Assessment of the risk of polyomavirus JC reactivation in patients with immune-mediated diseases during long-term treatment with infliximab.

Polyomavirus JC (JCV) reactivation causing progressive multifocal leukoencephalopathy is a main concern during biological therapies. Here, JCV reactivation in patients suffering from immune-mediated diseases after a long-term treatment with anti-tumor necrosis factor alpha (TNF-α) inhibitor infliximab was investigated. Peripheral mononuclear blood cells (PBMC), plasma and urine samples were obtained from 61 immune-mediated diseases patients treated or not with infliximab in combination with steroid and other immunomodulators and from 20 healthy donors. JCV DNA was transiently detected in 12 PBMC of 40 patients at different doses of infliximab with a higher prevalence than that of the 21 patients untreated. Conversely, a stable JCV positivity in urine of treated and untreated patients was detected. Sequencing the noncoding control region (NCCR), all samples exhibited the archetype structure with few mutations in transcriptional factor binding regions. The consequence of anti-TNF-α treatment on viral persistence was examined monitoring Torquetenovirus viremia and investigating the TNF-α-induced microRNA regulators of transcriptional factors, with a binding site on NCCR. Although infliximab treatment in this study did not affect directly JCV reactivation, further investigation on host factor(s) regulated by it will be of warranty in the understanding the mechanism(s) that may affect viral persistence.

Increased pathogenicity and shedding in chickens of a wild bird-origin low pathogenicity avian influenza virus of the H7N3 subtype following multiple in vivo passages in quail and turkey.

In order to investigate viral adaptation mechanisms to poultry, we performed serial in vivo passages of a wild bird low pathogenicity avian influenza isolate of the H7N3 subtype (A/mallard/Italy/33/01) in three different domestic species (chicken, turkey, and Japanese quail). The virus under study was administered via natural routes at the dose of 10(6) egg infective dose50/ 0.1 ml to chickens, turkeys, and quails in order to investigate the clinical susceptibility and the shedding levels after infection. Multiple in vivo passages of the virus were performed by serially infecting groups of five naive birds of each species, with samples collected from a previously infected group. Quails and turkeys were susceptible to infection for 10 serial passages, whereas chickens were susceptible to two cycles of infection only. Infection of chicken with the quail- and turkey-adapted viruses showed an increased pathogenicity and/or shedding, causing more severe clinical signs and/or higher levels of viral excretion compared to the original strain. The data obtained herein suggest that infection of selected avian species may facilitate the adaptation of avian influenza viruses originating from the wild bird reservoir to chicken. This is the first time turkey has been shown to act as a species in which a virus from the wild reservoir can increase its replication activity in other domestic species.

Oligonucleotides derived from the packaging signal at the 5' end of the viral PB2 segment specifically inhibit influenza virus in vitro.

The development of new antiviral molecules to fight the possible emergence of influenza viruses with pandemic potential is needed. In this study, phosphorothioate oligonucleotides (S-ONs) derived from the packaging signals in the 3' and 5' ends of the viral PB2 RNA were associated with liposomes and tested against influenza virus in vitro. A 15-mer S-ON derived from the 5' end of the viral PB2 RNA, complementary to the 3' end of its coding region (nucleotides 2279-2293) and designated 5-15b, proved markedly inhibitory. The antiviral activity of 5-15b was dose- and time-dependent but was independent of the cell substrate and multiplicity of infection used. Importantly, inhibition of influenza A and B viruses required S-ONs reproducing the respective packaging signals. Furthermore, 5-15b and its antisense derivative S-ON activity did not affect intracellular accumulation of viral RNA. Confocal microscopy showed that 5-15b is clearly nucleophilic. These findings indicate that the packaging signal at the 5' end of the PB2 RNA is an interesting target for the design of new anti-influenza-virus compounds.

Polyomavirus JC microRNA expression after infection in vitro.

The in vitro expression of the Polyomavirus JC (JCPyV) microRNAs, JC-miRNA-3p and -5p, at early time points post-infection was investigated. The expression of the JCPyV microRNAs was monitored in hematopoietic progenitor KG-1 cells and in kidney fibroblast-like COS-7 cells transformed with SV40 after infection with a JCPyV CY archetype viral clone. The JCPyV DNA viral load was low in KG-1 cells compared with that in COS-7 cells, which showed productive viral replication. The expression of the JCPyV microRNAs was observed from 12h after the viral infection of both cell types and in the exosomes present in their cell supernatant. Additionally, this study verified that the JCPyV microRNAs in the exosomes present in the supernatants produced by the infected cells might be carried into uninfected cells. These findings suggest that additional investigations of the expression of JCPyV microRNAs and their presence in exosomes are necessary to shed light on their regulatory role during viral reactivation.

Markers of JC virus infection in patients with multiple sclerosis under natalizumab therapy.

OBJECTIVE: To evaluate the frequency of JC polyomavirus (JCPyV) infection and anti-JCPyV antibodies in patients with multiple sclerosis under natalizumab therapy. METHODS: Presence of anti-JCPyV antibodies and JCPyV DNA was analyzed in 39 patients with relapsing-remitting multiple sclerosis undergoing natalizumab therapy. Anti-JCPyV antibodies were evaluated in serum by a 2-step virus-like particle-based ELISA assay (Stratify), and JCPyV DNA was evaluated in peripheral blood mononuclear cells, plasma, and urine by quantitative PCR. The anti-JCPyV antibodies were evaluated in serum samples collected at the same time or later than those collected for DNA analysis. RESULTS: JCPyV DNA was detected in 59% of patients, and anti-JCPyV antibodies were present in 67%. JCPyV DNA occurred more often in blood than in urine. Anti-JCPyV antibodies were observed in 70% of the JCPyV-infected patients, and JCPyV DNA was detected in 50% of the patients without anti-JCPyV antibodies. When JCPyV DNA was investigated in blood and urine the frequency of infection was higher than previously described. CONCLUSION: Under these experimental conditions, with respect to the observed frequency of JCPyV infection, the sensitivity of the anti-JCPyV antibody assay was lower than expected.

Reassortment ability of the 2009 pandemic H1N1 influenza virus with circulating human and avian influenza viruses: public health risk implications.

Exploring the reassortment ability of the 2009 pandemic H1N1 (A/H1N1pdm09) influenza virus with other circulating human or avian influenza viruses is the main concern related to the generation of more virulent or new variants having implications for public health. After different coinfection experiments in human A549 cells, by using the A/H1N1pdm09 virus plus one of human seasonal influenza viruses of H1N1 and H3N2 subtype or one of H11, H10, H9, H7 and H1 avian influenza viruses, several reassortant viruses were obtained. Among these, the HA of H1N1 was the main segment of human seasonal influenza virus reassorted in the A/H1N1pdm09 virus backbone. Conversely, HA and each of the three polymerase segments, alone or in combination, of the avian influenza viruses mainly reassorted in the A/H1N1pdm09 virus backbone. Of note, A/H1N1pdm09 viruses that reassorted with HA of H1N1 seasonal human or H11N6 avian viruses or carried different combination of avian origin polymerase segments, exerted a higher replication effectiveness than that of the parental viruses. These results confirm that reassortment of the A/H1N1pdm09 with circulating low pathogenic avian influenza viruses should not be misjudged in the prediction of the next pandemic.

Packaging signals in the 5'-ends of influenza virus PA, PB1, and PB2 genes as potential targets to develop nucleic-acid based antiviral molecules.

In a previous study a 15-mer phosphorothioate oligonucleotide (S-ON) derived from the packaging signal in the 5' end of segment 1 (PB2) of influenza A virus (designated 5-15b) proved markedly inhibitory to virus replication. Here we investigated whether analogous inhibitory S-ONs targeting the 5' end of segments 2 (PB1) and 3 (PA) could be identified and whether viral resistance to S-ONs can be developed. Similar to our earlier result, 20-mer S-ONs reproducing the 5' ends of segments 2 or 3 (complementary to the 3'-coding regions of PB1 and PA, respectively) exerted a powerful antiviral activity against a variety of influenza A virus subtypes in MDCK cells. Serial passage of the A/Taiwan/1/86 H1N1 strain in the presence of S-ON 5-15b or its antisense as5-15b analogue showed that mutant viruses with reduced susceptibility to the S-ON could indeed be generated, although the resistant viruses displayed reduced replicative fitness. Sequencing the resistant viruses identified mutations in the PB1, PB2, PA and M1 genes. Introduction of these changes into the A/PR/8/34 H1N1 strain by reverse genetics, suggested that alterations to RNA function in the packaging regions of segments 2 and 3 were important in developing resistance to S-ON inhibition. However, many of the other sequence changes induced by S-ON treatment were markedly deleterious to virus fitness. We conclude that packaging signals in the influenza A virus polymerase segments provide feasible targets for nucleic acid-based antivirals that may be difficult for the virus to evade through resistance mutations.

Molecular adaptation of an H7N3 wild duck influenza virus following experimental multiple passages in quail and turkey.

To investigate the molecular adaptation of influenza viruses during natural interspecies transmission, we performed a phenotypic and genotypic analysis of a low-pathogenic duck H7N3 influenza virus after experimental passages in turkey and quail. Results obtained showed differences in the HA receptor-binding and in NA enzyme activities in viruses recovered after passages in quail, compared to those obtained from passages in turkey. Sequencing of the HA, NA and genes of internal proteins of the viruses obtained from quail and turkey, identified several amino acid substitutions in comparison with the progenitor virus. Of note, in the quail-adapted viruses the emergence of a 23-amino acid deletion in the stalk of the NA and the introduction of a glycosylation site in the HA were a reminiscence of changes typically observed in nature confirming a potential role of the quail in the adaptation of wild birds viruses to domestic poultry.