Efficient Marek's disease virus (MDV) and herpesvirus of turkey infection of the QM7 cell line that does not contain latent MDV genome.

Marek's disease virus (MDV; also known as Gallid herpesvirus 2, MDV-1) causes oncogenic disease in chickens producing clinical signs that include lymphomas, visceral tumours, nerve lesions, and immunosuppression. MDV vaccines are widely used and mostly produced using primary cells: chicken embryo fibroblast cells, duck embryo fibroblast cells, chicken embryo kidney cells or chicken kidney cells. An immortalized cell line that can be used to manufacture the virus has long been desired. In this report, we demonstrate that QM7 cells were susceptible to infection with either MDV or herpesvirus of turkey (HVT; also known as Meleagrid herpesvirus 1, MDV-3). Polymerase chain reaction analysis with primers amplifying selected MDV genes revealed that QM7 cells did not contain these sequences. However, MDV genes were detected in QT35 cells, which have been reported to harbour latent MDV virus. Transfection of naked MDV DNA initiated efficient infection of QM7 cells. In addition, QM7 cell lysate, clarified supernatant, and QM7 cell pellet infected with MDV were negative for reverse transcriptase activity, indicating absence of endogenous retrovirus. QM7 cells were also found to be free of other avian pathogens in a chick embryo inoculation test. In vivo studies of MDV growing in QM7 cells showed the virus retained its pathogenicity and virulence. In ovo experiments demonstrated that both HVT and MDV propagated in QM7 cells did not interfere with hatchability of injected eggs, and viruses could be re-isolated from hatched chicks. The results suggest that QM7 could be a good host cell line for growing both MDV and HVT.

Characterization of an avirulent FCV strain with a broad serum cross-neutralization profile and protection against challenge of a highly virulent vs feline calicivirus.

Highly virulent, systemic strains of Feline calicivirus (vs FCV) have been described in recent years. These vs FCV isolates cause severe edema, cutaneous ulcers, lameness and other upper respiratory and oral clinical signs typically associated with FCV infection in cats. Vs FCV isolates can cause high mortality even in cats vaccinated with currently available commercial vaccines. This study reports identification and characterization of an avirulent FCV strain (FCV 21). This strain offers a broader serum cross-neutralization profile in comparison with the commonly used vaccine strain (FCV F9), as tested with two separate viral panels of FCV isolates. The first viral panel consists of 45 FCV strains isolated around 1993. The second viral panel consists of 26 FCV strains with most isolated around 2003. The potential of using this strain as a vaccine, in a 3-way (FCV+FHV+FPV) or 4-way (FCV+FHV+FPV+FCp) format, was tested by using a highly virulent vs FCV strain (FCV-33585) as a challenge virus. The mortality induced by this vs FCV in unvaccinated control cats was 78% (7 out of 9 cats). The mortality decreased to 44% (4 out of 9 cats) in cats vaccinated with a 4-way vaccine containing FCV F9. However, when this novel FCV vaccine strain (FCV 21) was used, either in combination with FCV F9 or by itself, the mortality decreased to 0% (0 out of 10 cats). The 3-way vaccine (FCV+FHV+FPV) that contained both FCV 21 and FCV F9 also had mortality of 0% (0 out of 10 cats). The clinical scores, as calculated taking into consideration the frequency and severity of various clinical signs, correlated with mortality data. The results suggested this FCV vaccine has the potential to be broadly protective against newly emergent FCV isolates, including complete protection against challenge with a highly virulent vs FCV 33585.

Vaccination with cancer- and HIV infection-associated endogenous retrotransposable elements is safe and immunogenic.

The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.

Cloning and apoptosis-inducing activities of canine and feline TRAIL.

The apoptosis process is crucial to various biological processes including embryo development and organism homeostasis. Inducing apoptosis of cancer cells has become a very attractive field for cancer therapy in the recent years. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also called Apo2L, TNFSF10, CD253, or TL2) is a member of tumor necrosis factor family. Preclinical studies showed that human TRAIL induced apoptosis of various tumor cell lines, whereas nontransformed normal cell lines were not affected. We have cloned both canine and feline TRAIL full-length genes by using Rapid Amplification of cDNA Ends-PCR technology. Truncated soluble versions of the canine and feline TRAIL genes were also constructed. The degree of identity between canine TRAIL protein and the human, mouse, chicken, porcine, and rat homologues is 81.3%, 61.7%, 54.3%, 82.9%, and 63.2%, respectively. The degree of identity between the feline TRAIL protein and the human, mouse, chicken, porcine, and rat homologues is 84.2%, 64.2%, 54.4%, 86.8% and 65.7%, respectively. The identity between the canine and feline TRAIL proteins is 93.2%. The canine and feline soluble TRAIL proteins were expressed in both mammalian and bacterial expression systems. Western immunoblot assays with TRAIL-specific antibody confirmed the identity of expressed protein. Both canine and feline TRAIL proteins were shown to specifically induce apoptosis and inhibit cell growth of cancer cells at a level comparable with their human counterpart.

Characterization of a highly virulent feline calicivirus and attenuation of this virus.

Feline calicivirus (FCV) is a common cause of upper respiratory and oral disease in cats. Highly virulent, systemic strains of FCV (vs FCV) have been recently described. These vs FCV isolates cause edema, cutaneous ulcers and high mortality in affected cats. This study reports a disease model with such a vs FCV isolate (FCV-33585). It also describes a full-length capsid gene sequence of this vs FCV isolate and the capsid sequence comparison of this strain with 35 other virulent and non-virulent FCV strains. In addition, sequence comparison of this strain with other 114 known sequences in the hyper-variable region of capsid gene was analyzed. Two amino acids were identified within the hyper-variable region as potentially unique signature for this vs FCV strain. This study also describes the attenuation of FCV-33585 by two methods: serial passaging at low temperature, and the generation of a temperature sensitive (ts) mutant by UV irridiation. Moreover, the potential use of attenuated vs FCV as vaccine was also explored. Monoclonal antibodies were also identified which could differentiate commonly used FCV vaccine strain from this vs strain (FCV-33585). And two monoclonal antibodies were found to react specifically the wild-type, not the attenuated FCV-33585.