Subjective experience of parent-child relationship in adolescents with congenital heart disease: A qualitative study.

PURPOSE: To explore the parent-child relationship through the subjective experience of adolescents with congenital heart disease (CHD). DESIGN AND METHODS: A descriptive phenomenology approach was adopted. Twelve adolescents aged from 12 to 18 years with CHD were recruited from the pediatric cardiology clinics at two medical centers in Taiwan. Data were collected through in-depth interviews. Data were analyzed using Colaizzi's phenomenological analysis method, and results were reported in accordance with the Consolidated Criteria for Reporting Qualitative Research guidelines. RESULTS: The experiences of the adolescents with CHD revealed five themes: 1. the enhancement of self-worth through parents' love; 2. the importance of parental support in desperate situations; 3. the development of a sense of security through mutual understanding; 4. growth under parental expectations; and 5. parental overcontrol disguised as love. CONCLUSIONS: The parent-child relationship encompasses both positive and negative experiences. Adolescents prioritize their relationship with parents over that with peers. PRACTICE IMPLICATIONS: Nurses caring for adolescents with CHD can improve care by recognizing the influence of parental love, support in challenges, mutual understanding, parental expectations, and potential negative consequences of overcontrol. This insight guides effective guidance for adolescents, enhancing parent-child interactions and overall well-being.

"Warrior who transcends adversity": A qualitative examination of resilience among mothers caring for adolescents with congenital heart disease.

PURPOSE: Parenting resilience is essential for the well-being and development of children with chronic illnesses. Given the importance of parenting resilience in this context, this study explored the nature of parenting resilience among mothers caring for adolescents with congenital heart disease (CHD). DESIGN AND METHODS: We adopted Husserl's phenomenological approach and conducted semistructured in-depth interviews. In addition, we conducted purposive sampling at the pediatric cardiology outpatient departments of 2 medical centers in Taiwan to recruit 11 mothers of adolescents with CHD; all of these adolescents had received open-heart surgery. Furthermore, we analyzed data by using Colaizzi's approach, and we adhered to the COnsolidated criteria for REporting Qualitative research checklist. RESULTS: Mothers caring for adolescents with CHD was a dynamic process involving problem solving. The 11 mothers in this study employed resilience to remain strong, provided a sense of normalcy for their children, and approached challenges calmly and bravely. We uncovered three major themes among these mothers: "providing support for the child, "facing challenges with equanimity," and "overcoming adversity through positivity and gratitude." CONCLUSIONS: The present results provide a deeper understanding of how mothers caring for adolescents with CHD can cultivate resilience. PRACTICE IMPLICATIONS: The study's findings can inform transitional programs for adolescents with CHD and their families, with nursing professionals supporting mothers' resilience.

How grit mediates depressive symptoms with life and job satisfaction in full-time employed adults with congenital heart disease.

BACKGROUND: Technological advancements have extended the lifespan of individuals with congenital heart disease (CHD), but physical and mental health issues can affect their life and job satisfaction. AIMS: This study examined whether grit can protect full-time employed adults with CHD from depression by exploring its mediating role between depressive symptoms and life or job satisfaction. METHODS: A cross-sectional design involved 181 adults with CHD assessed using the Patient Health Questionnaire, Grit-10, Satisfaction with Life Scale, and Basic Psychological Need Satisfaction at Work Scale. Data analysis was conducted with SPSS and Smart PLS software for partial least squares structural equation modeling, adhering to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. RESULTS: Indicated that depressive symptoms were negatively correlated with grit, life satisfaction, and job satisfaction, while grit was positively correlated with both satisfaction measures. Grit partially mediated the relationship between depressive symptoms and life/job satisfaction, accounting for 30.70 % and 29.11 % of the variance, respectively. CONCLUSIONS: Grit significantly mitigates the negative impact of depressive symptoms on life and job satisfaction in full-time employed adults with CHD. Nurses should identify signs of depression in adults with CHD and evaluate their grit levels. Interventions to increase grit and reduce depressive symptoms could enhance life and job satisfaction.

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

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.

Expectant management of prolonged hemolysis following complete transcatheter coil closure of a patent ductus arteriosus after previous pulmonary artery banding: a case report.

BACKGROUND: Transcatheter coil occlusion has been the treatment of choice for closure of small patent ductus arteriosus (PDA). In spite of its safety, complications such as hemolysis still occasionally occur. And the hemolysis almost always occurs following partial transcatheter closure of PDA; hence, it occurs extremely rarely following complete transcatheter closure of PDA without residual ductal flow. CASE PRESENTATION: Here, we describe a male newborn who developed prolonged hemolysis following complete transcatheter coil closure of his PDA after previous palliative pulmonary artery banding. Over the following days, we corrected his refractory anemia by repeated blood transfusion with packed red blood cells and frequently monitored his hemoglobin, serum total bilirubin, and serum lactate dehydrogenase. We speculated that the high-velocity pulmonary blood flow jet coming into contact with the extruded part of the coil led to red blood cell mechanical injury, thereby resulting in the hemolysis. We adopted expectant management in expectation of the endothelialization of the coil with a resultant reduction in the hemolysis. The hemolysis, as expected, was reduced gradually until it spontaneously resolved 81 days after coil implantation. CONCLUSIONS: This case reminds us that hemolysis can still potentially occur following complete transcatheter coil closure of PDA. It also highlights the importance of preventing coils from extruding into the pulmonary artery in patients after previous pulmonary artery banding.

Unilateral renal artery stenosis presented with hyponatremic-hypertensive syndrome - case report and literature review.

BACKGROUND: Renal artery stenosis is one of the secondary causes of pediatric hypertension. Cases with critical unilateral renal artery stenosis manifesting with the hyponatremic hypertensive syndrome are rare and a comprehensive description of this disorder in the pediatric population is lacking in the literature. CASE PRESENTATION: We describe a 4-year-old boy who presented with severe hypertension, profound hyponatremia, hypokalemia, nephrotic range proteinuria, and polyuria. Distinctly, the diagnosis of hyponatremic hypertensive syndrome secondary to unilateral renal artery stenosis was confirmed in light of laboratory and radiographic findings of severe natriuresis, elevated renin, and unilateral small kidney. Two weeks following nephrectomy, there was resolution of hyponatremia, hypokalemia, nephrotic range proteinuria and hypertension. CONCLUSIONS: Findings of hyponatremia, hypokalemia, hypertension, polyuria, and unilateral renal hypoplasia can be attributed to a unifying pathology of unilateral renal artery stenosis.

Efficacy of a Novel Palivizumab Prophylaxis Protocol for Respiratory Syncytial Virus Infection in Congenital Heart Disease: A Multicenter Study.

OBJECTIVE: To analyze the efficacy of a novel palivizumab protocol for hemodynamically significant congenital heart disease (hsCHD) in subtropical areas without clear respiratory syncytial virus seasonality. STUDY DESIGN: Since July 2013, the National Health Insurance program has provided reimbursement for palivizumab prophylaxis with a novel monthly protocol in selected patients with hsCHD under 1 year of age. We performed a multicenter study to assess the trend of respiratory syncytial virus hospitalizations in patients with hsCHD from 2010 to 2016 during the prepalivizumab, transition, and postpalivizumab periods, and compared treatment and propensity-matched control groups. RESULTS: A total of 747 patients were enrolled in the study group and 809 in the control group. The male:female was 836:720. Cyanotic CHD was observed in 42.9% of patients. The mean age at diagnosis of CHD was 32.9 days. After 516 685 patient-days of follow-up and a mean of 3.9 doses of palivizumab in the treatment group, respiratory syncytial virus hospitalization rates decreased by 53% and 49% before and after match compared with the control group (P = .009 and .029, respectively). Hospitalization days and intensive care unit admission rate also decreased similarly in the treatment group. The efficacy of this protocol was more prominent in patients with cyanotic hsCHD. The annual respiratory syncytial virus-associated hospitalization rates also decreased significantly from the prepalivizumab to the palivizumab period (from 4.8% to 2.0%; P = .038). CONCLUSION: Palivizumab prophylaxis through the novel monthly protocol for patients with hsCHD is effective in reducing respiratory syncytial virus-related hospitalizations.

Initial validation of a healthcare needs scale for young people with congenital heart disease.

AIM: To validate the initial psychometric properties of a Healthcare Needs Scale for Youth with Congenital Heart Disease. BACKGROUND: As the number of patients with congenital heart disease surviving to adulthood increases, the transitional healthcare needs for adolescents and young adults with congenital heart disease require investigation. However, few tools comprehensively identify the healthcare needs of youth with congenital heart disease. DESIGN: A cross-sectional study was employed to examine the psychometric properties of the Healthcare Needs Scale for Youth with Congenital Heart Disease. METHODS: The sample consisted of 500 patients with congenital heart disease, aged 15-24 years, from paediatric cardiology departments and covered the period from March-August 2015. The patients completed the 25-item Healthcare Needs Scale for Youth with Congenital Heart Disease, the questionnaire on health needs for adolescents and the WHO Quality of Life-BREF. Reliability and construct, concurrent, predictive and known-group validity were examined. RESULTS: The Healthcare Needs Scale for Youth with Congenital Heart Disease includes three dimensions, namely health management, health policy and individual and interpersonal relationships, which consist of 25 items. It demonstrated excellent internal consistency and sound construct, concurrent, predictive and known-group validity. CONCLUSION: The Healthcare Needs Scale for Youth with Congenital Heart Disease is a psychometrically robust measure of the healthcare needs of youth with congenital heart disease. It has the potential to provide nurses with a means to assess and identify the concerns of youth with congenital heart disease and to help them achieve a successful transition to adult care.

Communicating printed headings to the ear.

Two experiments compared three methods of translating printed headings into an auditory format. In both experiments, college students listened to a text with instructions to stop the recording whenever they heard a heading and type the hierarchical level and exact wording of the heading. Listeners were poor at identifying headings and their levels if the headings were not distinguished from the rest of the text. However, listeners were very good at identifying headings if any method of signalling was used to distinguish headings and communicate their hierarchical level. The methods included: (1) tones preceding headings, (2) changes of speaker to indicate headings or (3) verbal labels preceding headings. Although all three signalling methods improved identification of a heading's hierarchical level, the labelling method was the most effective means of communicating hierarchical level. Thus, the study identifies a simple method of effectively communicating headings in spoken text. Practitioner Summary: The study attempted to identify effective ways of communicating heading information in spoken text. College students listened to texts in order to detect headings and record their wording and hierarchical level. Performance was excellent when headings were preceded by verbal phrases that signalled the upcoming headings and their levels.

Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus.

An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen-specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) -based vaccines expressing the MERS-CoV spike (S) protein. Intragastric administration of either Ad5-S or Ad41-S induced antigen-specific IgG and neutralizing antibody in serum; however, antigen-specific T-cell responses were not detected. In contrast, after a single intramuscular dose of Ad5-S or Ad41-S, functional antigen-specific T-cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd-based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5-S- or Ad41-S-based vaccines represents an appealing strategy for the control of MERS-CoV infection and transmission.

Influenza A virus utilizes a suboptimal Kozak sequence to fine-tune virus replication and host response.

The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome play important roles in controlling viral RNA transcription, replication and genome packaging. In this report, we present, for the first time to our knowledge, a full view of the segment-specific NCRs of all influenza A viruses by bioinformatics analysis. Our systematic functional analysis revealed that the eight segment-specific NCRs identified could differentially regulate viral RNA synthesis and protein expression at both transcription and translation levels. Interestingly, a highly conserved suboptimal nucleotide at -3 position of the Kozak sequence, which downregulated protein expression at the translation level, was only present in the segment-specific NCR of PB1. By reverse genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence at the -3 position in PB1 resulted in a significant multiple-cycle replication reduction that was independent of PB1-F2 expression. Our detailed dynamic analysis of virus infection revealed that the mutant virus displays slightly altered dynamics from the wild-type virus on both viral RNA synthesis and protein production. Furthermore, we demonstrated that the level of PB1 expression is involved in regulating type I IFN production. Together, these data reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host antiviral response through regulating PB1 protein expression.

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody.

The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

Clinical implications of dextrocardia based on four visceroatrial situs studies.

BACKGROUND: Previous studies on congenital heart diseases (CHD) associated with dextrocardia were based on selective patient databases and did not reflect the full spectrum of dextrocardia in the general population. Additionally, these studies had complex classification and presentation. Nor did these studies elaborate on the distribution of the associated CHD's complexity, the various segmental connections, and associated CHD among the four visceroatrial situs. METHODS: We retrospectively reviewed the medical records of 211 children with primary dextrocardia. We used a segmental approach to diagnose CHD. We then analyzed and compared the distribution of the above-mentioned issues among the four visceroatrial situs. RESULTS: Dextrocardia occurred most commonly with situs inversus (52.6%), followed by situs solitus (28.4%), asplenia (17.1%), and polysplenia (1.9%). Although some patients had a structurally normal heart (22.7%) or they were associated with simple CHD (17.5%), most patients had complex CHD (59.7%) consisting of a single ventricle (34.6%) or conotruncal anomaly (25.1%) (double-outlet right ventricle [7.6%], corrected transposition of the great arteries [6.2%], complete transposition of the great arteries [5.7%], tetralogy of Fallot [4.7%], etc.). Situs inversus or polysplenia had a higher prevalence of a structurally normal heart or associated with simple CHD, two patent atrioventricular (AV) valves connections, and biventricular AV connections. Situs solitus or asplenia had a higher prevalence of associated complex CHD, common AV valve connection, univentricular AV connection, pulmonary outflow tract obstruction, and anomalous pulmonary venous drainage. CONCLUSION: Our study finds that situs inversus is the most common visceroatrial situs in dextrocardia. Although some patients had a structurally normal heart or were associated with simple CHD, most patients have associated complex CHD consisting of a single ventricle or conotruncal anomaly. Dextrocardia is associated with a higher incidence of complex CHD in situs solitus and asplenia groups than in situs inversus and polysplenia groups.

In-hospital care of children with COVID-19.

Children have been reported to be less affected and to have milder severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection than adults during the coronavirus disease 2019 (COVID-19) pandemic. However, children, and particularly those with underlying disorders, are still likely to develop critical illnesses. In the case of SARS-CoV-2 infection, most previous studies have focused on adult patients. To aid in the knowledge of in-hospital care of children with COVID-19, this study presents an expert review of the literature, including the management of respiratory distress or failure, extracorporeal membrane oxygenation (ECMO), multisystem inflammatory syndrome in children (MIS-C), hemodynamic and other organ support, pharmaceutical therapies (anti-viral drugs, anti-inflammatory or antithrombotic therapies) and management of cardiopulmonary arrest.

Analysis of Fowl Adenovirus 4 Transcriptome by De Novo ORF Prediction Based on Corrected Nanopore Full-Length cDNA Sequencing Data.

The transcriptome of fowl adenovirus has not been comprehensively revealed. Here, we attempted to analyze the fowl adenovirus 4 (FAdV-4) transcriptome by deep sequencing. RNA samples were extracted from chicken LMH cells at 12, 18 or 26 h post-FAdV-4 infection, and subjected to Illumina strand-specific RNA-seq or nanopore full-length PCR-cDNA sequencing. After removing the reads of host cells, the data of FAdV-4 nanopore full-length cDNAs (transcripts) were corrected with reads from the Illumina RNA-seq, mapped to the viral genome and then used to predict viral open reading frames (ORFs). Other than 42 known ORFs, 39 novel ORFs were annotated to the FAdV-4 genome. Different from human adenovirus 5, one FAdV-4 ORF was often encoded by several transcripts, and more FAdV-4 ORFs were located on two exons. With these data, 18 major transcription start sites and 15 major transcription termination sites were defined, implying 18 viral promoters and 15 polyadenylation signals. The temporal cascade of viral gene transcription was observed in FAdV-4-infected cells, with six promoters possessing considerable activity in the early phase. Unexpectedly, four promoters, instead of one major late promoter, were engaged in the transcription of the viral genus-common genes on the forward strand. The clarification of the FAdV-4 transcriptome laid a solid foundation for the study of viral gene function, virulence and virus evolution, and it would help construct FAdV-4 as a gene transfer vehicle. The strategy of de novo ORF prediction could be used to parse the transcriptome of other novel adenoviruses.

Health-promoting lifestyle and life satisfaction in full-time employed adults with congenital heart disease: grit as a mediator.

AIMS: Enhancing life satisfaction is vital for adults with congenital heart disease (ACHD). Life satisfaction potentially correlates with a health-promoting lifestyle and grit. However, the mediating role of grit-a trait marked by perseverance and long-term goal-oriented passion-between a health-promoting lifestyle and life satisfaction in ACHD remains unverified. This study aimed to examine the relationships between health-promoting lifestyle, grit, and life satisfaction in full-time employed ACHD and to confirm the mediating role of grit. METHODS AND RESULTS: A total of 181 full-time employed ACHD aged 20-59 years with patient-reported New York Heart Association class I-IV were recruited from two medical centers in northern Taiwan. Participants completed questionnaires, including the short version of the Chinese Health-Promoting Lifestyle Profile scale, Grit-10 scale, and Satisfaction with Life Scale between February and December 2022. Data analysis employed the Hayes PROCESS macro. Health-promoting lifestyle, grit, and life satisfaction were discovered to be positively correlated. Overall grit score was a partial mediator between a health-promoting lifestyle and life satisfaction, accounting for 20% of the total variation. Of the grit domains, perseverance served as a partial mediator between a health-promoting lifestyle and life satisfaction, accounting for 32% of the total variation, whereas passion had a nonsignificant mediation effect. CONCLUSION: A health-promoting lifestyle boosts ACHD individuals' life satisfaction through grit, particularly perseverance. Integrating grit into such a lifestyle could benefit from comprehending support systems and influential factors aiding grit development for enhanced life satisfaction.

Construction of an infectious clone of human adenovirus type 41.

Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K-expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.

Modified tenodesis method for treatment of mallet fractures.

INTRODUCTION: Mallet fractures are avulsions of the terminal extensor tendon from the base of the distal phalangeal bone with a bony fragment. Surgical treatment, which provides accurate anatomical reduction and rigid fixation, is recommended for mallet fractures with involvement of more than one third of the base of the distal phalangeal bone. Various surgical methods have been reported, but there is still no standard treatment modality. The purpose of this investigation was to assess the results of our modified tenodesis method for mallet fractures. MATERIALS AND METHODS: Using our method, the dorsal fracture fragment was reduced and fixed to the main part of the distal phalangeal bone by 2 stitches of "figure-of-eight" 4-0 Prolene sutures. We reviewed 12 consecutive patients with 13 mallet fractures treated with our modified tenodesis method between January 2009 and March 2012. This retrospective study was composed of 7 male and 5 female patients, with a mean age of 35.7 years (range, 25-56 years). All patients underwent surgical treatment and sequent 3-week finger splinting. Patient follow-up lasted 3 to 6 months, with a mean period of 5.2 months. RESULTS: The modified tenodesis method allowed accurate anatomical reconstruction of the injured extensor mechanism. Grading by Crawford criteria showed that the outcome was "excellent" in 8 of 13 digits and "good" in 5 of 13 digits. The follow-up hand x-rays also revealed congruent joint surfaces of distal interphalangeal joints with no evidence of joint space narrowing. No wound complications were encountered, and all the patients returned to normal activities without any disability. CONCLUSIONS: The modified tenodesis method is simple and effective to provide accurate anatomical reduction and fixation for treatment of type I mallet fractures. No device penetration of the small bony fragment or pinning through the distal interphalangeal joint is required, and the surgical complication rate is low. We recommend this treatment modality for all but chronic cases.

No Genus-Specific Gene Is Essential for the Replication of Fowl Adenovirus 4 in Chicken LMH Cells.

Essential genus-specific genes have not been discovered for fowl adenovirus (FAdV), which hampers the development of FAdV-based vectors and attenuated FAdV vaccines. Reverse genetics approaches were employed to construct FAdV-4 mutants carrying deletions or frameshift mutations covering the whole left and right ends of the viral genome. The results of virus rescue and plaque forming experiments illustrated that all the 22 designated ORFs (open reading frames) were dispensable for the replication of FAdV-4 in chicken hepatoma Leghorn male hepatoma (LMH) cells and primary embryo hepatocytes. RNA-seq data demonstrated that ORF28 and ORF29 were not protein-encoding genes, and suggested a promoter (RP1) and an intron in these regions, respectively. The promoter activity of RP1 was further confirmed by reporter gene expression experiments. GAM-1-deleted FAdV-4 formed small plaques, while deletion of GAM-1 together with ORF22 resulted in even smaller ones in LMH cells. Simultaneous deletion of ORF28, ORF29, and GAM-1 led to growth defect of FAdV-4. These facts implied that genus-specific genes contributed to and synergistically affected viral replication, although no single one was essential. Notably, replication of FAdV-4 mutants could be different in vitro and in vivo. XGAM1-CX19A, a GAM-1-deleted FAdV-4 that replicated efficiently in LMH cells, did not kill chicken embryos because virus propagation took place at a very low level in vivo. This work laid a solid foundation for FAdV-4 vector construction as well as vaccine development, and would benefit viral gene function study. IMPORTANCE Identification of viral essential genes is important for adenoviral vector construction. Deletion of nonessential genes enlarges cloning capacity, deletion of essential genes makes a replication-defective vector, and expression of essential genes in trans generates a virus packaging cell line. However, the genus-specific essential genes in FAdV have not been identified. We constructed adenoviral plasmid carrying deletions covering all 22 genus-specific ORFs of FAdV-4, and found that all virus mutants could be rescued and amplified in chicken LMH cells except those that had defects in key promoter activity. These genus-specific genes affected virus growth, but no single one was indispensable. Dysfunction of several genus-specific genes at the same time could make FAdV-4 vectors replication-defective. In addition, the growth of FAdV-4 mutants could be different in LMH cells and in chicken embryos, suggesting the possibility of constructing attenuated FAdV-4 vaccines.

Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.