Molecular aspects of interferon induction by adenoviruses

The capacity of adenovirus to induce interferon in the infected cells was studied. The examined adenovirus strains of early types were grouped in 2 groups according to their capacity to induce interferon and to the sensitivity of the infecting and interferon-inducing activity to UV-irradiation. A common property of adenoviruses, potent interferon inducers, is their high sensitivity to UV-irradiation.     

Sensitivity of subgroup F adenoviruses to interferon

Summary The subgroup F adenoviruses were tested for their ability to induce interferon in semi-permissive human cells (Chang conjunctiva) and non-permissive cells (HEF, human embryo lung fibroblasts). These cells did not produce interferon spontaneously or in response to infection by either of these adenoviruses. It was concluded that interferon induction in response to subgroup F adenovirus infection is not a likely explanation of limited virus growth in culture. Adenovirus 40 and Ad 41, unlike Ad 2, were found to be sensitive to human lymphoblastoid interferon in Chang conjunctival cells. The addition of Ad 2 to cells before pretreatment with interferon resulted in the partial and complete abrogation of Ad 40 and Ad 41 interferon sensitivity, respectively. The suppressive effect of Ad 2 on the inhibitory action of interferon and the modulatory function of Ad 2 in mixed infection with either Ad 40 or Ad 41 suggests the inadequate functioning of a subgroup F adenovirus gene product or products involved in suppression of the interferon-induced antiviral state.     

Growth analysis of adenoviruses isolated from pigeons in chicken cells and serological characterization of the isolates.

The susceptibility of chicken embryo liver (CEL) and chicken kidney (CK) cells to eight adenoviruses isolated from different pigeons were investigated. Isolation and propagation was most successful in CEL cells. The cytopathic effect, i.e. cell rounding and detachment of the cells was typical for adenoviruses. Titres of up to 10(6.6) plaque-forming units/ml could be reached on CEL cells. Transferring CEL-grown virus onto CK cells also resulted in a cytopathic effect but with much lower titres, whereas the isolation of pigeon adenoviruses on these cells was not successful. Antibodies against fowl adenovirus reference strains (FAV1-12) were used to serotype the isolates in neutralisation tests. Six were identified as FAV serotypes 2,5,6,10 and 12. Two isolates could not be typed. An antiserum produced in chickens against one of these untypable strains was able to neutralize both. The neutralization indexes of these strains were very similar, indicating that they are probably the same serotype. A cross neutralization test confirmed that this serotype is different from known fowl adenoviruses.     

Some characteristics of three porcine adenoviruses

Summary Three strains of cloned porcine adenoviruses (25RCl, 6618Cl and A47C1) were shown to be serologically distinct in cross neutralization tests with specific antisera prepared in rabbits. They were not neutralized by reference antisera against the known human adenoviruses. Neutralizing antibodies to each of the three serotypes were demonstrated in samples of pig serum. Guinea pig, human 0, rat, monkey and mouse red cells were haemagglutinated by strain 25RCl, but fowl, ox, pig, rabbit and sheep cells were not haemagglutinated. Strains 6618Cl and A47Cl did not haemagglutinate these cells. Specific rabbit antiserum and normal pig sera contained HI antibodies to strain 25RCl adenovirus. Each of the three strains was resistant to ether and pH 4.0 buffer and growth in PK cells was inhibited by FUDR. In infected PK cells stained with haemotoxylin and eosin, typical basophilic central nuclear masses were produced by each strain of porcine adenovirus. In PK cells infected with strain 6618Cl multiple eosinophilic intranuclear inclusions in some cells were a prominent feature of the CPE. The provisional designation of strains 25RCl, 6618Cl and A47Cl as porcine adenovirus types 1, 2, and 3, respectively, is proposed.     

Development and evaluation of monoclonal antibody-based immune electron microscopy for diagnosis of adenovirus types 40 and 41

Immune electron microscopy based on monoclonal antibodies was developed and evaluated for diagnosis of adenovirus type 40 and adenovirus type 41 directly from clinical specimens. One adenovirus type 40 monoclonal (5-8) and one adenovirus type 41 monoclonal (5-15) were found to react to high titre with homotypic but not heterotypic antigen. These monoclonals were tested on a coded batch of 20 stools which contained adenovirus type 40 or adenovirus type 41. The results showed that 5/6 adenovirus type 40 and 13/14 adenovirus type 41 strains were correctly serotyped but one strain of each type failed to react with either serum. A wide variation in the numbers of virions bound to positive grids was observed. A further coded batch of 27 specimens, a mixture of subgenus F (i.e. type 40 or 41) or non-subgenus F adenoviruses, was then tested. There was complete serotype concordance with reference results for 16/19 subgenus F strains and all 8 non-subgenus F adenoviruses gave negative results. However, three subgenus F adenoviruses also gave negative results. In conclusion, monoclonal antibody-based immune electron microscopy accurately distinguished adenovirus type 40 from adenovirus type 41 and both viruses from other adenovirus serotypes in clinical specimens and will therefore be useful in the diagnosis of adenovirus gastroenteritis, but some strains may be missed, presumably because of antigenic variation in surface epitopes.     

Comparative sequence analysis of the inverted terminal repetition in the genomes of animal and avian adenoviruses

The nucleotide sequences at the inverted terminal repetitions from two animal adenoviruses, infectious canine hepatitis virus and equine adenovirus, and from one avian adenovirus, CELO, were analyzed. DNAs from infectious canine hepatitis virus and equine adenovirus contain a homologous region which is 23 nucleotides long from the terminus. The first 17 nucleotides of this region are identical to the ones in human adenovirus type 2 DNA. The striking homologous sequence of 14 nucleotides, conserved in the inverted terminal repetitions of several human adenovirus strains and in simian adenovirus type 7, is only partially conserved in the two animal and one avian adenoviruses reported here.     

Antigenically intermediate human adenovirus strain associated with conjunctivitis.

Two strains of an antigenically intermediate human adenovirus were isolated from eye specimens from cases of conjunctivitis. The viruses were doubly neutralized by antisera to adenoviruses 15 and 29 but possessed the hemagglutinins of adenovirus 8 or 9.     

Impaired humoral responses to subgenus D adenovirusenovirus infections in HIV-positive patients

HIV-positive patients are at increased risk of developing adenovirus infection, particularly of the gastrointestinal tract and with unusual subgenus D strains. To investigate humoral immunity to these strains of adenoviruses, the humoral immune response was examined in longitudinal samples of serum against isolates collected from a prospective study of HIV-positive patients with subgenus D adenovirus infection. Of 10 HIV-positive patients developing adenovirus infection, 3 had chronic infection (8->27 months) with one serotype, 3 had chronic infection (>/=10 months) with changing serotypes and 4 had acute and self-limiting adenovirus infection (<1 month). Fifty-one sera were tested, and samples collected before adenovirus infection were available in 8 patients. Neutralising assays were performed against the patient's own isolate (adenoviruses 9, 17, 19, 19/23, 19/37, 23, 26, 23/26, 43 and 46) and common circulating strains of adenovirus 1-5. Indirect immunofluorescence tests were carried out against the autologous isolate and complement-fixation tests were undertaken using a standard assay. Immunofluorescence test antibodies were detected (titre >/=160) in all patients, and present to high titre (>/=320) in 8/10 patients. Complement-fixing antibodies were not detected in significant titre. Of particular note, there was no significant neutralising antibody response to the patient's own isolate after acute infection. Neutralising antibody to adenovirus 3 (titre 20) was transiently detected in two patients. In the remaining patients neutralising antibody directed against adenoviruses 1-5 was not detected. Persistent carriage of subgenus D adenoviruses in HIV-positive patients is probably the result of failure of cell-mediated immune responses to clear primary infection. Nevertheless, there is marked impairment of B cell responses resulting in poor neutralising and complement-fixing antibody production even though immunofluorescence test determined antibodies are produced in high titre. These possibly reflect impairment of effective B cell priming mechanisms within the germinal centres of lymph nodes, or the polyclonal activation of B cells driven by HIV infection.     

Mouse adenovirus: growth of plaque-purified FL virus in cell lines and characterization of viral DNA

The FL strain of mouse adenovirus (AdFL) was plaque-purified and grown in mouse 3T6 cells. Purified virions resemble human adenoviruses by electron microscopy, and viral DNA shares physical properties with human adenovirus DNA. AdFL-DNA is a linear duplex with a molecular weight of 20 × 10⁶, shows evidence of protein covalently linked at each end, and forms single-strand circles after denaturation, indicating inverted repeat sequences at or near the ends of the molecule. However, less than 10% nucleotide sequence homology was found between AdFL-DNA and the DNAs of Ad2, 7, or 12. Also, the C + G content of AdFL-DNA (44%) is lower than that of human adenovirus DNAs. While there was reactivity between extracts of AdFL-infected cells and antiserum against human adenovirus, sera against the T antigens of human adenoviruses of groups A, B, and C did not react with AdFL-infected cell lysates. Six restriction endonucleases were used to cleave AdFL-DNA and to construct cleavage maps of the DNA. These maps differed from those of Ad2 or Ad5.     

Rapid Shell Vial Culture Technique for Detection of Enteroviruses and Adenoviruses in Fecal Specimens: Comparison with Conventional Virus Isolation Method

Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.     

Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.     

Synergism between aphidicolin and adenoviruses in the induction of breaks at fragile sites on human chromosomes.

Infection of human embryonic kidney cells with adenoviruses results in the induction of gaps and breaks in cell chromosomes. With adenovirus type 12, cytogenetic damage is known to occur primarily at fragile sites on chromosomes 1 and 17. We have mapped adenovirus type 5-induced breaks and have observed that, although they occur on all chromosomes, they are localized primarily on bands where fragile sites have been mapped. The susceptibility of fragile sites to adenovirus led us to investigate their expression upon combined treatments with virus and aphidicolin, a frequently used inducer of fragile sites. Under these experimental conditions, the frequency of damage at all sites was found to increase significantly, and the magnitude of such increases indicated a synergistic effect between drug and virus.     

人轮状病毒G2和G3型主要中和抗原VP7基因在重组腺病毒中的表达

目的 研究我国G2和G3型轮状病毒主要中和抗原VP7基因在重组腺病毒中的表达。方法 在前期成功表达Gl型VP7的基础上，选用我国G2和G3型主要流行株97S43和97S48 VP7基因，用非复制型腺病毒载体对上述基因进行表达。结果 获得了表达我国G2型和G3型人轮状病毒流行株VP7基因的非复制型重组腺病毒rvAdG2VP7和rvAdG3VP7，应用PCR及Southem blot技术证实在重组腺病毒中整合有轮状病毒G2型VP7和G3型VP7基因，RT-PCR证明重组腺病毒在感染的293细胞内均能有效地转录插入基因，Western blot检测到轮状病毒VP7基因的表达。结论 这一工作为进一步进行动物实验，发展多价轮状病毒疫苗打下了基础。     

Persistence of vesicular stomatitis virus in interferon-treated cell cultures.

A significant difference was observed in the functional stability of the vesicular stomatitis virus (VSV) genome in mouse L cells and the RK-13 line of rabbit kidney cells pretreated with homologous interferon. By utilizing the ability of vaccinia to rescue VSV from the inhibitory effects of interferon in these cell lines, it was demonstrated that there was a loss of a rescuable form of VSV genome in RK-13 cells pretreated with interferon; superinfection with vaccinia 24 hr after VSV infection did not result in a significant increase in VSV yield. In contrast, significant rescue of VSV occurred in interferon-treated L cells superinfected with vaccinia as late as 72 hr after VSV infection.The present study also provides evidence that in interferon-treated RK-13 cells doubly infected with VSV and vaccinia there was a correlation of the rescuability of the VSV genome and its ability to direct RNA synthesis.     

Serological classification of avian adenoviruses

Summary One hundred and sixty-seven agents recovered from domestic hens using chick kidney cell cultures were identified as adenoviruses and cross neutralization tests showed that they belong to 7 serological types. Cross neutralization tests between our agents and the 8 serotypes of avian adenovirus recovered in Japan indicated that 6 of our prototype strains were closely related to the Japanese agents (which included strains similar to CELO and GAL) whereas one strain (764) was serologically distinct. Electron microscopic examination, physico-chemical tests and gel precipitation tests confirm that strain 764 is an adenovirus and that it has an antigen which is common to the avian adenoviruses but does not cross-react with that of the mammalian agents.We conclude that strains of avian adenoviruses other than CELO and GAL are more widespread than is commonly assumed and that a numbering system for the various serotypes would be useful.     

Investigation of the immunological properties of the bovine viral diarrhea virus protein NS3 expressed by an adenovirus vector in mice

Summary.  Two replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-γ in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2. Received November 4, 1998 Accepted February 18, 1999     

Epidemic keratoconjunctivitis. Laboratory study of patients infected with adenovirus type 8

The authors tried to detect, using different methods, the causal agent of the disease in eight patients with epidemic keratoconjunctivitis. From the conjunctival sacs of one ambulatory patient and six of seven patients hospitalized on account of other eye diseases they isolated 11 strains of adenovirus type 8 on cell cultures from human embryonic lungs. Using direct and indirect ELISA, they detected adenovirus antigen in four of seven examined smears from the conjunctival sac without previous cultivation. In smears from the nasopharynx they did not detect adenoviruses by either of the two methods. Antibodies were assessed by micromodifications of the virus neutralizing test, by the complement fixation reaction and ELISA. A significant rise of antibodies against adenovirus type 8 and adenovirus hexone resp. was found in different six of eight examined pairs of sera. The combination of these three serological methods made it possible to confirm adenovirus infection in all eight patients.     

Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses

Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attractive candidate as an oncolytic virus for tumor therapy. Previous studies have suggested that VSV selectively infects tumor cells due to defects in their antiviral responses making them more susceptible to VSV infection than normal cells. We tested this hypothesis in the prostate tumor system by comparing LNCaP and PC-3 prostate tumor cells to benign human prostatic epithelial cells from patient prostatectomy specimens. We compared the cell killing ability of a recombinant virus containing a wild-type (wt) M protein (rwt) and an isogenic M protein mutant virus (rM51R-M) that induces interferon (IFN) in infected cells and should display a greater selectivity for tumor cells. Our results showed that in single-cycle infection experiments, LNCaP cells were sensitive to killing by both wt and mutant viruses, while PC-3 cells were highly resistant to VSV-induced cell killing. LNCaP and benign prostate cells were similarly susceptible to both viruses, indicating that normal prostate cells are not inherently resistant to killing by VSV. In each of the cell lines, the rM51R-M virus induced similar levels of apoptosis to rwt virus, showing that the M protein does not play a significant role in apoptosis induction by VSV in these cells. In multiple-cycle infection experiments, LNCaP cells were more sensitive than benign prostatic epithelial cells to virus-induced cell killing by rM51R-M virus, but not rwt virus. Both viruses were equally effective at reducing LNCaP tumor volume in vivo following intratumoral and intravenous inoculation in nude mice, while PC-3 tumors were resistant to VSV treatment. None of the mice treated with rM51R-M virus died as a result of virus infection, while 50-71% of mice treated with rwt virus succumbed to virus infection. Similarly, when inoculated by the more sensitive intranasal route, the rM51R-M virus was less pathogenic than the rwt virus from which it was derived. These results indicate that M protein mutant viruses are superior candidates as oncolytic viruses for therapies of prostate tumors, but future strategies for use of VSV will require testing individual tumors for their susceptibility to virus infection.     

Pseudotypes of vesicular stomatitis virus-bearing envelope antigens of certain HIV-1 strains permissively infect human syncytiotrophoblasts cultured in vitro: implications for in vivo infection of syncytiotrophoblasts by cell-free HIV-1

Intrauterine infection of the fetus is clearly an important mode of vertical transmission of human immunodeficiency virus type 1 (HIV-1). The syncytiotrophoblast layer of the human placenta must be traversed by HIV-1 in order to reach underlying cells and fetal capillaries. Although HIV-1 has been detected in the syncytiotrophoblast layer in situ, there is conflicting evidence regarding infection of syncytiotrophoblast cells with cell-free virus. The phenotypic mixing between HIV-1 and vesicular stomatitis virus (VSV) has been exploited to assay the susceptibility of human term syncytiotrophoblast cells to penetration by various strains of HIV-1. VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) pseudotypes were found to enter syncytiotrophoblast cells. In contrast, VSV pseudotyped with envelope glycoproteins of RF, MN, or Ada-M strains of HIV-1 did not infect syncytiotrophoblasts. Plating efficiency of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) was 10-fold lower on syncytiotrophoblasts than on T-cells and macrophages, respectively. Incubation of VSV(HIV-1(IIIB)) and VSV(HIV-1(Ba-L)) viruses with appropriate HIV-1 neutralizing sera before infection strongly inhibited entry of pseudotyped VSV into syncytiotrophoblast cells. These findings demonstrated that infection of syncytiotrophoblasts with VSV(HIV-1) pseudotypes was mediated by Env from IIIB and Ba-L strains of HIV-1. Monoclonal antibodies (MAb) to CD4, CXCR4, CCR5, and CCR3 were tested for their ability to block VSV(HIV-1) infection of syncytiotrophoblast cells. Neither the anti-CD4 nor the anti-CXCR4, anti-CCR5, and anti-CCR3 MAb had any inhibitory effect on infection of syncytiotrophoblast cells with VSV(HIV-1) pseudotypes. Results from this study suggest that cell-free HIV-1 can enter syncytiotrophoblasts and the susceptibility of these cells to penetration by the virus is strain dependent. Pseudotype infection merely demonstrates that the first steps in HIV-1 replication are possible in syncytiotrophoblast cells.