User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome

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.

Download Full-text

Fiber1, but not fiber2, is the essential fiber gene for fowl adenovirus 4 (FAdV-4)

Journal of General Virology ◽

10.1099/jgv.0.001559 ◽

2021 ◽

Author(s):

Xiaohui Zou ◽

Yejing Rong ◽

Xiaojuan Guo ◽

Wenzhe Hou ◽

Bingyu Yan ◽

...

Keyword(s):

Reverse Genetics ◽

Growth Curves ◽

Dependent Manner ◽

Helper Plasmid ◽

Fowl Adenovirus ◽

Virus Adsorption ◽

One Step ◽

Step Growth ◽

Lmh Cells

Fibre is the viral protein that mediates the attachment and infection of adenovirus to the host cell. Fowl adenovirus 4 (FAdV-4) possesses two different fibre trimers on each penton capsomere, and roles of the separate fibres remain elusive. Here, we attempted to investigate the function of FAdV-4 fibres by using reverse genetics approaches. Adenoviral plasmids carrying fiber1 or fiber2 mutant genes were constructed and used to transfect chicken LMH cells. Fiber1-mutated recombinant virus could not be rescued. Such defective phenotype was complemented when a fiber1-bearing helper plasmid was included for co-transfection. The infection of fiber-intact FAdV-4 (FAdV4-GFP) to LMH cells could be blocked with purified fiber1 knob protein in a dose-dependent manner, while purifed fiber2 knob had no such function. On the contrary, fiber2-mutated FAdV-4, FAdV4XF2-GFP, was successfully rescued. The results of one-step growth curves showed that proliferative capacity of FAdV4XF2-GFP was 10 times lower than that of the control FAdV4-GFP. FAdV4XF2-GFP also caused fewer deaths of infected chicken embryos than FAdV4-GFP did, which resulted from poorer virus replication in vivo. These data illustrated that fiber1 mediated virus adsorption and was essential for FAdV-4, while fiber2 was dispensable although it significantly contributed to the virulence.

Download Full-text

Reverse Genetic Generation of Recombinant Zaire Ebola Viruses Containing Disrupted IRF-3 Inhibitory Domains Results in Attenuated Virus Growth In Vitro and Higher Levels of IRF-3 Activation without Inhibiting Viral Transcription or Replication

Journal of Virology ◽

10.1128/jvi.00044-06 ◽

2006 ◽

Vol 80 (13) ◽

pp. 6430-6440 ◽

Cited By ~ 72

Author(s):

Amy L. Hartman ◽

Jason E. Dover ◽

Jonathan S. Towner ◽

Stuart T. Nichol

Keyword(s):

Reverse Genetics ◽

Ebola Virus ◽

Viral Transcription ◽

Type I ◽

Virus Growth ◽

Recombinant Viruses ◽

Inhibitory Function ◽

C Terminus ◽

Inhibitory Domain

ABSTRACT The VP35 protein of Zaire Ebola virus is an essential component of the viral RNA polymerase complex and also functions to antagonize the cellular type I interferon (IFN) response by blocking activation of the transcription factor IRF-3. We previously mapped the IRF-3 inhibitory domain within the C terminus of VP35. In the present study, we show that mutations that disrupt the IRF-3 inhibitory function of VP35 do not disrupt viral transcription/replication, suggesting that the two functions of VP35 are separable. Second, using reverse genetics, we successfully recovered recombinant Ebola viruses containing mutations within the IRF-3 inhibitory domain. Importantly, we show that the recombinant viruses were attenuated for growth in cell culture and that they activated IRF-3 and IRF-3-inducible gene expression at levels higher than that for Ebola virus containing wild-type VP35. In the context of Ebola virus pathogenesis, VP35 may function to limit early IFN-β production and other antiviral signals generated from cells at the primary site of infection, thereby slowing down the host's ability to curb virus replication and induce adaptive immunity.

Download Full-text

Fowl Adenovirus (FAdV) Recombination with Intertypic Crossovers in Genomes of FAdV-D and FAdV-E, Displaying Hybrid Serological Phenotypes

Viruses ◽

10.3390/v11121094 ◽

2019 ◽

Vol 11 (12) ◽

pp. 1094 ◽

Cited By ~ 6

Author(s):

Anna Schachner ◽

Gabriel Gonzalez ◽

Lukas Endler ◽

Kimihito Ito ◽

Michael Hess

Keyword(s):

In Vivo Studies ◽

Parental Origin ◽

Fowl Adenovirus ◽

Inclusion Body Hepatitis ◽

New Type ◽

Cross Neutralization ◽

Species Specific ◽

The Right ◽

Important Disease

After analyzing 27 new genomes from fowl adenovirus (FAdV) field isolates and so-far unsequenced prototypes, we report the first evidence for recombination in FAdVs. Recombination was confined to species FAdV-D and FAdV-E, accommodating the largest number of, and the intraspecies-wise most differentiated, types. The majority of detected events occurred in FAdV-E, involving segments with parental origin of all constitutive types. Together with the diversity of breakpoints, this suggests widespread recombination in this species. With possible constraints through species-specific genes and diversification patterns, the recombinogenic potential of FAdVs attains particular interest for inclusion body hepatitis (IBH), an important disease in chickens, caused by types from the recombination-prone species. Autonomously evolving, recombinant segments were associated with major sites under positive selection, among them the capsid protein hexon and fiber genes, the right-terminal ORFs 19, 25, and the ORF20/20A family. The observed mosaicism in genes indicated as targets of adaptive pressures points toward an immune evasion strategy. Intertypic hexon/fiber-recombinants demonstrated hybrid neutralization profiles, retrospectively explaining reported controversies on reference strains B3-A, T8-A, and X11-A. Furthermore, cross-neutralization supported sequence-based evidence for interdomain recombination in fiber and contributed to a tentatively new type. Overall, our findings challenge the purported uniformity of types responsible for IBH, urging more complete identification strategies for FAdVs. Finally, important consequences arise for in vivo studies investigating cross-protection against IBH.

Download Full-text

Requirement of Cellular Protein CCT7 for the Replication of Fowl Adenovirus Serotype 4 (FAdV-4) in Leghorn Male Hepatocellular Cells Via Interaction with the Viral Hexon Protein

Viruses ◽

10.3390/v11020107 ◽

2019 ◽

Vol 11 (2) ◽

pp. 107 ◽

Cited By ~ 2

Author(s):

Junfeng Gao ◽

Mingliang Zhao ◽

Xueyan Duan ◽

Yongqiang Wang ◽

Hong Cao ◽

...

Keyword(s):

Molecular Mechanisms ◽

Ectopic Expression ◽

Cellular Protein ◽

Economic Losses ◽

Adenovirus Serotype ◽

Hydropericardium Syndrome ◽

Fowl Adenovirus ◽

Infected Cells ◽

Lmh Cells ◽

Fowl Adenovirus Serotype 4

Fowl adenovirus serotype 4 (FAdV-4) causes hepatitis-hydropericardium syndrome (HHS), leading to severe economic losses in the poultry industry. Although the pathogenesis of FAdV-4 infection has caused much attention, the underlying molecular mechanisms remain poorly understood. Here, we identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the FAdV-4 capsid protein hexon. We found that ectopic expression of CCT7 in leghorn male hepatocellular (LMH) cells enhanced hexon expression in pRK5-flag-hexon transfected cells. On the contrary, knockdown of cellular CCT7 by RNAi markedly reduced hexon expression in FAdV-4-infected cells and suppressed viral replication. These data suggest that CCT7 is required for FAdV-4 replication and may serve as a potential target for controlling FAdV-4 infection.

Download Full-text

FAST Proteins: Development and Use of Reverse Genetics Systems for Reoviridae Viruses

Annual Review of Virology ◽

10.1146/annurev-virology-091919-070225 ◽

2021 ◽

Vol 8 (1) ◽

pp. 515-536

Author(s):

Yuta Kanai ◽

Takeshi Kobayashi

Keyword(s):

Reverse Genetics ◽

Rna Viruses ◽

Syncytium Formation ◽

Successful Development ◽

Recombinant Viruses ◽

Novel Technique ◽

Fast Proteins ◽

Viral Genes ◽

Severe Gastroenteritis ◽

The Individual

Reverse genetics systems for viruses, the technology used to generate gene-engineered recombinant viruses from artificial genes, enable the study of the roles of the individual nucleotides and amino acids of viral genes and proteins in infectivity, replication, and pathogenicity. The successful development of a reverse genetics system for poliovirus in 1981 accelerated the establishment of protocols for other RNA viruses important for human health. Despite multiple efforts, rotavirus (RV), which causes severe gastroenteritis in infants, was refractory to reverse genetics analysis, and the first complete reverse genetics system for RV was established in 2017. This novel technique involves use of the fusogenic protein FAST (fusion-associated small transmembrane) derived from the bat-borne Nelson Bay orthoreovirus, which induces massive syncytium formation. Co-transfection of a FAST-expressing plasmid with complementary DNAs encoding RV genes enables rescue of recombinant RV. This review focuses on methodological insights into the reverse genetics system for RV and discusses applications and potential improvements to this system.

Download Full-text

Interdependence of Hemagglutinin Glycosylation and Neuraminidase as Regulators of Influenza Virus Growth: a Study by Reverse Genetics

Journal of Virology ◽

10.1128/jvi.74.14.6316-6323.2000 ◽

2000 ◽

Vol 74 (14) ◽

pp. 6316-6323 ◽

Cited By ~ 171

Author(s):

Ralf Wagner ◽

Thorsten Wolff ◽

Astrid Herwig ◽

Stephan Pleschka ◽

Hans-Dieter Klenk

Keyword(s):

Influenza Virus ◽

Receptor Binding ◽

Reverse Genetics ◽

Mdck Cells ◽

Influenza Virus Infection ◽

Simian Virus 40 ◽

Host Cells ◽

Receptor Binding Site ◽

Virus Growth ◽

Recombinant Viruses

ABSTRACT The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377–8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.

Download Full-text

Transcriptome Analysis Reveals the Potential Role of Long Noncoding RNAs in Regulating Fowl Adenovirus Serotype 4-Induced Apoptosis in Leghorn Male Hepatocellular Cells

Viruses ◽

10.3390/v13081623 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1623

Author(s):

Bo Wen ◽

Xueping Wang ◽

Lulu Yang ◽

Ting Wang ◽

Xiaolan Hou ◽

...

Keyword(s):

Signaling Pathway ◽

Noncoding Rnas ◽

Long Noncoding Rnas ◽

Host Cells ◽

Economic Losses ◽

Adenovirus Serotype ◽

Fowl Adenovirus ◽

Induced Apoptosis ◽

Lmh Cells ◽

Fowl Adenovirus Serotype 4

Hepatitis-hydropericardium syndrome (HHS) is caused by fowl adenovirus serotype 4 (FAdV-4) and has resulted in considerable economic losses to the poultry industry globally. FAdV-4 elicits apoptosis in host cells. Long noncoding RNAs (lncRNAs) have emerged as important regulatory RNAs with profound effects on various biological processes, including apoptosis. However, it remains unknown whether lncRNAs participate in FAdV-4-induced apoptosis. In this study, RNA sequencing was applied to determine the transcription of cellular lncRNA in leghorn male hepatocellular (LMH) cells infected with FAdV-4. Cellular RNA transcription analysis demonstrated that FAdV-4 infection elicited 1798 significantly differentially expressed (DE) lncRNAs in infected LMH cells at 24 h post-infection (hpi) compared to mock control infection. In addition, 2873 DE mRNAs were also found. Target prediction and analyses revealed that 775 DE lncRNAs whose 671 target mRNAs were among the DE mRNAs were involved in several signaling pathways, including the AMPK signaling pathway, p53 signaling pathway and insulin signaling pathway. From these 775 DE lncRNAs, we identified 71 DE lncRNAs related to apoptosis based on their target gene functions. Subsequently, lncRNA 54128 was selected from the 71 identified DE lncRNAs, and its role in FAdV-4-induced apoptosis was verified. LncRNA 54128 interference significantly suppressed the rate of apoptosis, which was accompanied by reduced BMP4 transcription levels. To the best of our knowledge, this is the first study to analyze host lncRNA transcription during FAdV-4 infection. Our findings provide a better understanding of host responses to FAdV-4 infection and provide new directions for understanding the potential association between lncRNAs and FAdV-4 pathogenesis.

Download Full-text

Metabolomic Profiling Reveals New Insight of Fowl Adenovirus Serotype 4 Infection

Frontiers in Microbiology ◽

10.3389/fmicb.2021.784745 ◽

2022 ◽

Vol 12 ◽

Author(s):

Haiying Ma ◽

Yujuan Niu

Keyword(s):

Host Cell ◽

High Performance ◽

Tricarboxylic Acid Cycle ◽

Amino Sugar ◽

Host Cells ◽

Economic Losses ◽

Adenovirus Serotype ◽

Fowl Adenovirus ◽

Lmh Cells ◽

Fowl Adenovirus Serotype 4

Highly pathogenic fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium syndrome (HPS), which is characterized by pericardial effusion and hepatitis, and is one of the foremost causes of economic losses to the poultry industry over the last 30 years. However, the metabolic changes in cells in response to FAdV-4 infection remain unclear. In order to understand the metabolic interactions between the host cell and virus, we utilized ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry to analyze the metabolic profiles with hepatocellular carcinoma cell line (LMH) infected with FAdV-4. The results showed that FAdV-4 could restore metabolic networks in LMH cells and tricarboxylic acid cycle, glycolysis, and metabolism of purines, pyrimidines, alanine, aspartate, glutamate, and amino sugar and nucleotide sugar moieties. Moreover, FAdV-4 production was significantly reduced in LMH cells cultured in glucose or glutamine-deficient medium. These observations highlighted the importance of host cell metabolism in virus replication. Therefore, similarities and disparities in FAdV-4-regulation of the metabolism of host cells could help improve targeted drug and reduce infection.

Download Full-text

Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

Journal of Virology ◽

10.1128/jvi.01561-21 ◽

2021 ◽

Author(s):

Makoto Ujike∗ ◽

Yuka Etoh ◽

Naoya Urushiyama ◽

Fumihiro Taguchi ◽

Hideki Asanuma ◽

...

Keyword(s):

Viral Replication ◽

Cdna Clone ◽

Recombinant Virus ◽

Reverse Genetics ◽

Full Length ◽

Egfp Expression ◽

Egfp Gene ◽

Virus Growth ◽

Recombinant Viruses ◽

Gene Retention

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.

Download Full-text

Recent Progress in Torovirus Molecular Biology

Viruses ◽

10.3390/v13030435 ◽

2021 ◽

Vol 13 (3) ◽

pp. 435

Author(s):

Makoto Ujike ◽

Fumihiro Taguchi

Keyword(s):

Molecular Biology ◽

Recent Progress ◽

Reverse Genetics ◽

Cultured Cells ◽

Host Adaptation ◽

Economic Losses ◽

New Family ◽

Enteric Diseases ◽

Long Time ◽

Medical Importance

Torovirus (ToV) has recently been classified into the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. ToVs are associated with enteric diseases in animals and humans. In contrast to CoVs, which are recognised as pathogens of veterinary and medical importance, little attention has been paid to ToVs because their infections are usually asymptomatic or not severe; for a long time, only one equine ToV could be propagated in cultured cells. However, bovine ToVs, which predominantly cause diarrhoea in calves, have been detected worldwide, leading to economic losses. Porcine ToVs have also spread globally; although they have not caused serious economic losses, coinfections with other pathogens can exacerbate their symptoms. In addition, frequent inter- or intra-recombination among ToVs can increase pathogenesis or unpredicted host adaptation. These findings have highlighted the importance of ToVs as pathogens and the need for basic ToV research. Here, we review recent progress in the study of ToV molecular biology including reverse genetics, focusing on the similarities and differences between ToVs and CoVs.

Download Full-text