Nonstructural Research Articles

Minicircle-based vaccine induces potent T-cell and antibody responses against hepatitis C virus

An effective vaccine against hepatitis C virus (HCV) should elicit both humoral and cellular immune responses. Previously, we characterized a bivalent vaccine candidate against hepatitis B (HBV) and HCV using chimeric HBV-HCV virus-like particles (VLP), in which the highly conserved epitope of HCV E2 glycoprotein (residues 412–425) was inserted into the hydrophilic loop of HBV small surface antigen (sHBsAg). While sHBsAg_412–425 elicited cross-neutralizing antibodies, it did not trigger a T-cell response against HCV. Thus, this study aimed to develop a vaccine candidate engaging both arms of adaptive immune response, potentially offering stronger protection against HCV. We evaluated the immunogenicity of minicircle (MC) DNA vaccines encoding sHBsAg_412–425 and HCV nonstructural (NS) proteins in BALB/c mice. Co-administration of sHBsAg_412–425 and NS induced a potent T-cell response, especially against NS3 and high titers of antibodies specific to HCV E2. Additionally, these antibodies recognized native HCV envelope glycoprotein heterodimers (E1E2) across multiple HCV genotypes and showed binding profiles to E1E2 alanine mutants comparable to the broadly neutralizing AP33 antibody. Overall, the findings demonstrate that MC DNA vaccine incorporating both sHBsAg_412–425 and HCV NS protein sequences induces robust, T-cell and AP33-like antibody responses, highlighting its potential as pan-genotypic prophylactic vaccine against HCV.

Exploring the Targets of Dengue Virus and Designs of Potential Inhibitors.

Dengue, a mosquito-borne viral disease spread by the dengue virus (DENV), has become one of the most alarming health issues in the global scenario in recent days. The risk of infection by DENV is mostly high in tropical and subtropical areas of the world. The mortality rate of patients affected with DENV is ever-increasing, mainly due to a lack of anti-dengue viral-specific synthetic drug components. Repurposing synthetic drugs has been an effective tool in combating several pathogens, including DENV. However, only the Dengvaxia vaccine has been developed so far to fight against the deadly disease despite the grave situation, mainly because of the limitations of understanding the actual pathogenicity of the disease. To address this particular issue and explore the actual disease pathobiology, several potential targets, like three structural proteins and seven non-structural (NS) proteins, along with their inhibitors of synthetic and natural origin, have been screened using docking simulation. Exploration of these targets, along with their inhibitors, has been extensively studied in culmination with molecular docking-based screening to potentiate the treatment. These screened inhibitors could possibly be helpful for the designing of new congeneric potential compounds to combat dengue fever and its complications.

Identification of a Novel Parvovirus in the Arctic Wolf (Canis lupus arctos).

A novel virus, temporarily named "Arctic wolf parvovirus" (AWPV), was discovered in a pharyngeal metagenomic library derived from an Arctic wolf (Canis lupus arctos) in China. The genome sequence was assigned GenBase accession number C_AA071902.1. AWPV has a genome comprised of 4,920 base pairs with a nucleotide composition of 36.4% A, 23.4% T, 18.2% G, and 22.0% C, with a GC content of 40.2%. Its structure resembles parvoviruses, containing two open reading frames: the nonstructural (NS) region encoding replication enzymes and the structural (VP) region encoding capsid protein. Pairwise sequence comparison and phylogenetic analysis suggest AWPV may represent a novel species within the genus Protoparvovirus. This discovery enhances our understanding of mammalian virus ecology and potential future infectious diseases.

Analysis of the susceptibility of refractory hepatitis C virus resistant to nonstructural 5A inhibitors

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs.

Usutu virus NS4A suppresses the host interferon response by disrupting MAVS signaling

Usutu virus (USUV) is an emerging flavivirus that can infect birds and mammals. In humans, in severe cases, it may cause neuroinvasive disease. The innate immune system, and in particular the interferon response, functions as the important first line of defense against invading pathogens such as USUV. Many, if not all, viruses have developed mechanisms to suppress and/or evade the interferon response in order to facilitate their replication. The ability of USUV to antagonize the interferon response has so far remained largely unexplored. Using dual-luciferase reporter assays we observed that multiple of the USUV nonstructural (NS) proteins were involved in suppressing IFN-β production and signaling. In particular NS4A was very effective at suppressing IFN-β production. We found that NS4A interacted with the mitochondrial antiviral signaling protein (MAVS) and thereby blocked its interaction with melanoma differentiation-associated protein 5 (MDA5), resulting in reduced IFN-β production. The TM1 domain of NS4A was found to be essential for binding to MAVS. By screening a panel of flavivirus NS4A proteins we found that the interaction of NS4A with MAVS is conserved among flaviviruses. The increased understanding of the role of NS4A in flavivirus immune evasion could aid the development of vaccines and therapeutic strategies.

Residue K28 of Zika Virus NS5 Protein Is Implicated in Virus Replication and Antagonism of STAT2.

The identification of four potential nonstructural 5 (NS5) residues-K28, K45, V335, and S749-that share the same amino acid preference in STAT2-interacting flaviviruses [Dengue virus (DENV) and Zika virus (ZIKV)], but not in STAT2-non-interacting flaviviruses [West Nile virus (WNV) and/or Yellow fever virus (YFV)] from an alignment of multiple flavivirus NS5 sequences, implied a possible association with the efficiency of ZIKV to antagonize the human signal transducer and activator of transcription factor 2 (STAT2). Through site-directed mutagenesis and reverse genetics, mutational impacts of these residues on ZIKV growth in vitro and STAT2 antagonism were assessed using virus growth kinetics assays and STAT2 immunoblotting. The results showed that mutations at the residue K28 significantly reduced the efficiency of ZIKV to antagonize STAT2. Further investigation involving residue K28 demonstrated its additional effects on the phenotypes of ZIKV-NS5 nuclear bodies. These findings demonstrate that K28, identified from sequence alignment, is an important determinant of replication and STAT2 antagonism by ZIKV.

Different viral genes modulate virulence in model mammal hosts and Culex pipiens vector competence in Mediterranean basin lineage 1 West Nile virus strains.

West Nile virus (WNV) is a single-stranded positive-sense RNA virus (+ssRNA) belonging to the genus Orthoflavivirus. Its enzootic cycle involves mosquito vectors, mainly Culex, and wild birds as reservoir hosts, while mammals, such as humans and equids, are incidental dead-end hosts. It was first discovered in 1934 in Uganda, and since 1999 has been responsible for frequent outbreaks in humans, horses and wild birds, mostly in America and in Europe. Virus spread, as well as outbreak severity, can be influenced by many ecological factors, such as reservoir host availability, biodiversity, movements and competence, mosquito abundance, distribution and vector competence, by environmental factors such as temperature, land use and precipitation, as well as by virus genetic factors influencing virulence or transmission. Former studies have investigated WNV factors of virulence, but few have compared viral genetic determinants of pathogenicity in different host species, and even fewer have considered the genetic drivers of virus invasiveness and excretion in Culex vector. In this study, we characterized WNV genetic factors implicated in the difference in virulence observed in two lineage 1 WNV strains from the Mediterranean Basin, the first isolated during a significant outbreak reported in Israel in 1998, and the second from a milder outbreak in Italy in 2008. We used an innovative and powerful reverse genetic tool, e.g., ISA (infectious subgenomic amplicons) to generate chimeras between Israel 1998 and Italy 2008 strains, focusing on non-structural (NS) proteins and the 3'UTR non-coding region. We analyzed the replication of these chimeras and their progenitors in mammals, in BALB/cByJ mice, and vector competence in Culex (Cx.) pipiens mosquitoes. Results obtained in BALB/cByJ mice suggest a role of the NS2B/NS3/NS4B/NS5 genomic region in viral attenuation in mammals, while NS4B/NS5/3'UTR regions are important in Cx. pipiens infection and possibly in vector competence.

Characterizing the West Nile Virus's polyprotein from nucleotide sequence to protein structure – Computational tools

ObjectivesWest Nile virus (WNV) belongs to the Flaviviridae family and causes West Nile fever. The mechanism of transmission involves the culex mosquito species. Infected individuals are primarily asymptomatic, and few exhibit common symptoms. Moreover, 10 % of neuronal infection caused by this virus cause death. The proteins encoded by these genes had been uncharacterized, although understanding their function and structure is important for formulating antiviral drugs. MethodsHerein, we used in silico approaches, including various bioinformatic tools and databases, to analyse the proteins from the WNV polyprotein individually. The characterization included GC content, physicochemical properties, conserved domains, soluble and transmembrane regions, signal localization, protein disorder, and secondary structure features and their respective 3D protein structures. ResultsAmong 11 proteins, eight had >50 % GC content, eight proteins had basic pI values, three proteins were unstable under in vitro conditions, four were thermostable according to >100 AI values and some had negative GRAVY values in physicochemical analyses. All protein-conserved domains were shared among Flaviviridae family members. Five proteins were soluble and lacked transmembrane regions. Two proteins had signals for localization in the host endoplasmic reticulum. Non-structural (NS) 2A showed low protein disorder. The secondary structural features and tertiary structure models provide a valuable biochemical resource for designing selective substrates and synthetic inhibitors. ConclusionsWNV proteins NS2A, NS2B, PM, NS3 and NS5 can be used as drug targets for the pharmacological design of lead antiviral compounds.

Analysis of Cleavage Activity of Dengue Virus Protease by Co-transfections.

The genome of the dengue virus codes for a single polypeptide that yields three structural and seven non-structural (NS) proteins upon post-translational modifications. Among them, NS protein-3 (NS3) possesses protease activity, involved in the processing of the self-polypeptide and in the cleavage of host proteins. Identification and analysis of such host proteins as substrates of this protease facilitate the development of specific drugs. In vitro cleavage analysis has been applied, which requires homogeneously purified components. However, the expression and purification of both S3 and erythroid differentiation regulatory factor 1 (EDRF1) are difficult and unsuccessful on many occasions. EDRF1 was identified as an interacting protein of dengue virus protease (NS3). The amino acid sequence analysis indicates the presence of NS3 cleavage sites in this protein. As EDRF1 is a high-molecular-weight (~138 kDa) protein, it is difficult to express and purify the complete protein. In this protocol, we clone the domain of the EDRF1 protein (C-terminal end) containing the cleavage site and the NS3 into two different eukaryotic expression vectors containing different tags. These recombinant vectors are co-transfected into mammalian cells. The cell lysate is subjected to SDS-PAGE followed by western blotting with anti-tag antibodies. Data suggest the disappearance of the EDRF1 band in the lane co-transfected along with NS3 protease but present in the lane transfected with only EDRF1, suggesting EDRF1 as a novel substrate of NS3 protease. This protocol is useful in identifying the substrates of viral-encoded proteases using ex vivo conditions. Further, this protocol can be used to screen anti-protease molecules. Key features • This protocol requires the cloning of protease and substrate into two different eukaryotic expression vectors with different tags. • Involves the transfection and co-transfection of both the above recombinant vectors individually and together. • Involves western blotting of the same PVDF membrane containing total proteins of the cell lysate with two different antibodies. • Does not require purified proteins for the analysis of cleavage of any suspected substrate by the protease.

Direct-acting antiviral resistance of Hepatitis C virus is promoted by epistasis

Direct-acting antiviral agents (DAAs) provide efficacious therapeutic treatments for chronic Hepatitis C virus (HCV) infection. However, emergence of drug resistance mutations (DRMs) can greatly affect treatment outcomes and impede virological cure. While multiple DRMs have been observed for all currently used DAAs, the evolutionary determinants of such mutations are not currently well understood. Here, by considering DAAs targeting the nonstructural 3 (NS3) protein of HCV, we present results suggesting that epistasis plays an important role in the evolution of DRMs. Employing a sequence-based fitness landscape model whose predictions correlate highly with experimental data, we identify specific DRMs that are associated with strong epistatic interactions, and these are found to be enriched in multiple NS3-specific DAAs. Evolutionary modelling further supports that the identified DRMs involve compensatory mutational interactions that facilitate relatively easy escape from drug-induced selection pressures. Our results indicate that accounting for epistasis is important for designing future HCV NS3-targeting DAAs.

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

Flavivirus Concentrates Host ER in Main Replication Compartments to Facilitate Replication.

Flavivirus remodels the host endoplasmic reticulum (ER) to generate replication compartments (RCs) as the fundamental structures to accommodate viral replication. Here, a centralized replication mode of flavivirus is reported, i.e., flavivirus concentrates host ER in perinuclear main replication compartments (MRCs) for efficient replication. Superresolution live-cell imaging demonstrated that flavivirus MRCs formed via a series of events, including multisite ER clustering, growth and merging of ER clusters, directional movement, and convergence in the perinuclear region. The dynamic activities of viral RCs are driven by nonstructural (NS) proteins and are independent of microtubules and actin. Moreover, disrupting MRCs formation by small molecule compounds inhibited flavivirus replication. Overall, the findings reveal unprecedented insight into dynamic ER reorganization by flavivirus and identify a new inhibition strategy.

NS5 hijacks TRAF3 to inhibit type I interferon signaling during duck Tembusu virus infection

The tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) is a key signaling molecule in the retinoic acid-inducible gene I (RIG-I) signaling pathway and plays an important role in host innate immune regulation. The function of TRAF3 has been extensively studied in mammals, however, the role of TRAF3 in ducks remains unclear. In order to reveal the function of duck TRAF3 (duTRAF3) in the innate immune response induced by virus infection, the TRAF3 homologue of mallard (Anas platyrhynchos) has been cloned and the function of duTRAF3 is investigated in this study. We sequenced duTRAF3 and found that the open reading frame (ORF) region of duTRAF3 is 1704 bp long and encodes 567 amino acids (aa), which has a similar functional domain to the mammalian gene. Analysis of tissue distribution of duTRAF3 in 7-day-old ducks showed that the expression of duTRAF3 was highest in harderian gland, followed by heart and lung. Subsequently, duck Tembusu virus (DTMUV) has been shown to enhance duTRAF3 expression, and overexpression of duTRAF3 inhibits DTMUV replication in a dose-dependent manner. In addition, duTRAF3 activates the transcriptional activity of IFN-α and its downstream interferon-stimulating genes (ISGs) induced after DTMUV infection. In this process, DTMUV non-structural (NS) protein 5 resists this innate immune process by interacting with TRAF3 and inhibiting TRAF3 expression. These data support the conclusion that duTRAF3 is an antiviral protein that plays a key role in the defense against DTMUV invasion. These results lay a theoretical foundation for developing new anti-DTMUV strategies.

Assessing the inhibitory potential of anti-dengue compounds against Japanese encephalitis virus RNA dependent RNA polymerase: an in silico study

Japanese encephalitis (JE), a neurological infection of severe nature, is caused by the Japanese encephalitis virus (JEV) and is transmitted by the mosquito vector. The polymerase domain of Non-structural 5 (NS5), which is also referred to as RdRp (RNA-dependent RNA polymerase), is considered a potential therapeutic target for JEV. The present study employed molecular dynamics modelling and high-throughput virtual screening to evaluate the possible antiviral activity of anti-dengue drugs against JEV RdRp. Furthermore, a ranking was performed utilising the MM/GBSA analysis to identify the three most promising compounds. Compound ID 57409246 exhibited the highest binding affinity with the protein, as evidenced by its minimum binding free energy of −72.96 kcal/mole. In contrast, the other two compounds had minimum binding free energies of −67.57 and −59.19 kcal/mole, respectively. Upon conducting a 100 nanosecond molecular dynamics simulation to confirm the binding of the chemical complexes, it was observed that the three hits, namely 57409246, 70683874, and 44577154, exhibited a consistent and stable RMSD. Subsequently, the binding strength of the trajectory was confirmed through MM/GBSA analysis. The compounds 70683874 and 57409246 exhibited the lowest binding free energies, which were −97.58 kcal/mol and −96.38 kcal/mol, respectively. The binding free energy (ΔG Bind) values for the native ligand ATP and molecule 44577154 were −65.64 kcal/mol and −69.44 kcal/mol, respectively. Overall, compared to the native ligand ATP, all three compounds exhibited higher binding affinity. The study proposes three anti-dengue molecules as a potential remedy for JE, which can be confirmed through in vitro and in vivo investigations. Communicated by Ramaswamy H. Sarma

Packaging defects in pestiviral NS4A can be compensated by mutations in NS2 and NS3.

The non-structural (NS) proteins of the Flaviviridae members play a dual role in genome replication and virion morphogenesis. For pestiviruses, like bovine viral diarrhea virus, the NS2-3 region and its processing by the NS2 autoprotease is of particular importance. While uncleaved NS2-3 in complex with NS4A is essential for virion assembly, it cannot replace free NS3/4A in the viral replicase. Furthermore, surface interactions between NS3 and the C-terminal cytosolic domain of NS4A were shown to serve as a molecular switch between RNA replication and virion morphogenesis. To further characterize the functionality of NS4A, we performed an alanine-scanning mutagenesis of two NS4A regions, a short highly conserved cytoplasmic linker downstream of the transmembrane domain and the C-terminal domain. NS4A residues critical for polyprotein processing, RNA replication, and/or virion morphogenesis were identified. Three double-alanine mutants, two in the linker region and one close to the C-terminus of NS4A, showed a selective effect on virion assembly. All three packaging defective mutants could be rescued by a selected set of two second-site mutations, located in NS2 and NS3, respectively. This phenotype was additionally confirmed by complementation studies providing the NS2-3/4A packaging molecules containing the rescue mutations in trans. This indicates that the linker region and the cytosolic C-terminal part of NS4A are critical for the formation of protein complexes required for virion morphogenesis. The ability of the identified sets of second-site mutations in NS2-3 to compensate for diverse NS4A defects highlights a surprising functional flexibility for pestiviral NS proteins. IMPORTANCE Positive-strand RNA viruses have a limited coding capacity due to their rather small genome size. To overcome this constraint, viral proteins often exhibit multiple functions that come into play at different stages during the viral replication cycle. The molecular basis for this multifunctionality is often unknown. For the bovine viral diarrhea virus, the non-structural protein (NS) 4A functions as an NS3 protease cofactor, a replicase building block, and a component in virion morphogenesis. Here, we identified the critical amino acids of its C-terminal cytosolic region involved in those processes and show that second-site mutations in NS2 and NS3 can compensate for diverse NS4A defects in virion morphogenesis. The ability to evolve alternative functional solutions by gain-of-function mutations highlights the astounding plasticity of the pestiviral system.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1β, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.

Dengue virus serotypic replacement of NS3 protease or helicase domain causes chimeric viral attenuation but can be recovered by a compensated mutation at helicase domain or NS2B, respectively.

Mosquito-borne dengue viruses (DENVs) have evolved to four serotypes with 69%-78% amino acid identities, resulting in incomplete immunity, where one serotype's infection does not cross-protect against secondary infections by other serotypes. Despite the amino acid differences, structural and nonstructural (NS) proteins among serotypes play similar functions. NS3 is an enzyme complex: NS3 has N-terminal protease (PRO) and C-terminal helicase (HEL) activities in addition to 5' RNA triphosphatase (5'RTP), which is involved in the RNA capping process. In this study, the effects of NS3 replacements among serotypes were tested. The replacement of NS3 full-length (FULL), PRO or HEL region suppressed viral replication in BHK-21 mammalian cells, while the single compensatory mutation improved the viral replications; P364S mutation in HEL revived PRO (DENV3)-replaced DENV1, while S68T alteration in NS2B recovered HEL (DENV1)-replaced DENV2. The results suggest that the interactions between PRO and HEL as well as HEL and NS2B are required for replication competence. Lower-frequency mutations also appeared at various locations in viral proteins, although after infecting C6/36 mosquito cells, the mutations' frequencies changed, and/or new mutations appeared. In contrast, the inter-domain region (INT, 12 amino acids)-replaced chimera quickly replicated without mutation in BHK-21 cells, although extended cell culture accumulated various mutations. These results suggest that NS3 variously interacts with DENV proteins, in which the chimeric NS3 domain replacements induced amino acid mutations, irrespective of replication efficiency. However, the viral sequences are further adjusted for replication efficiency, to fit in both mammalian cells and mosquito cells. IMPORTANCE Enzyme activities for replicating DENV 5' cap positive (+) sense RNA have been shown to reside in NS3 and NS5. However, it remains unknown how these enzymes coordinately synthesize negative (-) sense RNA, from which abundant 5' cap (+) sense RNA is produced. We previously revealed that NS5 dimerization and NS5 methyltransferase(MT)-NS3HEL interaction are important for DENV replication. Here, we found that replication incompetence due to NS3PRO or HEL replacement was compensated by a mutation at HEL or NS2B, respectively, suggesting that the interactions among NS2B, NS3PRO, and HEL are critical for DENV replication.

Isolation and identification of Eurasian avian-like H1N1 swine influenza virus and evaluation of their pathogenicity and immune protective effects in pigs

Swine influenza (SI) is a severe disease affecting pigs, with a huge economic impact on pig farmers. Currently, available SIV vaccines do not meet the requirements for Swine influenza prevention and control, indicating the need for vaccine development using predominant strains. Here, we isolated and identified the swine influenza virus in farms and slaughterhouses in nine provinces in China to determine the most prevalent strain. A total of 8383 samples were collected between 2013 and 2022, from which 87 swine influenza virus strains were isolated. Genome sequencing identified 62 strains of the H1N1 subtype, three strains of the H1N2 subtype, and 22 strains of the H3N2 subtype. The 521# strain virus possesses the viral ribonucleoprotein (vRNP) and matrix (M) genes from the pdm/09 lineage, the HA, NA from the original Eurasian avian-like (EA) H1N1 lineage, and the nonstructural (NS) gene from the triple-reassortant (TR) lineage. The 431# strain was also a TR, except its M-gene was derived from the original EA H1N1 lineage. The pathogenicity of two 431# strains and one typical 521# strain was evaluated in mice, and the 431# strain exhibited higher pathogenicity. Therefore, a new 521# strain was selected for vaccine production because it is the current circulating strain. The vaccine produced using the 521# strain and pre-evaluated adjuvants was effective against the homologous H05 strain, as evidenced by the normal body temperature of vaccinated pigs and low virus titer of nasal swabs. In contrast, infection with the H05 strain significantly increased the body temperature of unvaccinated pigs and increased the virus titer of nasal swabs. Notably, vaccination with the 521#-based vaccine conferred some level of protection against the heterologous B15 strain (H3N2 subtype), thus reducing the viral load in pigs.

Molecular identification and in vitro assessment of zoonotic-potential of a novel Orthobunyavirus isolated from broiler chicken in Malaysia.

We have previously isolated a novel avian Orthobunyavirus, Kedah Fatal Kidney Syndrome (KFKS) virus from a broiler farm in Kedah, Malaysia in 2020 with a severe kidney lesion in chickens. The virus was designated as KFKS2_CS virus. Sequence analysis of partial nucleocapsid (N) and nonstructural (NSs) sequence of this virus showed the highest sequence identity with previous KFKS1 from Malaysia (100%) and 97% with a zoonotic Umbre (UMB) virus, which was reported to cause encephalitis in immunocompromised humans in India. Phylogenetic analysis revealed that this virus was clustered together with previous KFKS1 virus from Malaysia, UMB and Cristoli viruses. This study aimed to assess the zoonotic potential of this KFKS2_CS virus in vitro by determining its ability to inhibit the production of interferon (IFN) in human glioblastoma multiforme (GBM) brain cells using reverse-transcriptase polymerase reaction (RT-PCR). This virus blocked the production of interferon-a in this human brain cells. In conclusion, this KFKS2_CS virus may have a zoonotic potential and become a public health concern in the future.

Bipartite genome and structural organization of the parvovirus Acheta domesticus segmented densovirus

Parvoviruses (family Parvoviridae) are currently defined by a linear monopartite ssDNA genome, T = 1 icosahedral capsids, and distinct structural (VP) and non-structural (NS) protein expression cassettes within their genome. We report the discovery of a parvovirus with a bipartite genome, Acheta domesticus segmented densovirus (AdSDV), isolated from house crickets (Acheta domesticus), in which it is pathogenic. We found that the AdSDV harbors its NS and VP cassettes on two separate genome segments. Its vp segment acquired a phospholipase A2-encoding gene, vpORF3, via inter-subfamily recombination, coding for a non-structural protein. We showed that the AdSDV evolved a highly complex transcription profile in response to its multipartite replication strategy compared to its monopartite ancestors. Our structural and molecular examinations revealed that the AdSDV packages one genome segment per particle. The cryo-EM structures of two empty- and one full-capsid population (3.3, 3.1 and 2.3 Å resolution) reveal a genome packaging mechanism, which involves an elongated C-terminal tail of the VP, “pinning” the ssDNA genome to the capsid interior at the twofold symmetry axis. This mechanism fundamentally differs from the capsid-DNA interactions previously seen in parvoviruses. This study provides new insights on the mechanism behind ssDNA genome segmentation and on the plasticity of parvovirus biology.