Identification of different respiratory viruses, after a cell culture step, by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

In this study matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a reliable identification method for the diagnosis of bacterial and fungal infections, is presented as an innovative tool to investigate the protein profile of cell cultures infected by the most common viruses causing respiratory tract infections in humans. MALDI-TOF MS was applied to the identification of influenza A and B viruses, adenovirus C species, parainfluenza virus types 1, 2 and 3, respiratory syncytial virus, echovirus, cytomegalovirus and metapneumovirus. In this study MALDI-TOF MS was proposed as a model to be applied to the identification of cultivable respiratory viruses using cell culture as a viral proteins enrichment method to the proteome profiling of virus infected and uninfected cell cultures. The reference virus strains and 58 viruses identified from respiratory samples of subjects with respiratory diseases positive for one of the above mentioned viral agents by cell culture were used for the in vitro infection of suitable cell cultures. The isolated viral particles, concentrated by ultracentrifugation, were used for subsequent protein extraction and their spectra profiles were generated by MALDI-TOF MS analysis. The newly created library allowed us to discriminate between uninfected and respiratory virus infected cell cultures.

Human cytomegalovirus reactivation from latency: validation of a "switch" model in vitro.

BACKGROUND: Human cytomegalovirus (HCMV) is an opportunistic pathogen leading to severe and even fatal diseases in 'at-risk' categories of individuals upon primary infection or the symptomatic reactivation of the endogenous virus. The mechanisms which make the virus able to reactivate from latency are still matter of intense study. However, the very low number of peripheral blood monocytes (an important latent virus reservoir) harbouring HCMV DNA makes it very difficult to obtain adequate viral quantities to use in such studies. Thus, the aim of the present study was to demonstrate the usefulness of human THP-1 monocytes, mostly employed as HCMV latent or lytic infection system, as a reactivation model. METHODS: THP-1 monocytes were infected with HCMV TB40E strain (latency model) at multiplicities of infection (MOI) of 0.5, 0.25 or 0.125. After infection, THP-1 aliquots were differentiated into macrophages (reactivation model). Infections were carried out for 30 h, 4, 6 and 7 days. Viral DNA evaluation was performed with viable and UV-inactivated virus by q-Real-Time PCR. RNA extracted from latency and reactivation models at 7 days post-infection (p.i.) was subjected to RT-PCR to analyse viral latency and lytic transcripts. To perform viral progeny analysis and titration, the culture medium from infected THP-1 latency and reactivation models (7 days p.i.) was used to infect human fibroblasts; it was also checked for the presence of exosomes. For viral progeny analysis experiments, the Towne strain was also used. RESULTS: Our results showed that, while comparable TB40E DNA amounts were present in both latent and reactivation models at 30 h p.i., gradually increased quantities of viral DNA were only evident in the latter model at 4, 6, 7 days p.i.. The completion of the lytic cycle upon reactivation was also proved by the presence of HCMV lytic transcripts and an infectious viral yield at 7 days p.i. CONCLUSIONS: Our data demonstrate the effectiveness of THP-1 cells as a "switch" model for studying the mechanisms that regulate HCMV reactivation from latency. This system is able to provide adequate quantities of cells harbouring latent/reactivated virus, thereby overcoming the intrinsic difficulties connected to the ex vivo system.

Combined genetic variants of human cytomegalovirus envelope glycoproteins as congenital infection markers.

BACKGROUND: Human cytomegalovirus (HCMV) is still considered to be the main viral cause of birth defects and long-term neurological and sensory sequelae following congenital infection. Several Authors sustain a key role of HCMV envelope glycoproteins, such as gB, gN and gO - mainly involved in cell targeting, viral penetration and spread - as putative virulence factors. The genes coding for these glycoproteins possess hypervariable regions, resulting in a number of genetic variants in circulating clinical strains. Considering that the genetic polymorphisms underlying the specific differences between gB, gN and gO genotypes can influence the ability of HCMV to preferentially target specific host cells, it is very likely that they play an important role in defining HCMV infection outcome. In the present study, we analysed HCMV gB, gN and gO gene polymorphisms in viral strains isolated from paediatric patients with congenital or post-natal infection, to investigate whether specific genetic variants may be associated with congenital infection. METHODS: The restriction fragment polymorphisms of genes coding for HCMV gB (UL55), gN (UL73) and gO (UL74) were investigated by analysing viral DNA extracted from 40 urine samples of as many paediatric patients with congenital or post-natal HCMV infection. Randomly selected samples were subjected to DNA sequencing and phylogenetic analysis. Statistical analysis was performed using Fisher's exact test to assess the significance of single and combined glycoprotein genotypes frequency distribution. Statistical significance was considered at a P <0.05. RESULTS: While gB genomic variants were quite homogeneously represented in both paediatric groups, the gN4 genotype significantly prevailed in congenitally infected children (89.5 %) vs post-natally infected children (47.6 %), with a predominance of the gN4c variant (47.4 %). A similar trend was observed for gO3 (52.6 % vs 19 %). Concerning genotypes association, a statistically significant (P = 0.037) gN4-gO3 combination was found specifically in the congenitally infected group. CONCLUSIONS: The results indicate that the gN4 (mostly the gN4c variant) and gO3 combined genotypes could provide useful markers of congenital infection and represent suitable candidate molecules for prophylactic vaccine preparations.