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Introduction: Akabane virus (AKAV) is a worldwide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. Reporter-expressing recombinant virus represents a powerful tool to characterize the viral biology in vitro and in vivo. Methods: In this study, we have successfully established a reverse genetics system for AKAV. The recued virus possessed similar growth characteristics to the parental virus in vitro. Moreover, the recombinant AKAV reporter viruses expressing nanoluciferase (Nluc) or mWasabi were constructed by inserting into S segment, named rAKAV-Nluc and rAKAV-mWasabi, respectively. Results: We investigated the virological characteristics of rAKAV-Nluc and rAKAV-mWasabi and found that rAKAV-Nluc displayed similar growth kinetics as the parental virus and could stably produce the nano-luciferase even after 10 rounds of serial passages. rAKAV-mWasabi also exhibited comparable growth kinetics and genetic stability as the parental virus. We further used the two reporter viruses to test the susceptibility of different cell lines to AKAV and found that cell lines derived from various host species, including human, swine, cattle, and monkey enables AKAV replication efficiently, accelerating our understanding of the AKAV cell tropism range. Discussion: Taken together, our established reverse genetics system for AKAV provides more convenient screening tools and can be used to study AKAV virulence and tropism, and to elucidate the molecular biology of AKAV. [ABSTRACT FROM AUTHOR]

The diverse rhabdoviruses infecting plants and animals have conserved genome organizations, and the functions of viral structural proteins have been extensively studied. However, increasing number of rhabdoviruses have been found to encode various accessory proteins, whose specific roles during viral infection remain poorly understood. In this study, we investigated the function of the X proteins encoded by several members of the genus Alphanucleorhabdovirus. Using the recently established eggplant mottled dwarf virus (EMDV) reverse genetics system, we found that recombinant EMDV lacking the X gene was able to systemically infect Nicotiana benthamiana plants, albeit with reduced efficiency. However, this deletion mutant was largely restricted to the veinal tissues and caused asymptomatic infections. The EMDV X protein, which localized to the cytoplasm when expressed alone, was translocated to the nucleus via a specific interaction with the nucleocapsid (N) protein. Through analyzing the interactions of the X deletion mutants and the infection phenotypes of the derived EMDV deletion mutants, we demonstrated that the carboxyl-terminal region of the X protein (amino acids 71–83) is crucial for its interaction with the N protein and for viral pathogenesis. Moreover, the X proteins encoded by related alphanucleorhabdoviruses could fully or partially complement the functions of EMDV X in viral infection. These findings provide new insights into the roles of accessory proteins in plant rhabdovirus infection. [ABSTRACT FROM AUTHOR]

Abstract The diverse rhabdoviruses infecting plants and animals have conserved genome organizations, and the functions of viral structural proteins have been extensively studied. However, increasing number of rhabdoviruses have been found to encode various accessory proteins, whose specific roles during viral infection remain poorly understood. In this study, we investigated the function of the X proteins encoded by several members of the genus Alphanucleorhabdovirus. Using the recently established eggplant mottled dwarf virus (EMDV) reverse genetics system, we found that recombinant EMDV lacking the X gene was able to systemically infect Nicotiana benthamiana plants, albeit with reduced efficiency. However, this deletion mutant was largely restricted to the veinal tissues and caused asymptomatic infections. The EMDV X protein, which localized to the cytoplasm when expressed alone, was translocated to the nucleus via a specific interaction with the nucleocapsid (N) protein. Through analyzing the interactions of the X deletion mutants and the infection phenotypes of the derived EMDV deletion mutants, we demonstrated that the carboxyl-terminal region of the X protein (amino acids 71–83) is crucial for its interaction with the N protein and for viral pathogenesis. Moreover, the X proteins encoded by related alphanucleorhabdoviruses could fully or partially complement the functions of EMDV X in viral infection. These findings provide new insights into the roles of accessory proteins in plant rhabdovirus infection.

IntroductionAkabane virus (AKAV) is a worldwide epidemic arbovirus belonging to the Bunyavirales order that predominantly infects livestock and causes severe congenital malformations. Reporter-expressing recombinant virus represents a powerful tool to characterize the viral biology in vitro and in vivo.MethodsIn this study, we have successfully established a reverse genetics system for AKAV. The recued virus possessed similar growth characteristics to the parental virus in vitro. Moreover, the recombinant AKAV reporter viruses expressing nanoluciferase (Nluc) or mWasabi were constructed by inserting into S segment, named rAKAV-Nluc and rAKAV-mWasabi, respectively.ResultsWe investigated the virological characteristics of rAKAV-Nluc and rAKAV-mWasabi and found that rAKAV-Nluc displayed similar growth kinetics as the parental virus and could stably produce the nano-luciferase even after 10 rounds of serial passages. rAKAV-mWasabi also exhibited comparable growth kinetics and genetic stability as the parental virus. We further used the two reporter viruses to test the susceptibility of different cell lines to AKAV and found that cell lines derived from various host species, including human, swine, cattle, and monkey enables AKAV replication efficiently, accelerating our understanding of the AKAV cell tropism range.DiscussionTaken together, our established reverse genetics system for AKAV provides more convenient screening tools and can be used to study AKAV virulence and tropism, and to elucidate the molecular biology of AKAV.

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains. [ABSTRACT FROM AUTHOR]

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is a very severe disease characterized by high fatality rates and the development of permanent behavioral, psychiatric, and neurological sequelae among survivors. Japanese encephalitis virus (JEV), a flavivirus, is responsible for JE. In Asia, Genotype I (GI) has emerged as the dominant strain, replacing Genotype III (GIII). However, no clinically approved drug is available to treat JEV infection, and currently available commercial vaccines derived from JEV GIII strains provide only partial protection against GI. Utilizing a reverse genetics system, this study attempted to produce a novel chimeric JEV strain with high efficacy against JEV GI. Accordingly, a GI/GIII intertypic recombinant strain, namely SA14-GI env, was generated by substituting the E region of the GIII SA14-14-2 strain with that of the GI strain, K05GS. The neurovirulence of the mutant virus was significantly reduced in mice. Analysis of the immunogenicity of the chimeric virus revealed that it induced neutralizing antibodies against JEV GI in mice, and the protective efficacy of SA14-GI env was higher than that of SA14-14-2. These findings suggest that SA14-GI env may be a safe and effective live-attenuated vaccine candidate against JEV GI. [ABSTRACT FROM AUTHOR]

Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-CoV (SADS-CoV), cause severe diarrhea in neonatal piglets, and CoV infection is associated with significant economic losses for the swine industry worldwide. Reverse genetics systems realize the manipulation of RNA virus genome and facilitate the development of new vaccines. Thus far, five reverse genetics approaches have been successfully applied to engineer the swine CoV genome: targeted RNA recombination, in vitro ligation, bacterial artificial chromosome-based ligation, vaccinia virus -based recombination, and yeast-based method. This review summarizes the advantages and limitations of these approaches; it also discusses the latest research progress in terms of their use for virus-related pathogenesis elucidation, vaccine candidate development, antiviral drug screening, and virus replication mechanism determination. [ABSTRACT FROM AUTHOR]

Rotavirus A (RVA) is the leading cause of diarrhea requiring hospitalization in children and causes over 100,000 annual deaths in Sub-Saharan Africa. In order to generate next-generation vaccines against African RVA genotypes, a reverse genetics system based on a simian rotavirus strain was utilized here to exchange the antigenic capsid proteins VP4, VP7 and VP6 with those of African human rotavirus field strains. One VP4/VP7/VP6 (genotypes G9-P[6]-I2) triple-reassortant was successfully rescued, but it replicated poorly in the first cell culture passages. However, the viral titer was enhanced upon further passaging. Whole genome sequencing of the passaged virus revealed a single point mutation (A797G), resulting in an amino acid exchange (E263G) in VP4. After introducing this mutation into the VP4-encoding plasmid, a VP4 mono-reassortant as well as the VP4/VP7/VP6 triple-reassortant replicated to high titers already in the first cell culture passage. However, the introduction of the same mutation into the VP4 of other human RVA strains did not improve the rescue of those reassortants, indicating strain specificity. The results show that specific point mutations in VP4 can substantially improve the rescue and replication of recombinant RVA reassortants in cell culture, which may be useful for the development of novel vaccine strains.

Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes. [ABSTRACT FROM AUTHOR]

Abstract Background Newcastle disease virus (NDV) strain ZM10, a typical enterotropic avirulent vaccine strain, has been widely used in China for chickens against Newcastle disease. To elucidate its enterotropic mechanism and develop recombiant multivalent vaccines based on it, the reverse genetics system for NDV ZM10 is an indispensable platform. Results A full-length cDNA clone of NDV ZM10 and three supporting plasmids were constructed using the ligation-independent cloning method. Recombinant NDV rZM10 was successfully rescued after these plasmids were co-transfected into BHK-21 cells. Besides, the recombinant virus rZM10-RFP encoding the red fluorescent protein was generated by inserting the RFP gene into the full-length clone of NDV between the P and M genes. These rescued viruses were genetically and biologically identical to the parental strain and showed similar growth kinetics. Conclusion The recovery system of NDV ZM10 strain was established, and can be used as a foundation for research on the enterotropic mechanism and development of multivalent vaccines against viral diseases of livestock and poultry.

Orthotospoviruses, the plant-infecting bunyaviruses, cause serious diseases in agronomic crops and pose major threats to global food security. The family of Tospoviridae contains more than 30 members that are classified into two geographic groups, American-type and Euro/Asian-type orthotospovirus. However, the genetic interaction between different species and the possibility, during mixed infections, for transcomplementation of gene functions by orthotospoviruses from different geographic groups remains underexplored. In this study, minireplicon-based reverse genetics (RG) systems have been established for Impatiens necrotic spot virus (INSV) (an American-type orthotospovirus) and for Calla lily chlorotic spot virus and Tomato zonate spot virus (CCSV and TZSV) (two representative Euro/Asian orthotospoviruses). Together with the earlier established RG system for Tomato spotted wilt virus (TSWV), a type species of the Orthotospovirus American-clade, viral replicase/movement proteins were exchanged and analyzed on interspecies transcomplementation. Whereas the homologous RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) protein supported the replication of orthotospoviruses from both geographic groups, heterologous combinations of RdRp from one group and N from the other group were unable to support the replication of viruses from both groups. Furthermore, the NSm movement protein (MP), from both geographic groups of orthotospoviruses, was able to transcomplement heterologous orthotospoviruses or a positive-strand Cucumber mosaic virus (CMV) in their movement, albeit with varying efficiency. MP from Rice stripe tenuivirus (RSV), a plant-infecting bunyavirus that is distinct from orthotospoviruses, or MP from CMV also moves orthotospoviruses. Our findings gain insights into the genetic interaction/reassortant potentials for the segmented plant orthotospoviruses. IMPORTANCE Orthotospoviruses are agriculturally important negative-strand RNA viruses and cause severe yield-losses on many crops worldwide. Whereas the emergence of new animal-infecting bunyaviruses is frequently associated with genetic reassortants, this issue remains underexposed with the plant-infecting orthotospovirus. With the development of reverse genetics systems for orthotospoviruses from different geographic regions, the interspecies/intergroup replication/movement complementation between American- and Euro/Asian-type orthotospoviruses were investigated. Genomic RNAs from American orthotospoviruses can be replicated by the RdRp and N from those of Euro/Asia-group orthotospoviruses, and vice versa. However, their genomic RNAs cannot be replicated by a heterologous combination of RdRp from one geographic group and N from another geographic group. Cell-to-cell movement of viral entity is supported by NSm from both geographic groups, with highest efficiency by NSm from viruses belonging to the same group. Our findings provide important insights into the genetic interaction and exchange ability of viral gene functions between different species of orthotospovirus. [ABSTRACT FROM AUTHOR]

Rotavirus A (RVA) genome segments can reassort upon co-infection of target cells with two different RVA strains. However, not all reassortants are viable, which limits the ability to generate customized viruses for basic and applied research. To gain insight into the factors that restrict reassortment, we utilized reverse genetics and tested the generation of simian RVA strain SA11 reassortants carrying the human RVA strain Wa capsid proteins VP4, VP7, and VP6 in all possible combinations. VP7-Wa, VP6-Wa, and VP7/VP6-Wa reassortants were effectively rescued, but the VP4-Wa, VP4/VP7-Wa, and VP4/VP6-Wa reassortants were not viable, suggesting a limiting effect of VP4-Wa. However, a VP4/VP7/VP6-Wa triple-reassortant was successfully generated, indicating that the presence of homologous VP7 and VP6 enabled the incorporation of VP4-Wa into the SA11 backbone. The replication kinetics of the triple-reassortant and its parent strain Wa were comparable, while the replication of all other rescued reassortants was similar to SA11. Analysis of the predicted structural protein interfaces identified amino acid residues, which might influence protein interactions. Restoring the natural VP4/VP7/VP6 interactions may therefore improve the rescue of RVA reassortants by reverse genetics, which could be useful for the development of next generation RVA vaccines. [ABSTRACT FROM AUTHOR]

Siniperca chuatsi rhabdovirus (SCRV) is an important pathogen that infects mandarin fish. A reverse genetics system is an important technical platform for virus research. In this study, the minigenome in which the enhanced green fluorescent protein gene is flanked by the viral genomic ends of SCRV and transcribed using a T7 promoter‐terminator cassette was constructed. Co‐transfection of the minigenome construct with SCRV‐supporting plasmids of N, P, and L in BSRT7 cells resulted in the expression of the reporter gene. Transcription of a positive‐strand RNA copy from cDNA of the SCRV genome along with the viral N, P, and L proteins resulted in the recovery of infectious SCRV in cells. Viral titre up to 108 PFU/ml was achieved. Recombinant SCRV was verified by the detection of a unique restriction site engineered into the SCRV genome. The phenotypes of the recombinant SCRV and the parental virus were evaluated by plaque size, replication kinetics in vitro, and pathogenicity in vivo. The recovered SCRV from cDNA showed similar phenotypes compared to the parental virus. The established reverse genetics system is of great significance and value for the functional genome study of SCRV and for laying a foundation for the development of the viral vector and SCRV vaccine. [ABSTRACT FROM AUTHOR]

Feline caliciviruses can cause oral and upper respiratory tract infections in cats. However, a virulent and systemic feline calicivirus (VS-FCV) variant implicated in multisystem lesions and death in cats has emerged recently. To date, the mechanism underlying virulence variations in VS-FCV remains unclear. The aim of the present study was to provide a tool for exploring genetic variation in VS-FCV, by constructing an infectious clone of VS-FCV SH/2014. First, a full-length cDNA molecular clone of VS-FCV SH/2014 strain, which contains an Xba I recognition site generated by mutating one base (A→T) as a genetic marker, was constructed using the circular polymerase extension reaction (CPER) method. Second, the full-length cDNA clone was introduced into Crandell-Rees feline kidney cells using liposomes to rescue recombinant VS-FCV SH/2014 (rVS-FCV SH/2014). Third, the rescued viruses were identified by real-time PCR, immunofluorescence assay, western blotting, and electron microscopy. The full-length cDNA molecular clone of the VS-FCV SH/2014 strain was successfully constructed and that rVS-FCV SH/2014 could be rescued efficiently. rVS-FCV SH/2014 had the expected genetic markers and morphology and growth characteristics similar to those of the parental virus. The reverse genetics system provides a research platform for future studies on VS-FCV genetic variation and pathogenesis. • Full-length cDNA clone of the VS-FCV SH/2014 strain successfully constructed. • rVS-FCV SH/2014 could be rescued efficiently by circular polymerase extension reaction. • rVS-FCV SH/2014 had expected markers and parental virus morphology and growth traits. • A research platform for future studies on VS-FCV genetic variation and pathogenesis. [ABSTRACT FROM AUTHOR]

Japanese encephalitis (JE) is a very severe disease characterized by high fatality rates and the development of permanent behavioral, psychiatric, and neurological sequelae among survivors. Japanese encephalitis virus (JEV), a flavivirus, is responsible for JE. In Asia, Genotype I (GI) has emerged as the dominant strain, replacing Genotype III (GIII). However, no clinically approved drug is available to treat JEV infection, and currently available commercial vaccines derived from JEV GIII strains provide only partial protection against GI. Utilizing a reverse genetics system, this study attempted to produce a novel chimeric JEV strain with high efficacy against JEV GI. Accordingly, a GI/GIII intertypic recombinant strain, namely SA14-GI env, was generated by substituting the E region of the GIII SA14-14-2 strain with that of the GI strain, K05GS. The neurovirulence of the mutant virus was significantly reduced in mice. Analysis of the immunogenicity of the chimeric virus revealed that it induced neutralizing antibodies against JEV GI in mice, and the protective efficacy of SA14-GI env was higher than that of SA14-14-2. These findings suggest that SA14-GI env may be a safe and effective live-attenuated vaccine candidate against JEV GI.

Background: Newcastle disease virus (NDV) strain ZM10, a typical enterotropic avirulent vaccine strain, has been widely used in China for chickens against Newcastle disease. To elucidate its enterotropic mechanism and develop recombiant multivalent vaccines based on it, the reverse genetics system for NDV ZM10 is an indispensable platform. Results: A full-length cDNA clone of NDV ZM10 and three supporting plasmids were constructed using the ligation-independent cloning method. Recombinant NDV rZM10 was successfully rescued after these plasmids were co-transfected into BHK-21 cells. Besides, the recombinant virus rZM10-RFP encoding the red fluorescent protein was generated by inserting the RFP gene into the full-length clone of NDV between the P and M genes. These rescued viruses were genetically and biologically identical to the parental strain and showed similar growth kinetics. Conclusion: The recovery system of NDV ZM10 strain was established, and can be used as a foundation for research on the enterotropic mechanism and development of multivalent vaccines against viral diseases of livestock and poultry. [ABSTRACT FROM AUTHOR]

Emerging and re-emerging swine coronaviruses (CoVs), including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome-CoV (SADS-CoV), cause severe diarrhea in neonatal piglets, and CoV infection is associated with significant economic losses for the swine industry worldwide. Reverse genetics systems realize the manipulation of RNA virus genome and facilitate the development of new vaccines. Thus far, five reverse genetics approaches have been successfully applied to engineer the swine CoV genome: targeted RNA recombination, in vitro ligation, bacterial artificial chromosome-based ligation, vaccinia virus -based recombination, and yeast-based method. This review summarizes the advantages and limitations of these approaches; it also discusses the latest research progress in terms of their use for virus-related pathogenesis elucidation, vaccine candidate development, antiviral drug screening, and virus replication mechanism determination.

Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.

We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5′-cap structure is essential for the virus' generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic that emerged in December 2019 in Wuhan city, China. An effective vaccine is urgently needed to protect humans and to mitigate the economic and societal impacts of the pandemic. Despite standard vaccine development usually requiring an extensive process and taking several years to complete all clinical phases, there are currently 184 vaccine candidates in pre-clinical testing and another 88 vaccine candidates in clinical phases based on different vaccine platforms as of April 13, 2021. Moreover, three vaccine candidates have recently been granted an Emergency Use Authorization by the United States Food and Drug Administration (for Pfizer/BioNtech, Moderna mRNA vaccines, and Johnson and Johnson viral vector vaccine) and by the UK government (for University of Oxford/AstraZeneca viral vector vaccine). Here we aim to briefly address the current advances in reverse genetics system of SARS-CoV-2 and the use of this in development of SARS-CoV-2 vaccines. Additionally, we cover the essential points concerning the different platforms of current SARS-CoV-2 vaccine candidates and the advantages and drawbacks of these platforms. We also assess recommendations for controlling the COVID-19 pandemic and future pandemics using the benefits of genetic engineering technology to design effective vaccines against emerging and re-emerging viral diseases with zoonotic and/or pandemic potential.

The H9N2 avian influenza virus has been widely spread in poultry around the world. It is proved to the world that the avian influenza virus can directly infect human beings without any intermediate host adaptation in “1997 Hong Kong avian influenza case,” which shows that the avian influenza virus not only causes significant losses to the poultry industry but also affects human health. In this study, we aimed to address the problem of low protection of avian H9N2 subtype influenza virus vaccine against H9N2 wild-type virus. We have rescued the H9.4.2.5 branched avian influenza virus isolated in South China by reverse genetics technology. We have recombined these virus (rHA/NA-GD37 and rHA/NA-GD38) which contain hemagglutinin and neuraminidase genes from the H9N2 avian influenza virus (MN064850 or MN064851) and 6 internal genes from the avian influenza virus (KY785906). We compared the biological properties of the virus for example virus proliferation, virus elution, thermostability, and pH stability. Then, we evaluated the immune effects between rHA/NA-GD37 and GD37, which show that the recombinant avian influenza virus–inactivated vaccine can stimulate chickens to produce higher antibody titers and produce little inflammatory response after the challenge. It is noticeable that the recombinant virus-inactivated vaccine had better immune impact than the wild-type inactivated vaccine. Generally speaking, this study provides a new virus strain for the development of a H9N2 vaccine.

Avian infectious bronchitis virus (IBV) causes highly contagious respiratory reproductive and renal system diseases in chickens, and emergence of serotypic variants resulting from mutations in the viral S gene hampers vaccine management for IBV infection. In this study, to facilitate the molecular analysis of IBV pathogenesis and the development of a new-generation IBV vaccine, we established a reverse genetics system (RGS) for cloning the full-length cDNA of the IBV C-78E128 attenuated strain in a bacterial artificial chromosome (BAC). The BAC-cloned C-78E128 cDNA generated infectious viruses with biological properties of the parental C-78E128 strain with regard to an avirulent phenotype, tissue tropism and induction of virus neutralizing (VN) antibody in vivo. To assess the feasibility of genetic manipulation of the IBV genome using the BAC-based RGS, the S gene of the BAC-cloned C-78E128 cDNA was replaced with that of the IBV S95E4 virulent strain, which differs from the C-78E128 strain in serotype and tissue tropism, by bacteriophage lambda Red-mediated homologous recombination in Escherichia coli (E. coli). The resultant S gene recombinant virus was found to be avirulent and fully competent to induce a serotype-specific VN antibody against the S95 strain; however, the S gene recombinant virus did not fully recapitulate the tissue tropism of the S95E4 strain. These data imply that serotype-specific VN immunogenicity, but not tissue-tropism and pathogenicity, of IBV is determined by the viral S gene. The IBV BAC-based RGS that enables cloning and manipulation of the IBV virus genome entirely in E. coli provides a useful platform for the molecular analyses of IBV pathogenesis and the development of rationally designed IBV recombinant vaccines.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, tick-borne Bandavirus that causes lethal disease in humans. As there are no licensed vaccines and therapeutics for SFTSV, there is an urgent need to develop countermeasures against it. In this respect, a reverse genetics (RG) system is a powerful tool to help achieve this goal. Herein, we established a T7 RNA polymerase-driven RG system to rescue infectious clones of a Korean SFTSV human isolate entirely from complementary DNA (cDNA). To establish this system, we cloned cDNAs encoding the three antigenomic segments into transcription vectors, with each segment transcribed under the control of the T7 promoter and the hepatitis delta virus ribozyme (HdvRz) sequences. We also constructed two helper plasmids expressing the nucleoprotein (NP) or viral RNA-dependent RNA polymerase (RdRp) under the control of the T7 promoter and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). After co-transfection into BHK/T7-9 cells with three transcription and two helper plasmids, then passaging in Vero E6 or Huh-7 cells, we confirmed efficient rescue of the recombinant SFTSV. By evaluating the in vitro and in vivo virological properties of the parental and rescued SFTSVs, we show that the rescued virus exhibited biological properties similar to those of the parental virus. This system will be useful for identifying molecular viral determinants of SFTSV infection and pathogenesis and for facilitating the development of vaccine and antiviral approaches. [ABSTRACT FROM AUTHOR]

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, andRotavirus,whichhavegenomeswith10or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiTtagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae. [ABSTRACT FROM AUTHOR]

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, tick-borne Bandavirus that causes lethal disease in humans. As there are no licensed vaccines and therapeutics for SFTSV, there is an urgent need to develop countermeasures against it. In this respect, a reverse genetics (RG) system is a powerful tool to help achieve this goal. Herein, we established a T7 RNA polymerase-driven RG system to rescue infectious clones of a Korean SFTSV human isolate entirely from complementary DNA (cDNA). To establish this system, we cloned cDNAs encoding the three antigenomic segments into transcription vectors, with each segment transcribed under the control of the T7 promoter and the hepatitis delta virus ribozyme (HdvRz) sequences. We also constructed two helper plasmids expressing the nucleoprotein (NP) or viral RNA-dependent RNA polymerase (RdRp) under the control of the T7 promoter and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). After co-transfection into BHK/T7-9 cells with three transcription and two helper plasmids, then passaging in Vero E6 or Huh-7 cells, we confirmed efficient rescue of the recombinant SFTSV. By evaluating the in vitro and in vivo virological properties of the parental and rescued SFTSVs, we show that the rescued virus exhibited biological properties similar to those of the parental virus. This system will be useful for identifying molecular viral determinants of SFTSV infection and pathogenesis and for facilitating the development of vaccine and antiviral approaches.

The generation of genetically engineered recombinant viruses from modified DNA/RNA is commonly referred to as reverse genetics, which allows the introduction of desired mutations into the viral genome. Reverse genetics systems (RGSs) are powerful tools for studying fundamental viral processes, mechanisms of infection, pathogenesis and vaccine development. However, establishing RGS for coronaviruses (CoVs) and toroviruses (ToVs), which have the largest genomes among vertebrate RNA viruses, is laborious and hampered by technical constraints. Hence, little research has focused on animal CoVs and ToVs using RGSs, especially in large domestic animals such as pigs and cattle. In the last decade, however, studies of porcine CoVs and bovine ToVs using RGSs have been reported. In addition, the coronavirus disease-2019 pandemic has prompted the development of new and simple CoV RGSs, which will accelerate RGS-based research on animal CoVs and ToVs. In this review, we summarise the general characteristics of CoVs and ToVs, the RGSs available for CoVs and ToVs and the progress made in the last decade in RGS-based research on porcine CoVs and bovine ToVs. • Coronaviruses (CoVs) and toroviruses (ToVs) are key pathogens in domestic animals. • COVID-19 prompted the development of new CoV reverse genetics systems (RGSs). • Several RGSs are now available to create gene-engineered recombinant CoVs or ToVs. • Studies on CoVs and ToVs using RGSs have yielded novel and important findings. [ABSTRACT FROM AUTHOR]

We devised a reverse genetics system to generate an infectious human sapovirus (HuSaV) GII.3 virus. Capped/uncapped full-length RNAs derived from HuSaV GII.3 AK11 strain generated by in vitro transcription were used to transfect HuTu80 human duodenum carcinoma cells; infectious viruses were recovered from the capped RNA-transfected cells and passaged in the cells. Genome-wide analyses indicated no nucleotide sequence change in the virus genomes in the cell-culture supernatants recovered from the transfection or those from the subsequent infection. No virus growth was detected in the uncapped RNA-transfected cells, suggesting that the 5′-cap structure is essential for the virus’ generation and replication. Two types of virus particles were purified from the cell-culture supernatant. The complete particles were 39.2-nm-dia., at 1.350 g/cm3 density; the empty particles were 42.2-nm-dia. at 1.286 g/cm3. Two proteins (58-kDa p58 and 17-kDa p17) were detected from the purified particles; their molecular weight were similar to those of VP1 (~60-kDa) and VP2 (~16-kDa) of AK11 strain deduced from their amino acids (aa) sequences. Protein p58 interacted with HuSaV GII.3-VP1-specific antiserum, suggesting that p58 is HuSaV VP1. A total of 94 (57%) aa of p17 were identified by mass spectrometry; the sequences were identical to those of VP2, indicating that the p17 is the VP2 of AK11. Our new method produced infectious HuSaVs and demonstrated that VP2 is the minor protein of the virion, suggested to be involved in the HuSaV assembly.

Rabbit hepatitis E virus (HEV) is a novel zoonotic infectious agent. Although a cell culture system to grow the virus has been established, there is currently no reverse genetics system for generating the virus. In this study, capped genomic rabbit HEV RNAs generated by in vitro transcription were transfected into PLC/PRF/5 cells, and the recovered viruses were subsequently passaged in the cells. The cell culture supernatant was capable of infecting rabbits negative for anti‐HEV antibody by intravenous and oral inoculation, indicating that rabbit HEV generated by the reverse genetics system is infectious. Genome‐wide analyses indicated that no nucleotide sequence change occurred in the virus genomes that were recovered from the cell culture supernatant after transfection and passaged one time or in the virus genomes recovered from faecal specimens of the infected rabbits. Ribavirin, a broad‐spectrum anti‐viral inhibitor, efficiently abrogated virus replication ex vivo and transiently suppressed the virus growth in the virus‐infected rabbits, suggesting that this reagent is a candidate for therapeutic treatment. In addition, transmission of rabbit HEV to rabbits caused persistent infection, suggesting that the virus‐infected rabbit could be an animal model for virus‐induced hepatitis. The infectious rabbit HEV produced by a reverse genetics system would be useful to elucidate the mechanisms of HEV replication and the pathogenesis of viral hepatitis. [ABSTRACT FROM AUTHOR]

Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs. • Recombinant rotaviruses harboring HiBiT tag were generated. • The function of viral NSP1 was not affected by addition of HiBiT tag. • Replication of HiBiT reporter rotaviruses was monitored by NanoLuc activity. • HiBiT reporter rotavirus system is a powerful tool for antiviral screening. [ABSTRACT FROM AUTHOR]

Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine- glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1- modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin V. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo. This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy. [ABSTRACT FROM AUTHOR]

Negative-stranded/ambisense RNA viruses (NSVs) include not only dangerous pathogens of medical importance but also serious plant pathogens of agronomic importance. Tomato spotted wilt virus (TSWV) is one of the most important plant NSVs, infecting more than 1,000 plant species, and poses major threats to global food security. The segmented negative-stranded/ambisense RNA genomes of TSWV, however, have been a major obstacle to molecular genetic manipulation. In this study, we report the complete recovery of infectious TSWV entirely from complementary DNA (cDNA) clones. First, a replication- and transcription-competent minigenome replication system was established based on 35S-driven constructs of the S(-)-genomic (g) or S(+)-antigenomic (ag) RNA template, flanked by the 5′ hammerhead and 3′ ribozyme sequence of hepatitis delta virus, a nucleocapsid (N) protein gene and codon-optimized viral RNA-dependent RNA polymerase (RdRp) gene. Next, a movement-competent minigenome replication system was developed based on M(-)-gRNA, which was able to complement cell-to-cell and systemic movement of reconstituted ribonucleoprotein complexes (RNPs) of S RNA replicon. Finally, infectious TSWV and derivatives carrying eGFP reporters were rescued in planta via simultaneous expression of full-length cDNA constructs coding for S(+)-agRNA, M(-)-gRNA, and L(+)-agRNA in which the glycoprotein gene sequence of M(-)-gRNA was optimized. Viral rescue occurred with the addition of various RNAi suppressors including P19, HcPro, and γb, but TSWV NSs interfered with the rescue of genomic RNA. This reverse genetics system for TSWV now allows detailed molecular genetic analysis of all aspects of viral infection cycle and pathogenicity. [ABSTRACT FROM AUTHOR]

Group A rotavirus (RV) is a major cause of acute gastroenteritis in infants and young children worldwide. Recently, we established an entirely plasmid-based reverse genetics system for simian RV strain SA11. Although that system was robust enough to generate reassortant RVs, including human RV gene segments, and enabled better understanding of the biological differences between animal and human RV strains, a complete reverse genetics system for human RV strains is desirable. Here, we established a plasmid-based reverse genetics system for G4P[8] human RV strain Odelia. This technology was used to generate a panel of monoreassortant viruses between human and simian RV strains for all of the 11 gene segments demonstrating full compatibility between human and simian RV strains. Furthermore, we generated recombinant viruses lacking the C-terminal region of the viral nonstructural protein NSP1 and used it to define the biological function of the interaction between NSP1 and its target protein β-transducin repeat-containing protein (β-TrCP) during viral replication. While the NSP1 truncation mutant lacking the C-terminal 13 amino acids displayed lower β-TrCP degradation activity, it replicated as efficiently as the wildtype virus. In contrast, the truncation mutant lacking the C-terminal 166 amino acids of NSP1 replicated poorly, suggesting that the C-terminal region of NSP1 plays critical roles in viral replication. The system reported here will allow generation of engineered recombinant virus harboring desired mutations, increase our understanding of the molecular biology of human RV, and facilitate development of novel therapeutics and vaccines. [ABSTRACT FROM AUTHOR]

Highly pathogenic porcine reproductive and respiratory syndrome virus PRRSV (HP-PRRSV) was one of the most devastating diseases of the pig industry, among various strategies, vaccination was one of the most useful tools for PRRS control. Attenuated live vaccine was used worldwide, however, the genetic basis of HP-PRRSV virulence change during attenuation remain to be determined. Here, to identify virulence associated regions of HP-PRRSV during attenuation in vitro, six full-length infectious cDNA clones with interchanges of 5′UTR + ORF1a, ORF1b, and ORF2-7 + 3′UTR regions between HP-PRRSV strain HuN4-F5 and its attenuated vaccine strain HuN4-F112 were generated, and chimeric viruses were rescued. Piglets were inoculated with chimeric viruses and their parental viruses, and rectal temperature were recorded daily, and serum were collected for future experiments. Our results showed that ORF1a played an important role on virus replication, cytokine response and lung damage, the exchange of ORF1b and ORF2-7 in different backbone led to different exhibition on virus replication in vivo/vitro and cytokine response. Among 9 PRRSV attenuated series, consistent amino acid changes during PRRSV attenuation were found in NSP4, NSP9, GP2, E, GP3 and GP4. Our study provides a fundamental data for the investigation of PRRSV attenuation, the different results of the virulence change among different studies indicated that different mechanisms might be used during PRRSV virulence enhancement in vivo and attenuation in vitro.

Porcine epidemic diarrhea virus (PEDV) is the predominant cause of an acute, highly contagious enteric disease in neonatal piglets. There are currently no approved drugs against PEDV infection. Here, we report the development of a nanoluciferase (NLuc)-based high-throughput screening (HTS) platform to identify novel anti-PEDV compounds. We constructed a full-length cDNA clone for a cell-adapted PEDV strain YN150. Using reverse genetics, we replaced the open reading frame 3 (ORF3) in the viral genome with an NLuc gene to engineer a recombinant PEDV expressing NLuc (rPEDV-NLuc). rPEDV-NLuc produced similar plaque morphology and showed similar growth kinetics compared with the wild-type PEDV in vitro. Remarkably, the level of luciferase activity could be stably detected in rPEDV-NLuc-infected cells and exhibited a strong positive correlation with the viral titers. Given that NLuc expression represents a direct readout of PEDV replication, anti-PEDV compounds could be easily identified by quantifying the NLuc activity. Using this platform, we screened for the anti-PEDV compounds from a library of 803 natural products and identified 25 compounds that could significantly inhibit PEDV replication. Interestingly, 7 of the 25 identified compounds were natural antioxidants, including Betulonic acid, Ursonic acid, esculetin, lithocholic acid, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and grape seed extract. As expected, all of the antioxidants could potently reduce PEDV-induced oxygen species production, which, in turn, inhibit PEDV replication in a dose-dependent manner. Collectively, our findings provide a powerful platform for the rapid screening of promising therapeutic compounds against PEDV infection.

Abstract Background Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. Methods We established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3′-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. Results Targeted RNA recombination allowed for the modification of the 3′-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52. Conclusions Targeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general.

Bovine parainfluenza virus type 3 (BPIV3) is a key pathogen associated with bovine respiratory disease complex (BRDC). However, its specific pathogenesis mechanisms have not been fully elucidated. Reverse genetics provides a useful method for understanding the pathogenic mechanism of BPIV3. To ensure the functionality of the rescue platforms, we first constructed a minigenome (MG) system of BPIV3 utilizing a 5-plasmid system in this investigation. Then, a full-length infection clone of BPIV3 was obtained from the SX-2021 strain, and different methods were employed to identify the rescued virus. Additionally, we recovered a recombinant BPIV3 using the reverse genetics system that could express enhanced green fluorescence protein (eGFP). Through the growth curve assays, the replicate capability of rBPIV3-SX-EGFP was found to be similar to that of the parental virus. Subsequently, the rBPIV3-SX-EGFP was used to determine the antiviral activity of ribavirin. The results showed that ribavirin had an anti-BPIV3 effect in MDBK cells. In conclusion, the successful development of a reverse genetic system for the SX-2021 strain establishes a foundation for future studies on BPIV3, including investigations into its pathogenic mechanism, gene function, and antiviral screening properties., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Han, Lu, Zhang, Wei, Guo, Tong, Wang, Xiao, Liu and Yang.)

The hepatitis E virus (HEV) can cause hepatitis E in humans. Recently, the occurrence of HEV strains carrying insertions in their hypervariable genome region has been described in chronically infected patients. The insertions originate from human genes or from the HEV genome itself. Although their distinct functions are largely unknown, an involvement in efficient cell culture replication was shown for some strains. The HEV strain 47832c, originally isolated from a chronically infected transplant patient, carries a bipartite insertion composed of HEV genome duplications. Here, several mutants with deletions and substitutions of the insertion were generated and tested in cell culture. Complete deletion of the insertion abolished virus replication and even a single glycine to arginine substitution led to reduced cell culture growth. A mutant encoding a frameshift of the inserted sequence was not infectious, whereas a mutant carrying synonymous codons in this region replicated similar like the wild type. Substitution of the insertion with the S17 insertion from HEV strain Kernow C1-p6 did not result in viable virus, which might indicate strain- or cell type-specificity of the insertions. Generally, the translated amino acid sequence of the insertion, but not the RNA sequence, seems to be responsible for the observed effect.

A novel fowl adenovirus 4 (FAdV-4) has caused significant economic losses to the poultry industry in China since 2015. We established an easy-to-use reverse genetics system for modification of the whole right and partial left ends of the novel FAdV-4 genome, which worked through cell-free reactions of restriction digestion and Gibson assembly. Three recombinant viruses were constructed to test the assumption that species-specific viral genes of ORF4 and ORF19A might be responsible for the enhanced virulence: viral genes of ORF1, ORF1b and ORF2 were replaced with GFP to generate FAdV4-GFP, ORF4 was replaced with mCherry in FAdV4-GFP to generate FAdV4-GX4C, and ORF19A was deleted in FAdV4-GFP to generate FAdV4-CX19A. Deletion of ORF4 made FAdV4-GX4C form smaller plaques while ORF19A deletion made FAdV4-CX19A form larger ones on chicken LMH cells. Coding sequence (CDS) replacement with reporter mCherry demonstrated that ORF4 had a weak promoter. Survival analysis showed that FAdV4-CX19A-infected chicken embryos survived one more day than FAdV4-GFP- or FAdV4-GX4C-infected ones. The results illustrated that ORF4 and ORF19A were non-essential genes for FAdV-4 replication although deletion of either gene influenced virus growth. This work would help function study of genes on the right end of FAdV-4 genome and facilitate development of attenuated vaccines.

The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.

Ibaraki virus (IBAV) is a member of the epizootic hemorrhagic disease virus (EHDV) serogroup, which belongs to theOrbivirus genus of theReoviridae family. Although EHDV, including IBAV, represents an ongoing threat to livestock in the world, molecular mechanisms of EHDV replication and pathogenesis have been unclear. The reverse genetics (RG) system is one of the strong tools to understand molecular mechanisms of virus replication. Here, we developed a RG system for IBAV to identify the nonessential region of a minor structural protein, VP6, by generating VP6‐truncated IBAV. Moreover, several tags were inserted into the truncated region to produce VP6‐tagged IBAV. We demonstrated that all VP6‐tagged IBAV could replicate in BHK cells in the absence of any helper VP6 protein. Further, tagged‐VP6 proteins were first assembled into puncta in cells infected with VP6‐tagged IBAV. Our data suggests that, in order to initiate primary replication, IBAV VP6 is likely to accumulate in some parts of infected cells to assemble efficiently into the primary replication complex (subcore).

Highlights • We developed a DNA-launched infectious clone for ZIKV isolate ZG01 (Asian lineage). • Two substitutions deactivating E. coli promoter enable the cloning of ZG01 genome. • Four mutations (4m) efficiently adapted recombinant ZG01 in vitro and in vivo. • ZG01_4m clone was stable after repetitive transformation-purification cycles in bacteria. • 5′-3′UTR base-paring may exist and the nucleotides involved were critical for ZIKV. Abstract Zika virus (ZIKV) is an Aedes mosquitoes-transmitted flavivirus, and its infection may cause severe neurological diseases. A genetically stable infectious clone is essential for ZIKV research, however the toxicity and instability of the viral cDNA in bacteria potentially due to its bacterial promoter activity are major challenges. Here, we constructed a full-length cDNA clone for isolate ZG01 by introducing non-coding changes T1865C/A1868G to reduce the bacterial promoter activity. Wild-type and recombinant ZG01 were highly attenuated in Vero cells, thus we serially passaged wild-type ZG01 through neonatal mice and Vero cells to generate high-titer virus, from which four mutations (4m, C2178T/G2913A/T4991C/T10561C) were identified. Addition of 4m greatly enhanced the infectivity, as ZG01_4m released ZIKV of 107.0–107.5 plaque-forming unit (PFU)/ml in infected Vero and A549 cells. ZG01_4m resembled the infectivity of high-titer ZG01 in vitro and in vivo. Notably, ZG01_4m plasmid was genetically stable after multiple rounds of transformation-purification in bacteria. Using ZG01_4m, we identified a potential RNA-RNA interaction between 5′UTR and 3′UTR and demonstrated that the nucleotides involved were essential for ZIKV production. The genetically stable ZG01 cDNA clone provides a reliable tool for the study of this important virus, and the strategy used here is feasible for the development of reverse genetics systems for other ZIKV isolates and related flaviviruses. [ABSTRACT FROM AUTHOR]

Recently, a newly emerged avian flavivirus, duck Tembusu virus (TMUV), was identified as the causative agent of a serious duck viral disease in Asia. Its rapid spread and expanded host range have raised substantial concerns regarding its potential threat to non-avian hosts, including humans. In this study, we report an infectious cDNA clone for a clinical strain CQW1 isolated from Southwest China, which is representative of the disease outbreak in the Chinese mainland. We generated a full-length cDNA clone pACYC FL-TMUV, which is infectious, and this cDNA clone-derived recombinant TMUV (rTMUV) showed comparative growth kinetics in both BHK21 cells and DEF cells compared with parental TMUV (pTMUV). In addition, rTMUV also showed the same high virulence in 9-day-old duck embryos as that in pTMUV, suggesting that rTMUV possessed similar properties to the natural virus both in vitro and in vivo . Based on the cDNA-clone, we first generated a reporter TMUV (TMUV-RLuc) carrying a Renilla luciferase (RLuc) gene. The luciferase kinetics of TMUV-RLuc were determined both in BHK21 and DEF cells. It seems that TMUV-RLuc grew well in vitro ; however, the insertion of the RLuc gene attenuated viral replication in vitro . The higher viral titres of TMUV-RLuc were observed in BHK21 compared with that in DEF cells. The antiviral effects of exogenous-expressed duck RIG-I, MDA5, STING, MAVS, TBK1, IFNα and IFNγ were studied in vitro by using TMUV-RLuc. Our reverse genetics system will provide a multicomponent platform for the pathogenesis study of duck TMUV and the development of molecular countermeasures against duck TMUV infection. [ABSTRACT FROM AUTHOR]

Among the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus. BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation. IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that represents as a serious health threat to both domestic animals and humans. The viral protein NSs is the key virulence factor of RVFV, and has been proposed that NSs nuclear filament formation is critical for its virulence. However, the detailed mechanisms are currently unclear. Here, we generated a T7 RNA polymerase-driven RVFV reverse genetics system based on a strain imported into China (BJ01). Several NSs mutations (T1, T3 and T4) were introduced into the system for investigating the correlation between NSs filament formation and virulence in vivo. The NSs T1 mutant showed distinct NSs filament in the nuclei of infected cells, the T3 mutant diffusively localized in the cytoplasm and the T4 mutant showed fragmented nuclear filament formation. Infection of BALB/c mice with these NSs mutant viruses revealed that the in vivo virulence was severely compromised for all three NSs mutants, including the T1 mutant. This suggests that NSs filament formation is not directly correlated with RVFV virulence in vivo. Results from this study not only shed new light on the virulence mechanism of RVFV NSs but also provided tools for future in-depth investigations of RVFV pathogenesis and anti-RVFV drug screening.

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

Background: Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. Methods: We established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3'-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. Results: Targeted RNA recombination allowed for the modification of the 3'-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52. Conclusions: Targeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general. [ABSTRACT FROM AUTHOR]