infected cats in Kunshan, China.MethodsThe occulo-nasal swabs were collected from diseased cats showing respiratory distress, conjunctivitis, and corneal ulcers at different veterinary clinics in Kunshan from 2022 to 2023. Clinical data and general information were recorded. Swab samples were processed for preliminary detection of FHV-1. Thymidine kinase (TK), glycoprotein B (gB) and glycoprotein D (gD) genes were sequenced and analyzed to investigate genetic diversity and evolution of FHV-1.ResultsThe FHV-1 genome was detected in 43 (43/200, 21.5%) samples using RT-PCR targeting the TK gene. Statistical analysis showed a significant correlation between age, vaccination status and living environment (p < 0.05) with FHV-1 positivity, while a non-significant correlation was observed for FHV-1 positivity and sex of cats (p > 0.05). Additionally, eight FHV-1 positive cats were co-infected with feline calicivirus (8/43,18.6%). FHV-1 identified in the present study was confirmed as FHV-1 based on phylogenetic analyses. The sequence analyses revealed that 43 FHV-1 strains identified in the present study did not differ much with reference strains within China and worldwide. A nucleotide homology of 99-100% was determined among gB, TK and gD genes nucleotide sequences when compared with standard strain C-27 and vaccine strains. Amino acid analysis showed some amino acid substitutions in TK, gB and gD protein sequences. A potential N-linked glycosylation site was observed in all TK protein sequences. Phylogenetic analyses revealed minor variations and short evolutionary distance among FHV-1 strains detected in this study.ConclusionsOur findings indicate that genomes of 43 FHV-1 strains are highly homogenous and antigenically similar, and the degree of variation in major envelope proteins between strains is low. This study demonstrated some useful data about prevalence, genetic characteristics, and evolution of FHV-1 in Kunshan, which may aid in future vaccine development.

Tecovirimat for the treatment of severe Mpox in Germany

BackgroundIn May 2022, a multi-national mpox outbreak was reported in several non-endemic countries. The only licensed treatment for mpox in the European Union is the orally available small molecule tecovirimat, which in Orthopox viruses inhibits the function of a major envelope protein required for the production of extracellular virus.MethodsWe identified presumably all patients with mpox that were treated with tecovirimat in Germany between the onset of the outbreak in May 2022 and March 2023 and obtained demographic and clinical characteristics by standardized case report forms.ResultsA total of twelve patients with mpox were treated with tecovirimat in Germany in the study period. All but one patient identified as men who have sex with men (MSM) who were most likely infected with mpox virus (MPXV) through sexual contact. Eight of them were people living with HIV (PLWH), one of whom was newly diagnosed with HIV at the time of mpox, and four had CD4+ counts below 200/µl. Criteria for treatment with tecovirimat included severe immunosuppression, severe generalized and/or protracted symptoms, a high or increasing number of lesions, and the type and location of lesions (e.g., facial or oral soft tissue involvement, imminent epiglottitis, or tonsillar swelling). Patients were treated with tecovirimat for between six and 28 days. Therapy was generally well-tolerated, and all patients showed clinical resolution.ConclusionsIn this cohort of twelve patients with severe mpox, treatment with tecovirimat was well tolerated and all individuals showed clinical improvement.

Identification and characterization of BmNPV Bm5 protein required for the formation of nuclear vesicle structures.

BmNPV infection induces nuclear vesicle-like structures and its Bm5 protein mediates the intranuclear lipid accumulation, which is thought to participate in the formation of nuclear vesicles. However, the relationship between viral-induced nuclear vesicles and Bm5 protein is still unclear. Here, our results indicated that BmNPV Bm5 protein participated in the baculovirus infection-induced nuclear vesicle-like structures' invagination thereby influencing the production of occlusion-derived virion (ODV) and occlusion body (OB). The process of nuclear vesicle-like structures' formation was dispensable for the transport or recruitment of ODV major envelope proteins, such as P74 and Bm14. Furthermore, baculovirus-induced nuclear F-actin might provide a direct mechanical force to mediate the scission of large vesicle-like structures from the nuclear membrane. Collectively, these findings illustrated a BmNPV Bm5 protein-induced nuclear membrane invagination pathway and revealed the function of nuclear vesicle-like structures in ODV production.

MARCO Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection through Intensifying Viral GP5-Induced Apoptosis.

Studying viral glycoprotein-host membrane protein interactions contributes to the discovery of novel cell receptors or entry facilitators for viruses. Glycoprotein 5 (GP5), which is a major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) virions, is a key target for the control of the virus. Here, the macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified as one of the host interactors of GP5 through a DUALmembrane yeast two-hybrid screening. MARCO was specifically expressed on porcine alveolar macrophages (PAMs), and PRRSV infection downregulated MARCO expression both in vitro and in vivo. MARCO was not involved in viral adsorption and internalization processes, indicating that MARCO may not be a PRRSV-entry facilitator. Contrarily, MARCO served as a host restriction factor for PRRSV. The knockdown of MARCO in PAMs enhanced PRRSV proliferation, whereas overexpression suppressed viral proliferation. The N-terminal cytoplasmic region of MARCO was responsible for its inhibitory effect on PRRSV. Further, we found that MARCO was a proapoptotic factor in PRRSV-infected PAMs. MARCO knockdown weakened virus-induced apoptosis, whereas overexpression aggravated apoptosis. MARCO aggravated GP5-induced apoptosis, which may result in its proapoptotic function in PAMs. The interaction between MARCO and GP5 may contribute to the intensified apoptosis induced by GP5. Additionally, the inhibition of apoptosis during PRRSV infection weakened the antiviral function of MARCO, suggesting that MARCO inhibits PRRSV through the regulation of apoptosis. Taken together, the results of this study reveal a novel antiviral mechanism of MARCO and suggest a molecular basis for the potential development of therapeutics against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the most serious threats to the global swine industry. Glycoprotein 5 (GP5) exposed on the surface of PRRSV virions is a major glycoprotein, and it is involved in viral entry into host cells. A macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified to interact with PRRSV GP5 in a DUALmembrane yeast two-hybrid screening. Further investigation demonstrated that MARCO may not serve as a potential receptor to mediate PRRSV entry. Instead, MARCO was a host restriction factor for the virus, and the N-terminal cytoplasmic region of MARCO was responsible for its anti-PRRSV effect. Mechanistically, MARCO inhibited PRRSV infection through intensifying virus-induced apoptosis in PAMs. The interaction between MARCO and GP5 may contribute to GP5-induced apoptosis. Our work reveals a novel antiviral mechanism of MARCO and advances the development of control strategies for the virus.

Evaluation of Virus-Free Manufacture of Recombinant Proteins Using CRISPR-Mediated Gene Disruption in Baculovirus-Infected Insect Cells.

The manufacture and downstream processing of virus-like particles (VLPs) using the baculovirus expression vector system (BEVS) is complicated by the presence of large concentrations of baculovirus particles, which are similar in size and density to VLPs, and consequently are difficult to separate. To reduce the burden of downstream processing, CRISPR-Cas9 technology was used to introduce insertion-deletion (indel) mutations within the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp64 open reading frame, which encodes the major envelope protein of AcMNPV. After comfirming the site-specific targeting of gp64 leading to reduced budded virus (BV) release, the gag gene of human immunodeficiency virus type 1 was expressed to produce Gag VLPs. This approach was effective for producing VLPs using the BEVS whilst simultaneously obstructing BV release.

Identification of phytochemical compounds to inhibit the matrix-like linker protein VP26 to block the assembles of white spot syndrome virus (WSSV) envelope and nucleocapsid protein of marine shrimp: In silico approach

White spot syndrome virus (WSSV) is an enveloped pathogenic virus of cultured shrimp that has a mortality rate of up to 100 % in intensive aquaculture systems. The virulent pathogen causes an economic loss estimated at US$1 billion annually to global shrimp and prawn production. To date, no effective antiviral or vaccine has been progress that can reduce the economic losses caused by the pathogen. The complex genomic structure of the nucleocapsid of a virus, consisting of double-stranded DNA as genetic material surrounded by lipid envelope protein. It has been found that VP26, a major envelope protein interacts with the virus VP51 capsid and makes a bridge between the envelope and nucleocapsid protein resulting in mature virion of the virus. Blocking the interaction by targeting VP26 can hinder the mature virion production and non-infectivity of the virus. Therefore, in order to blocking the function of VP26, an in-silico drug design approach has been utilized to screen the potential antiviral compounds from the medicinal plant, Withania somnifera (WS) of Saudi Arabia. Isolated thirty-nine (39) phytochemicals compounds from the plant have initially been screened by molecular docking method. The best three compounds, namely, withanolide (CID: 118701104), Coagulin Q (CID: 10100411), and withanolide D (CID: 23266161) have been selected based on their docking score −8.2 kcal/mol, −8.1 kcal/mol and −7.9 kcal/mol, respectively, which have been further evaluated based on pharmacokinetics including ADME (Absorption, Distribution, Metabolism and Excretion) and toxicity approaches. Additionally, 100 ns molecular dynamics (MD) simulation methods confirmed the structural stability of the compounds to the binding sites of the VP26 protein. Therefore, the selected compounds can be utilized as an antiviral drug to fight against the WSSV virus, which will bring remarkable advancement in the aquaculture sector.

Neuraminidase (NA) 370-Loop Mutations of the 2009 Pandemic H1N1 Viruses Affect NA Enzyme Activity, Hemagglutination Titer, Mouse Virulence, and Inactivated-Virus Immunogenicity.

Hemagglutinin (HA) and neuraminidase (NA) are the two major envelope proteins of influenza viruses. The spatial organization of HA and NA on the virus surface needs to be optimized to promote viral fitness, host specificity, transmissibility, infectivity, and virulence. We previously demonstrated that the recombinant NA protein of the 2009 pandemic H1N1 (pH1N1) with the I365T/S366N mutation in the NA 370-loop elicited higher NA-inhibition antibody titers against the homologous pH1N1 virus and three heterologous H5N1, H3N2, and H7N9 viruses in mice. In this study, we used PR8-based reverse genetics (RG) by replacing the HA and NA genes of A/Texas/05/2009 pH1N1 virus to obtain the wild-type pH1N1 and three NA 370-loop mutant viruses of pH1N1 (I365T/S366N), RG pH1N1 (I365E/S366D), and RG pH1N1 (I365T/S366A). Our results revealed that the viral NA enzyme activity increased for the RG pH1N1(I365T/S366N) and RG pH1N1 (I365E/S366D) viruses but reduced for the RG pH1N1 (I365T/S366A) virus. The increased or decreased NA enzyme activity was found to correlate with the increase or decrease in HA titers of these NA 370-loop mutant viruses. All of these three NA 370-loop mutant RG pH1N1 viruses were less virulent than the wild-type RG pH1N1 virus in mice. Immunizations with the inactivated viruses carrying the three NA 370-loop mutations and the wild-type RG pH1N1 virus were found to elicit approximately the same titers of NA-inhibition antibodies against H1N1 and H5N1 viruses. These results may provide information for developing NA-based influenza virus vaccines.

Minor and major envelope proteins of PRRSV play synergistic roles in inducing heterologous neutralizing antibodies and conferring cross protection

High genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) isolates is a major obstacle for the development of effective PRRS vaccines. A chimeric highly pathogenic PRRSV2 (HP-PRRSV2) strain containing the consensus sequence of ORF2-6 genes was constructed in our previous study, which could induce broadly neutralizing antibodies (bnAbs) and confer satisfied cross protection against virulent NADC30-like isolate. To further elucidate the roles of minor and major envelope proteins encoded by ORF2-4 and ORF5-6 genes in conferring cross protection, two chimeric HP-PRRSV2 strains (rJS-ORF2-4-CON and rJS-ORF5-6-CON) containing consensus sequences of ORF2-4 or ORF5-6 were constructed and rescued in this study. The rJS-ORF5-6-CON strain has similar replication efficiency as the backbone HP-PRRSV2 rJSTZ1712-12 virus, while rJS-ORF2-4-CON has significantly lower in vitro and in vivo replication efficiency comparing to rJS-ORF5-6-CON. Animal inoculation indicated that both rJS-ORF2-4-CON and rJS-ORF5-6-CON did not cause obvious clinical signs in piglets and could induce heterologous nAbs after immunization. Challenge with a virulent heterologous NADC30-like SD17-38 isolate showed that even though both immunized groups presented lower viremia, faster virus elimination, less fever and alleviated lung gross lesions when compared with the only challenged pigs, rJS-ORF2-4-CON and rJS-ORF5-6-CON could not confer enough cross protection. Considering the bnAbs and satisfied cross protection induced by the chimeric virus containing ORF2-6 consensus sequence, our results support that minor and major envelope proteins play synergistic roles in inducing broader nAbs and conferring better cross protection.

A New Enzyme-linked Immunosorbent Assay for Serological Diagnosis of Seal Parapoxvirus Infection in Marine Mammals.

IntroductionSeal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection.Material and MethodsThe gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA.ResultsPositive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal.ConclusionThe established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies.

An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles.

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called “stem” region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

Glyceraldehyde-3-Phosphate Dehydrogenase Restricted in Cytoplasmic Location by Viral GP5 Facilitates Porcine Reproductive and Respiratory Syndrome Virus Replication via Its Glycolytic Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important endemic swine pathogens, causing enormous losses in the global swine industry. Commercially available vaccines only partially prevent or counteract the virus infection and correlated losses. PRRSV's replication mechanism has not been well understood. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was screened to bind with the viral major envelope glycoprotein 5 (GP5) after PRRSV infection. The interacting sites are located within a 13-amino-acid (aa) region (aa 93 to 105) of GP5 and at Lys227 of GAPDH. Interestingly, viral GP5 restricts the translocation of GAPDH from the cytoplasm to the nucleus. Moreover, cytoplasmic GAPDH facilitates PRRSV replication by virtue of its glycolytic activity. The results suggest that PRRSV GP5 restricts GAPDH to the nucleus and exploits its glycolytic activity to stimulate virus replication. The data provide insight into the role of GAPDH in PRRSV replication and reveal a potential target for controlling viral infection. IMPORTANCE PRRSV poses a severe economic threat to the pig industry. PRRSV GP5, the major viral envelope protein, plays an important role in viral infection, pathogenicity, and immunity. However, interactions between GP5 and host proteins have not yet been well studied. Here, we show that GAPDH interacts with GP5 through binding a 13-aa sequence (aa 93 to 105) in GP5, while GP5 interacts with GAPDH at the K277 amino acid residue of GAPDH. We demonstrate that GP5 interacts with GAPDH in the cytoplasm during PPRSV infection, inhibiting GAPDH entry into the nucleus. PRRSV exploits the glycolytic activity of GAPDH to promote viral replication. These results enrich our understanding of PRRSV infection and pathogenesis and open a new avenue for antiviral prevention and PRRSV treatment strategies.

The genetic identification of camel contagious ecthyma virus as the causative agent of contagious ecthyma in dromedary camels (Camelus dromedarius) in Qatar.

Camel contagious ecthyma is a contagious viral disease of camels caused by either Orf virus (ORFV) or camel contagious ecthyma virus (CCEV). It has been previously reported and shown to cause economic losses in some camel-rearing countries in Asia and Africa, but has not been detected in Qatar. The purpose of this study was to identify and genetically characterize the contagious ecthyma causative agent in Qatari dromedary camels between 2017 and 2018. Accordingly, we made diagnoses of camel contagious ecthyma based on the clinical signs and genetic analysis of the entire major envelop protein (B2L) gene. The sequence analysis showed that CCEV was the infecting virus, and the B2L gene sequences were highly conserved between the locally infected camels with 100% similarity with isolates from Bahrain. This is the first study reporting the detection of CCEV in Qatar. We suggest that sequencing of the CCEV genome is necessary to determine the origin and relationship of this virus with other members of the parapoxvirus genus.

Adamantane derivatives as potential inhibitors of p37 major envelope protein and poxvirus reproduction. Design, synthesis and antiviral activity.

Currently, smallpox, caused by the variola virus belonging to the poxvirus family, has been completely eradicated according to the WHO. However, other representatives of poxviruses, such as vaccinia virus, cowpox virus, ectromelia virus, monkeypox virus, mousepox virus and others, remain in the natural environment and can infect both animals and humans. The pathogens of animal diseases, belonging to the category with a high epidemic risk, have already caused several outbreaks among humans, and can, in an unfavorable combination of circumstances, cause not only an epidemic, but also a pandemic. Despite the fact that there are protocols for the treatment of poxvirus infections, the targeted design of new drugs will increase their availability and expand the arsenal of antiviral chemotherapeutic agents. One of the potential targets of poxviruses is the p37 protein, which is a tecovirimat target. This protein is relatively small, has no homologs among proteins of humans and other mammals and is necessary for the replication of viral particles, which makes it attractive target for virtual screening. Using the I-TASSER modelling and molecular dynamics refinement the p37 orthopox virus protein model was obtained and its was confirmed by ramachandran plot analysis and superimposition of the model with the template protein with similar function. A virtual library of adamantane containing compounds was generated and a number of potential inhibitors were chosen from virtual library using molecular docking. Several compounds bearing adamantane moiety were synthesized and their biological activity was tested in vitro on vaccinia, cowpox and mousepox viruses. The new compounds inhibiting vaccinia virus replication with IC50 concentrations between 0.133 and 0.515 μM were found as a result of the research. The applied approach can be useful in the search of new inhibitors of orthopox reproduction. The proposed approach may be suitable for the design of new poxvirus inhibitors containing cage structural moiety.

A swine arterivirus deubiquitinase stabilizes two major envelope proteins and promotes production of viral progeny.

Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell’s exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly.

Detection of Myxoma Virus DNA in Ticks from Lagomorph Species in Spain Suggests Their Possible Role as Competent Vector in Viral Transmission.

Myxoma virus (MYXV) causes morbidity and mortality in European wild rabbits (Oryctolagus cuniculus) worldwide, and recently in Iberian hares (Lepus granatensis) in Spain. We aimed to assess the presence of MYXV-specific DNA in ixodid ticks collected from both hosts. A total of 417 ticks harvested from 30 wild lagomorphs, including wild rabbits and Iberian hares were collected from southern Spain. Enzyme-linked immunosorbent assay and PCR-sequencing were used to detect virus exposure and presence, respectively. Antibodies to MYXV were detected in 68% (17/25) of wild rabbits and in 67% (2/3) of Iberian hares. We detected MYXV DNA in 50.7% of pools of two different tick species (nymphs and adults of Rhipicephalus pusillus, and nymphs of Hyalomma lusitanicum) parasitizing rabbits and hares. The obtained partial sequence of the viral major envelope protein gene showed a mutation (G383A) within the MYXV_gp026 locus between the rabbit strain and Iberian hare strain (recently isolated in tissues of infected hares from Spain). However, in our study, the viral DNA presence was detected for the first time using tick DNA as the PCR-template, but the possible role of ticks as vectors of MYXV still needs to be elucidated.

Ipomoeassin-F inhibits the in vitro biogenesis of the SARS-CoV-2 spike protein and its host cell membrane receptor.

ABSTRACTIn order to produce proteins essential for their propagation, many pathogenic human viruses, including SARS-CoV-2, the causative agent of COVID-19 respiratory disease, commandeer host biosynthetic machineries and mechanisms. Three major structural proteins, the spike, envelope and membrane proteins, are amongst several SARS-CoV-2 components synthesised at the endoplasmic reticulum (ER) of infected human cells prior to the assembly of new viral particles. Hence, the inhibition of membrane protein synthesis at the ER is an attractive strategy for reducing the pathogenicity of SARS-CoV-2 and other obligate viral pathogens. Using an in vitro system, we demonstrate that the small molecule inhibitor ipomoeassin F (Ipom-F) potently blocks the Sec61-mediated ER membrane translocation and/or insertion of three therapeutic protein targets for SARS-CoV-2 infection; the viral spike and ORF8 proteins together with angiotensin-converting enzyme 2, the host cell plasma membrane receptor. Our findings highlight the potential for using ER protein translocation inhibitors such as Ipom-F as host-targeting, broad-spectrum antiviral agents.This article has an associated First Person interview with the first author of the paper.

Functional expression of human prostaglandin E2 receptor 4 (EP4) in E. coli and characterization of the binding property of EP4 with Gα proteins

Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E2 (PGE2) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein signaling cascade. To prepare an active form of EP4 for biochemical characterization and pharmaceutical application, this study designed a recombinant protein comprising human EP4 fused to the P9 protein (a major envelope protein of phi6 phage) and overexpressed the P9-EP4 fusion protein in the membrane fraction of E. coli. The solubilized P9-EP4 with sarkosyl (a strong anionic detergent) was purified by affinity chromatography. The purified protein was stabilized with amphiphilic polymers derived from poly-γ-glutamate. The polymer-stabilized P9-EP4 showed specific interaction with the alpha subunits of Gs or Gi proteins, and a high content of α-helical structure by a circular dichroism spectroscopy. Furthermore, the polymer-stabilized P9-EP4 showed strong heat resistance compared with P9-EP4 in detergents. The functional preparation of EP4 and its stabilization with amphiphilic polymers could facilitate both the biochemical characterization and pharmacological applications targeting EP4.

First detection and molecular characterisation of pseudocowpox virus in a cattle herd in Zambia

BackgroundPseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to rapidly identify the causative agents of poxvirus infections. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with Lumpy Skin Disease virus.MethodsWe used a High-Resolution Melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L gene) for comparative sequence and phylogenetic analysis.ResultsOur field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis, based on the HRM assay revealed PCPV DNA in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples and revealed genomic differences between samples collected in 2017 and 2018 from the same farm.ConclusionOur work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs. This rapid and accurate diagnosis improves the response time for more accurate veterinary interventions.

Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi

Lates calcarifer herpes virus (LCHV) is a novel virus of farmed barramundi in Southeast Asia. However, a rapid detection method is yet to be available for LCHV. This study, therefore, aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it timely available to public for disease diagnostics and surveillance in barramundi farming countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative major envelope protein encoding gene (MEP) was used for developing and optimizing a SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral copies per μl of DNA template in a reaction containing spiked host DNA. No cross-amplification with genomic DNA extracted from host as well as common aquatic pathogens (12 bacteria and 4 viruses) were observed. Validation test of the method with clinical samples revealed that the virus was detected in multiple organs of the clinically sick fish but not in the healthy fish. We thus recommend that barramundi farming countries should promptly initiate active surveillance for LCHV in order to understand their circulation for preventing possibly negative impact to the industry.

Structural Features of a Conformation-dependent Antigen Epitope on ORFV-B2L Recognized by the 2E4 mAb

Previously, we successfully prepared a monoclonal antibody (mAb) named 2E4, that directly recognizes the major envelope protein B2L of the orf virus (ORFV), but there is little information about its epitope. Here, we meticulously mapped the 2E4 epitope through combinatorial programs and identified the functional binding domain and a key amino acid residue. Briefly, the simulated epitope peptide closely resembles 84VDVQSKDKDADELR97 located at the N-terminus of B2L, strongly suggesting that the epitope is conformationally or spatially structure-dependent. Subsequently, we combined these findings with the results from the antigenicity prediction of B2L to design three truncated fragments of B2L (F1, F2 and F3) selected using 2E4, and only the F1 fragment was found to be eligible for the advanced stage. Alanine-scanning mutagenesis suggested that the D94 residue is structurally crucial for the 2E4 epitope. The other participating residues, including K61, E62, and D92, together with D94 were responsible for enabling 2E4 binding and served as factors that synergistically enabled binding to the whole 2E4 epitope. In this paper, we describe, for the first time, the architecture of an ORFV conformational epitope, and it is also expected that mAb 2E4 and its epitope can be used for applications relating to orf control.