Stable expression of shRNA for the control of recombinant adenovirus replication

Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

Classification of adenoviral conjunctivitis based on sequencing and phylogenetic analysis of hexon and fiber / 中华实验眼科杂志

Objective:To classify human adenovirus (HAdV) of adenoviral conjunctivitis by sequencing and phylogenetic analysis of hexon protein and fiber protein.Methods:A total of 256 conjunctival swabs were collected from the inferior conjunctival sac of 256 patients with viral conjunctivitis in Shanghai from January 2015 to August 2017.After DNA extraction, the whole length of hexon and fiber was amplified by PCR to perform gene sequencing and phylogenetic analysis.The study protocol was approved by the Ethics Committee of Shanghai General Hospital (No.2020-202).Written informed consent was obtained from each subject.Results:Among the samples, 89(34.76%) were positive for hexon gene amplification, including 1(1.12%) of HAdV-C1, 7(7.87%) of HAdV-C2, 20(22.47%) of HAdV-B3, 6(6.747%) of HAdV-E4, 23(25.84%) of HAdV-D8, 17(19.10%) of HAdV-D19, and 15(16.85%) of HAdV-D37.In phylogenetic analysis, sequenced hexon gene was clustered with the reference prototype correctly.In fiber phylogenetic analysis, 15 of HAdV-D19 and 1 of HAdV-D37 were cross clustered.Conclusions:The combined sequencing of hexon and fiber can provide abundant and effective biological information for the subtype and pathogenicity analysis of HAdV.

Identification of adenovirus epidemic strains and analysis of hexon gene characteristics in Anqing area from 2013 to 2015 / 中华微生物学和免疫学杂志

Objective To investigate the characteristics of human adenovirus ( HAdv) epidemic strains and the variations of hexon protein and amino acid residues in acute respiratory infections in Anqing. Methods A total of 596 throat swab samples of children with acute respiratory infections were collected from influenza surveillance sites between 2013 and 2015 and detected with real-time fluorescent PCR adeno-virus nucleic acid test kit. Hep cells were used to separate viruses from positive samples. PCR amplification of hexon gene and sequencing analysis were conducted. Homologous alignment and phylogenetic tree analysis were performed between the obtained sequences and those published in GenBank. Results HAdv-positive samples accounted for 11. 4% (68). Thirty-four viruses were successfully isolated, including nine HAdv-3 (26. 5% ), 12 HAdv-7 (35. 3% ), 12 HAdv-14 (35. 3% ) and one HAdv-55 (2. 9% ). The 9 strains of HAdv-3 had a close genetic relationship with KX384958, sharing a homology of 99. 8%-100% . Three muta-tions in main amino acid residues were found in them as compared with reference strains. The 12 strains of HAdv-7 were genetically related to KX897164 and KU361344 with a homology ranging from 99. 8% to 100% and had seven major amino acid residue mutations in comparison to reference strains. The 12 strains of HAdv-14 were highly similar to JF420883 with a homology of 99. 6%-99. 9% , and possessed three major variations in amino acid residues in comparison to reference strains. The HAdv-55 strain was closely related to KP279748 and KX289874, showing a homology of 100% in both nucleotide and amino acid sequences. HAdv-7 strains had the greatest variations, followed by HAdv-14 strains. Conclusion From 2013 to 2015, the epidemic adenovirus strains causing acute respiratory infections in Anqing area were mainly HAdv-3, HAdv-7 and HAdv-14 with a small number of HAdv-55. The hexon genes of HAdv-55 strains were stable and no variation occurred. However, HAdv-3, HAdv-7 and HAdv-14 strains all had some variations in nu-cleotides and amino acids. Amino acid variations in the antigenic determinants of HVR1, HVR2 and HVR7 regions were detected.

Construction and characterization of a hexon-chimeric human adenovirus type 3 vector expressing one major epitope of dengue virus type 1 / 中华微生物学和免疫学杂志

Objective To construct a recombinant human adenovirus type 3 ( HAd3 ) vector ex-pressing one major epitope of dengue virus type 1.Methods The gene encoding the envelope protein (304-314 aa) of dengue virus type 1 was inserted into the hypervariable region 1 ( HVR1 ) of HAd3 hexon by using overlap PCR.The recombinant gene was cloned into the shuttle plasmid, then linearized with AsisⅠrestriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas-mid for homologous recombination.The recombinant plasmid pBRAdΔE3GFP-DENV1 was transfected into AD293 cells to rescue recombinant adenovirus strains (rAdΔE3GFP-DENV1).ELISA and Western blot as-say were performed to evaluate the humoral responses induced in BALB/c mice after the immunization with rAdΔE3GFP-DENV1 strains.Results The recombinant adenovirus strains were successfully rescued. ELISA and Western blot assay showed that the antibodies in serum sample could recognize dengue virus type 1 strains.Conclusion The recombinant adenovirus strains expressing the epitope of dengue virus type 1 were successfully constructed.This study provided evidence for the development of multivalent vaccines against dengue virus.

Application of hexon gene sequencing of adenovirus in pathogen detection of epidemic keratoconjunctivitis / 中华实验眼科杂志

Background Epidemic keratoconjunctivitis is a common eye disease,and adenovirus is one of the common pathogens.The hexon protein,one main capsid protein of the virus,is an important target of antibody binding.Thus,sequencing the coding region of the hexon protein is an important way for adenovirus fast typing.Objective This study was to complete a molecular epidemiology survey of epidemic keratoconjunctivitis and investigate its association with adenovirus in Shanghai area by sequencing the coding region of hexon protein.Methods Two hundred and fourteen sacconjunctival swab specimens were collected from 214 patients with suspicious epidemic keratoconjunctivitis who visited Shanghai Eye Disease Prevention and Treatment Center and the clinical sites supervised by the Shanghai Prevention and Monitoring Office of Acute Hemerragic Conjunctivitis under the informed consent from January 2010 to December 2012.DNA was extracted from the specimens and then the 140 bp conserved sequence in hexon protein coding region was amplified by PCR initially to determine an adenovirus pathogen.Furtherly,956 bp conserved sequence of the hexon codind district was sequencied to clarify the serotype of adenovirus in the adenovirus-positive specimens.Results 50.93％ patients (109/214) were detected to be adenovirus-positive by generic PCR,in which AdV1 + was in 4 patiens,AdV2+ was in 33 patients,AdV3+ was in 15 patients,AdV4+ was in 12 patients,AdV8+ was in 19 patiens,AdV19+ was in 15 patients,AdV37+ was in 8 patients.The subgenus D adenoviruses,including AdV8+,AdV19+ and AdV37+ often resulted in corneal inflammation,pseudomembranous conjunctivitis and preauricular lymph nodes;while subgenus B adenovirus induced much frequent tract infection and less corneal response.Conclusions PCR-sequence of conserved region of hexon protein coding district is applicable for the detection and serotyping of adenovirus in epidemic keratoconjunctivitis.

Construction of a hexon-chimeric human adenovirus type 3 vector expressing two neutralizing epitopes of hepatitis B surface antigen preS1 and analysis of antigenicity of the chimeric epitopes / 中华微生物学和免疫学杂志

Objective To construct a hexon-chimeric human adenovirus type 3 ( HAd3 ) vector expressing two neutralizing epitopes of hepatitis B surface antigen preS 1 (HBsAg-preS1) and to analyze the antigenicity of the chimeric epitopes .Methods Two neutralizing epitopes of HBsAg-preS1 including KR359 and KR127 were inserted into hypervariable region 1 ( HVR1) and hypervariable region 2 ( HVR2) of HAd3 hexon .Chimeric hexon gene encoding the two epitopes was amplified by overlap PCR and then subcloned in -to shuttle plasmid pBR322-L/R containing the homologous recombination regions .The digested shuttle plas-mid containing chimeric hexon gene was co-transfected into Escherichia coli BJ5183 cells together with back-bone plasmid pBRAdΔE3GFP to construct pBRAdΔE3GFP-preS1 vector.Then pBRAdΔE3GFP-preS1 vector was digested with AsiSⅠand transfected into AD293 cells to construct recombinant virus (rAD3E-preS1). CsCl gradient centrifugation was used for purification .Glutathione S-transferase ( GST ) fusion protein KR359KR127 ( GST-KR359KR127 ) was expressed in Escherichia coli BL21 by using expression vector pGEX-4T3.Female BALB/c mice at age 6-8 weeks were intraperitoneally injected with 1010 virus particles or 80 μg GST fusion protein .Serum samples were collected to analyze the antigenicity of two epitopes by ELISA and Western blot .Results ELISA showed that KR 359 and KR127 were successfully exposed on viral sur-faces by using hexon-chimeric HAd3 vector .The induced polyclonal antibodies in serum samples could rec-ognize GST fusion protein and native HBsAg from patients infected with hepatitis B virus .Conclusion The antigen capsid-incorporation strategy could be used to display epitopes on viral surface .Enhanced antigen-specific responses could be achieved through inserting multiple foreign epitopes into hexon HVRs .This study provided evidence for further application of hexon -chimeric human adenovirus type 3 vector in the developmentof vaccine, especially for the development of multivalent hepatitis B vaccine .

Cloning and expression of hexon protein of human respiratory adenovirus of three serotypes and analysis of antigenicity of the recombinant proteins / 中华微生物学和免疫学杂志

Objective To clone and express the hexon protein of three prevalent human adenovi -rus strains causing respiratory disease and analyze the antigenic characteristics of the recombinant proteins . Methods The full length genes encoding hexon protein of human adenovirus serotype 3(HAdV3), serotype 4(HAdV4) and serotype 7(HAdV7) were cloned by PCR and sequenced , respectively.The alignment anal-ysis was performed by using hexon gene sequences from GenBank .The major antigenic regions of hexon pro-tein of the three serotypes were expressed in E.coli and purified.The antigenicity, immunogenicity and cross reactivity of the recombinant proteins were determined by ELISA and Western blot assay .Results The full length gene sequence encoding hexon protein of human adenovirus serotype 4 was firstly reported in China , which showed more than 99%homology in both nucleotide and amino acid sequences with the human adeno-virus type 4 NHRC3 strain.The partial hexon protein sequence of HAdV 3, HAdV4 and HAdV7 containing all of the 7 hyperviriable regions ( HVRs) were expressed in E.coli, respectively .The purified recombinant proteins could be recognized by antiserum of the three serotypes of adenovirus .The antiserum samples against the three recombinant proteins could cross-react with particles of the three serotypes of adenovirus . The possible type-and species-specific epitopes were predicted .Conclusion The major antigenic regions of hexon protein of the three serotypes were successfully expressed .The purified recombinant proteins contai-ning both intertypes and type-specific epitopes showed a strong immunogenicity .

Sequence analysis for Hexon genes of types 3,7 and 11 of adenoviruses identified in Beijing / 中华流行病学杂志

Objective The objective of this study was to develop a rapid,sensitive and specific method for identifying and typing for adenovirus from clinical specimens and to learn about the viruses identified in Beijing on the molecular bases.Methods Primers were designed using hexon gene of adenovirus as target.One primer pair was designed as universal primers for amplifying a 1278 bp gene fragment located at the hcxon gene of adenovirus types 3,7,11 and 21.Four primer pairs located within the region of this 1278 bp fragment were designed specifically for amplifying adenovirusea types 3,7,II and 21.Which were used for a multiplex nest-PCR in a single tube.The products from this multiplex nest-PCR were 502 bp(for type 3),311 bp(for type 7),880 bp(for type 11)and 237 bp(for type 21)，respectively.The type of the adenovirus being tested could be determined after agarose eleetrophoresis analysis of the PCR products．Sequencing was performed for part of the Hexon genes at the 5'end from types 3．7 and 11 strains isolated from clinical specimens and the sequences were compared with corresponding genes published in GenBank．Results PCR products with predicted sizes were visualized in the agarose gel for prototype strains of adenovirus types 3,7,11 and 21,but not for other respiratory viruses,indicating that the technique is specific for typing without cross reaction with other viruses.Out of 61 clinicaI specimens which had been proved to be adenovirus positive by tissue culture and/or immunofluerescence assay,37 were found as adenovirus type 3(37161,60.73％),17 8S adenovirus type 7 (17/61,27.9％),3 Was adenovirus type 11(3161,4.9％).1 Was positive for both type 3 and 7(1／61,1.6％),suggesting that the patient Was co-infected with type 3 and 7 adenoviruses．No adenovirns type 21 was detected.Out of the 61 positive specimens,three showed positive on both tissue culture and immunofluerescence but could not be identified under the methods we used,suggesting that these 3 strains (4.9％)were with the types other than types 3,7,11 and 21．Data from sequence analysis indicated that adeno~ruses types of 3.7 and 11 in this study shared high homology with corresponding types of the strains published in GenBank.Three of the type 3 adenovirus in this study shared highest homology with the adenovirus type 3 identified in Guangzhou,China in 2005.Three of the type 7 adenovirus shared highest homology with the adenovirus type 7 identified in Japan in 1998 and 3 of type 11 adenovirus shared highest homology with the adenovirus type 11 identified in Japan in 2004.Comparing with types 3 and 7.The type 11 in this study showed highest diversity with the corresponding type in GenBank,indicated by the dispersing of the varied amino acids within the region of HVRl and HVR, of the Hexon genes.Conclusion This multiplex nest-PCR method had the advantages of rapid,sensitive and specific and could be used for identilying types of adenoviruses in clinical specimens.Although adenovirus types 3.7 and 1l from Beijing strains shared high homology with the corresponding genes in GenBank,some variances were noticed,especially in type 11 strains.

Clone and Expression of Loop1 and Loop2 Gene of Hexonof Infectious Canine Hepatitis Virus / 中国生物工程杂志

The mainly antigenic sites for the adenovirus neutraliation are present on Loop1 and Loop2 of hexon.Majority research were focus in the human adenovirus.Little was known on infectious canine hepatitis virus (ICHV), which was also called canine adenovirus typeⅠ.Here,ICHV (the isolated strain) DNA was isolated and purified from the cultured MDCK cells.The Loop1 and Loop2 fragments were amplified by polymerase chain reaction(PCR) method,and then was connected by ligase T4.The target fragment was then connected with vector pET28a.The nucleotide sequence ecoding Loop1 and Loop2 was determined.The nucleotide sequence identity of Loop1 region between the isolated strain and CLL, RI261 and Toronto A26/61 strains is 100%, 100% and 83.8%, and the nucleotide sequence identity of Loop2 region between the isolated strain and CLL, RI261 and Toronto A26/61 strains is 88.1% , 88.1% and 99.3%, and amino acid identity is 93.6%, 93.6% and 98.6%.The recombinant Loop protein was expressed in E.coli and was approximately 36kDa in size,and then was purified. Then BALB/c mice were injected subcutaneously in the back and armpit with the recombinant Loop protein.The anti-ICHV antibody titers of immunized serum was tested by indirect ELISA and the titers were up to 1:320.Western blot demonstrated that immunized sera could specifically combine with ICHV. The research laid a foundation for creating new genetic engineering products of infectious canine hepatitis virus.