Two clusters of Toscana virus meningo-encephalitis in Livorno Province and Elba Island, July-September 2018.

BACKGROUND: Toscana virus (TOSV) is an arbovirus transmitted to humans by Phlebotomus spp sandflies. It causes aseptic meningitis and meningoencephalitis with marked seasonality. Here we describe the clinical, microbiological and epidemiological features of two clusters of cases occurred in Tuscany in 2018. METHODS: A confirmed case was defined as the detection of anti-TOSV IgM and IgG in serum sample, in presence of typical clinical manifestations. We consulted hospital records of hospitalized patients to collect clinical information and obtained epidemiological information from the local health authority investigation report. We telephonically interviewed patients using a standard questionnaire for a 6 months follow-up. RESULTS: A total of 12 cases of TOSV meningo-encephalitis with onset between 4th of July and 12th of September accessed health care services in the province of Livorno. Eight cases were males with median age 41,5 and four were not resident in the area. Serological investigations confirmed a recent TOSV infection. Eight cases reported visiting Elba Island and four had a possible occupational-related exposure. CONCLUSIONS: This surge of infection emphasizes the need of information campaigns coupled with adequate surveillance and control interventions against TOSV that, among other arboviruses, is a growing issue of concern in Italy.

Spinocerebellar ataxia type 10: common haplotype and disease progression rate in Peru and Brazil.

BACKGROUND AND PURPOSE: Spinocerebellar ataxia type 10 is a neurodegenerative disorder that is due to an expanded ATTCT repeat tract in the ATXN10 gene. Our aim was to describe clinical characteristics and intragenic haplotypes of patients with spinocerebellar ataxia type 10 from Brazil and Peru. METHODS: Expanded alleles were detected by repeat-primed polymerase chain reaction. Disease progression was measured by the Scale for the Assessment and Rating of Ataxia, and the Neurological Examination Score for Spinocerebellar Ataxias when possible. Haplotypes were constructed based on polymorphic markers within and outside the gene. RESULTS: Thirteen new families were diagnosed (three from Peru). Patients from three Brazilian families diagnosed previously were also reassessed. In total, 25 individuals (16 families) were evaluated. Mean (± SD) age at onset and disease duration were 34.8 ± 10.2 and 12 ± 8 years, respectively. Common findings were ataxia, dysarthria/dysphagia, nystagmus, pyramidal signs, ophthalmoparesis and seizures. No associations were found between clinical findings and geographical origins. Twelve patients living in remote regions were examined only once. In the remaining individuals, the Scale for the Assessment and Rating of Ataxia score, and Neurological Examination Score for Spinocerebellar Ataxias worsened by 0.444 (95% CI, -0.088 to 0.800) and 0.287 (95% CI, -0.061 to 0.635) points/year, respectively. A common haplotype, 19CGGC14, was found in 11/13 of Brazilian and in 1/3 of Peruvian families. CONCLUSIONS: The progression rate was slower than in other spinocerebellar ataxias. A consistently recurrent intragenic haplotype was found, suggesting a common ancestry for most, if not all, patients.

Graves' orbitopathy, idiopathic orbital inflammatory pseudotumor and Epstein-Barr virus infection: a serological and molecular study.

OBJECTIVE: One of the hypotheses on the pathogenesis of autoimmune diseases, including Graves' disease (GD) and Graves' orbitopathy (GO), involves bacterial or viral infections. Recently, Epstein-Barr virus (EBV) has been proposed to play a role in the pathogenesis of idiopathic orbital inflammatory pseudotumor (IOIP) in Asians. The aim of the present study was to investigate the possible association of GO with EBV infection/exposure, as compared with IOIP, using serum and tissue samples, as well as primary cultures of orbital fibroblasts. METHODS: Thirty-one patients were studied, including four with IOIP, ten with GO, nine with GD without GO and eight control patients without IOIP, GD and GO. All patients with IOIP and GO underwent orbital decompression. Control patients underwent palpebral surgery. Fibroadipose orbital tissue samples were collected. Serum anti-EBV antibodies were measured in all patients. EBV-DNA was measured in blood samples, orbital tissue samples and primary cultures of orbital fibroblasts. RESULTS: Serum assays showed that the vast majority of patients have had a previous exposure to EBV, but no one had an acute infection. EBV-DNA was detected in ~40% of blood samples from GO, GD and control patients, but in none of the IOIP samples. EBV-DNA was not detected in any of the orbital tissue samples tested or in primary cultures of orbital fibroblasts. CONCLUSIONS: EBV infection does not seem to be associated with GD, GO and IOIP in Caucasians. Whether EBV is involved in IOIP in Asians or other populations remains to be confirmed.

Absence of xenotropic murine leukemia virus-related virus in Italian patients affected by chronic fatigue syndrome, fibromyalgia, or rheumatoid arthritis.

The xenotropic murine leukemia virus-related virus (XMRV) has been recently linked to chronic fatigue syndrome in a US cohort in whom the virus was demonstrated in 67% patients vs 3.7% healthy controls. Albeit this finding was not substantiated by subsequent reports and eventually considered a laboratory contamination, the matter is still the object of intense debate and scrutiny in various cohorts of patients. In this work we examined well-clinically characterized Italian patients affected by chronic fatigue syndrome, and also fibromyalgia and rheumatoid arthritis, two chronic illnesses of basically unknown etiology which show quite a few symptoms in common with chronic fatigue syndrome. Although we used recently updated procedures and controls, the XMRV was not found in 65 patients with chronic fatigue syndrome diagnosis, 55 with fibromyalgia, 25 with rheumatoid arthritis, nor in 25 healthy controls. These results add to the ever-growing number of surveys reporting the absence of XMRV in chronic fatigue syndrome patients and suggest that the virus is also absent in fibromyalgia and rheumatoid arthritis.

Xenotropic murine leukaemia virus-related virus is not found in peripheral blood cells from treatment-naive human immunodeficiency virus-positive patients.

The human pathogen xenotropic murine leukaemia virus-related virus (XMRV) has been tentatively associated with prostate cancer and chronic fatigue syndrome. Unfortunately, subsequent studies failed to identify the virus in various clinical settings. To determine whether XMRV circulates in humans and the relationship with its host, we searched for the virus in 124 human immunodeficiency virus-infected patients who might have been exposed to XMRV, might be prone to infection as a result of progressive immunodeficiency, and had not yet been treated with antiretroviral drugs. Using nested PCR and single-step TaqMan real-time PCR, both designed on the XMRV gag gene, we could not find any positive samples. These findings add to the growing amount of scepticism regarding XMRV.

Effects of feline immunodeficiency virus on feline monocyte-derived dendritic cells infected by spinoculation.

During type 1 human immunodeficiency virus infection, not only can dendritic cells (DCs) prime T cells against the virus, but they can also infect them in trans. Feline AIDS is caused by feline immunodeficiency virus (FIV) and is considered a model for the human illness because the two diseases have many features in common. Little is known about the interaction of feline DCs with FIV; therefore, this study attempts to tackle such an issue. Infection of feline monocyte-derived DCs (MDDCs) was attempted by spinoculation with FIV strains Petaluma (FIV-Pet) and M2. FIV-Pet was released rapidly in the supernatants of both infected MDDCs and activated T cells after spinoculation. It is shown that FIV-Pet was produced by MDDCs by monitoring viral content in the supernatants of infected MDDCs, by intracellular staining for p25 and by showing its cytopathic effect. Although activated T cells were better substrates for FIV replication, leading to prolonged viral shedding, both immature MDDCs and MDDCs matured with lipopolysaccharide supported virus production, mostly during the first 2 days after infection. At later times, FIV induced syncytium formation by MDDCs. Concerning the FIV receptors, MDDCs were shown to be CD134-negative and CXCR4-positive, a phenotype compatible with permissiveness to FIV-Pet. These results also suggest that maturation is not hampered by FIV infection and that virus exposure itself does not induce MDDC maturation. It is also shown that infected MDDCs can infect activated PBMCs efficiently in trans. It is concluded that MDDCs can be infected by FIV, although infection does not appear to influence their functionality.

Evaluation of feline immunodeficiency virus ORF-A mutants as candidate attenuated vaccine.

Feline immunodeficiency virus (FIV) made defective in the accessory gene ORF-A were previously shown to be greatly attenuated in its ability to replicate in lymphocytes but to grow normally or near normally in other cell types. Here, we examined whether FIV thus mutated could protect specific pathogen-free cats against challenge with ex vivo fully virulent homologous virus. No reversion of the vaccinating infections to wild type ORF-A was noted over 22 months of in vivo infection. Following challenge, 6/6 unvaccinated control cats became readily and heavily infected. In contrast, 3/9 vaccinees showed no evidence of the challenge virus over a 15-month observation period. In the other vaccinees, the challenge virus was predominant for various periods of time, but pre-existing viral loads and CD4 lymphocyte counts were either unaffected or altered only marginally and transiently. These findings show that ORF-A-defective FIV should be further examined as a candidate live attenuated vaccine.

Development of feline immunodeficiency virus ORF-A (tat) mutants: in vitro and in vivo characterization.

A functional ORF-A is essential for efficient feline immunodeficiency virus replication in lymphocytes. We have characterized a series of mutants of the Petaluma strain, derived from p34TF10 and having different combinations of stop codons and increasingly long deletions in ORF-A. Six clones proved fully replicative in fibroblastoid Crandell feline kidney cells and monocyte-derived macrophage cultures but failed to replicate in T cell lines and primary lymphoblasts. Cats inoculated with three selected mutants had considerably milder infections than controls given intact ORF-A virus. In vivo, the mutants maintained growth properties similar to those in vitro for at least 7 months, except that replication in lymphoid cells was strongly reduced but not ablated. One mutant underwent extensive ORF-A changes without, however, reverting to wild-type. Antiviral immune responses were feeble in all cats, suggesting that viral loads were too low to represent a sufficiently powerful antigenic stimulus.

Densitometric analysis of Western blot assays for feline immunodeficiency virus antibodies.

Western blot (WB) strips for antibodies directed to feline immunodeficiency virus (FIV) were analysed using reflectance densitometry by a semiautomatic densitometer. This method was used to quantify the antibody responses to different FIV proteins in both vaccinated and naturally or experimentally-infected cats. In order to increase reproducibility, reagents and protocols were accurately standardised and internal controls were added. In a first format, an internal control band consisting of feline IgG was added to each blot to minimise the effect of band intensity variation. In a second format, antibody concentrations were calculated from the ratio of the densities produced by test sera and by positive and negative standard sera. The sera under scrutiny were also examined by standard enzyme-linked immunosorbent assay (ELISA) and the results obtained compared with those of the corresponding WB. A statistically significant positive correlation was found between the results obtained with the two methods, and this was especially evident when ELISA titres were compared to corrected WB values (P = 0.001). Densitometric analysis of WB assays allowed to quantify the antibodies against FIV proteins and might be useful to investigate possible humoral immune correlates of protection in FIV vaccination studies and antibody production in the early phase of infection. The quantitation of antibodies to Gag and Env FIV antigens might be used to obtain further informations on the course of FIV disease, as previously demonstrated in human immunodeficiency virus-1 (HIV-1) infections.

Immunogenicity of an anti-clade B feline immunodeficiency fixed-cell virus vaccine in field cats.

Attempts at vaccine development for feline immunodeficiency virus (FIV) have been extensive, both because this is a significant health problem for cats and because FIV may be a useful vaccine model for human immunodeficiency virus. To date, only modest success, producing only short-term protection, has been achieved for vaccine trials in controlled laboratory settings. It is unclear how relevant such experiments are to prevention of natural infection. The current study used a vaccine that employs cell-associated FIV-M2 strain fixed with paraformaldehyde. Subject cats were in a private shelter where FIV was endemic, a prevalence of 29 to 58% over an 8-year observation period. Cats roamed freely from the shelter through the surrounding countryside but returned for food and shelter. After ensuring that cats were FIV negative, they were immunized using six doses of vaccine over a 16-month period and observed for 28 months after the initiation of immunization. Twenty-six cats (12 immunized and 14 nonimmunized controls) were monitored for a minimum of 22 months. Immunized cats did not experience significant adverse effects from immunization and developed both antibodies and cellular immunity to FIV, although individual responses varied greatly. At the conclusion of the study, 0 of 12 immunized cats had evidence of FIV infection, while 5 of 14 control cats were infected. Thus, the vaccine was safe and immunogenic and did not transmit infection. Furthermore, vaccinated cats did not develop FIV infection in a limited clinical trial over an extended time period. Thus, the data suggest that a fixed, FIV-infected cell vaccine has potential for preventing natural FIV infection in free-roaming cats.

[The transplant coordinator]. / Il coordinatore ai prelievi.

Spain was the first European country adopting a strategy of organ procurement based on a specific health professional named transplant coordinator, who was first established in Catalunya in the middle eighties. In principle, the transplant coordinator is a doctor with hospital experience who is involved full time in organ procurement. The transplant coordination activity is available without interruption, due to a team work. Transplant coordination is based on four main functions: clinical, research, training and communication, management. The principles of transplant coordination according to the Spanish model are reported in the recently approved Italian law on transplantation (law 91/1999), indicating the coordinator's specific functions: a) communication to the regional reference centre of the data concerning the possible organ donors, b) preparation of the documents needed, c) relationship with the donors' family, d) information and education of the population on transplantation issues.

AIDS vaccination studies using feline immunodeficiency virus as a model: immunisation with inactivated whole virus suppresses viraemia levels following intravaginal challenge with infected cells but not following intravenous challenge with cell-free virus.

The feline immunodeficiency virus (FIV) provides an excellent model system for AIDS vaccination studies. In the present experiments we investigated the immunogenicity and the protective activity of two inactivated vaccines prepared from a primary virus isolate. One vaccine was composed of whole virus inactivated with paraformaldehyde and then purified (WIV) and the other of viral proteins extracted with Tween-ether (TEV). Both vaccines elicited robust antiviral responses, but neither conferred appreciable levels of resistance against systemic challenge with the homologous virus. In addition, we tested whether the WIV vaccine, that had appeared more immunogenic, could protect against nontraumatic intravaginal exposure to FIV-infected cells. Although the proportions of control and vaccinated animals that became infected following mucosal challenge were similar, the vaccinees had significantly lower viral burdens than the controls, thus suggesting that immunisation with the WIV vaccine had limited FIV replication following intravaginal challenge.

Polyneuropathy in autosomal dominant cerebellar ataxias: phenotype-genotype correlation.

Autosomal dominant cerebellar ataxias (ADCAs) are clinically and genetically heterogeneous neurodegenerative disorders. The aim of this study was to evaluate electrophysiologically peripheral nervous system involvement in each of the groups studied and its correlation with the number of CAG repeats. Forty patients with ADCA were clinically and electrophysiologically investigated. Thirty-five patients belonged to the ADCA type I group (SCA1, 12; SCA2, 10; SCA3, 13) and five to the ADCA type II group. Axonal sensory or sensorimotor polyneuropathy was found in 42% of the SCA1 patients, 80% of the SCA2 patients, and 54% of the SCA3 patients, whereas electrophysiological studies were normal in all those with ADCA type II. The number of CAG repeats was significantly higher in SCA1 patients with polyneuropathy than in those without polyneuropathy (P = 0.01), whereas the reverse was observed in SCA3/MJD (Machado-Joseph disease) patients (P = 0.05). We conclude that axonal polyneuropathy is often associated with ADCA type I, but its frequency varies according to factors such as the locus responsible and the number of CAG repeats.

Kinetics of replication of a partially attenuated virus and of the challenge virus during a three-year intersubtype feline immunodeficiency virus superinfection experiment in cats.

The effects of preinfecting cats with a partially attenuated feline immunodeficiency virus (FIV) on subsequent infection with a fully virulent FIV belonging to a different subtype were investigated. Eight specific-pathogen-free cats were preinfected with graded doses of a long-term in vitro-cultured cell-free preparation of FIV Petaluma (FIV-P, subtype A). FIV-P established a low-grade or a silent infection in the inoculated animals. Seven months later, the eight preinfected cats and two uninfected cats were challenged with in vivo-grown FIV-M2 (subtype B) and periodically monitored for immunological and virological status. FIV-P-preinfected cats were not protected from acute infection by FIV-M2, and the sustained replication of this virus was accompanied by a reduction of FIV-P viral loads in the peripheral blood mononuclear cells and plasma. However, from 2 years postchallenge (p.c.) until 3 years p.c., when the experiment was terminated, preinfected cats exhibited reduced total viral burdens, and some also exhibited a diminished decline of circulating CD4(+) T lymphocytes relative to control cats infected with FIV-M2 alone. Interestingly, most of the virus detected in challenged cats at late times p.c. was of FIV-P origin, indicating that the preinfecting, attenuated virus had become largely predominant. By the end of follow-up, two challenged cats had no FIV-M2 detectable in the tissues examined. The possible mechanisms underlying the interplay between the two viral populations are discussed.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: detailed analysis of the humoral immune response to a protective vaccine.

The feline immunodeficiency virus (FIV) cat model is extensively used to investigate possible vaccination approaches against AIDS in humans. Although consistent levels of protection have been achieved with FIV, as with other model systems, by immunizing with whole inactivated virus or fixed infected cells, the mechanisms responsible for protection are elusive. In previous studies we showed that cats immunized with a vaccine consisting of fixed infected cells were protected or unprotected against cell-free or cell-associated FIV challenge depending on the time interval between completion of vaccination and challenge. In an attempt to define possible humoral immune correlates of protection, selected sera harvested at the times of challenge from such cats were examined for anti-FIV-antibody titers and properties by using binding and functional immunological assays. Binding assays included quantitative Western blotting, enzyme-linked tests for antibodies to FIV glycoproteins and immunodominant linear epitopes, and tests for measuring conformation dependence and avidity of anti-viral-envelope antibodies. Functional assays included virus neutralization performed with two different cell substrates, complement- and antibody-dependent virolysis, blocking of reverse transcriptase, and an assay that measured the ability of sera to prevent FIV growth in cocultures of infected and uninfected cells. Despite the wide spectrum of parameters investigated, no correlation between vaccine-induced protection and the humoral parameters measured was noted.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: homologous erythrocytes as a delivery system for preferential immunization with putative protective antigens.

Feline immunodeficiency virus (FIV) is a useful model for testing of criteria for AIDS vaccine development. In the protocol we adopted, we used a primary isolate of FIV as a source of antigen and, for challenge, plasma from cats infected with the homologous virus never passaged in vitro. Cat erythrocytes (RBC) were coated with the surface components of freshly harvested and purified FIV by means of biotin-avidin-biotin bridges and used to immunize specific-pathogen-free cats (four doses at monthly intervals; total amount of FIV antigen administered per cat, approximately 14 microg). Immunized cats developed moderate levels of antibodies directed mainly to surface components of the virion and clearly evident lymphoproliferative responses. Four months after the last dose of immunogen, FIV-immunized cats and control cats immunized with bovine serum albumin-coated RBC were challenged. Judged from the results of the subsequent 12-month follow-up, FIV-immunized cats exhibited at least some degree of protection. However, following rechallenge, most of the FIV-immunized animals became virus positive in spite of a booster immunogen dose given 2 months before the second challenge.

Studies of AIDS vaccination using an ex vivo feline immunodeficiency virus model: protection conferred by a fixed-cell vaccine against cell-free and cell-associated challenge differs in duration and is not easily boosted.

Cats immunized with cells infected with a primary isolate of feline immunodeficiency virus (FIV) and fixed with paraformaldehyde were challenged with cell-free or cell-associated homologous virus obtained ex vivo. Complete protection was observed in animals challenged with cell-free virus 4 months after completion of vaccination (p.v.) or with cell-associated virus 12 months p.v. In contrast, no protection was observed in cats challenged with cell-free virus 12 or 28 months p.v. or with cell-associated virus 37.5 months p.v. Prior to the 28- and 37.5-month challenges, the animals had received a booster dose of vaccine that had elicited a robust anamnestic immune response. These results show that vaccine-induced protection against ex vivo FIV is achievable but is relatively short-lived and can be difficult to boost.

Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and distinct evolutionary rates among envelope sequence clades.

For the rapid genetic analysis of feline immunodeficiency virus (FIV), we developed a heteroduplex mobility assay (HMA) that utilizes a PCR-amplified fragment of the FIV envelope gene spanning the third and fourth variable regions of the envelope surface protein coding sequence. Viral sequences were successfully amplified from blood specimens from 98 naturally infected cats from Australia, Canada, Germany, Italy, South Africa, and the United States. Eighty were clearly assignable to the A or B envelope sequence subtypes. Three belonged to subtype C, one was dually infected with viruses harboring the A and B env subtypes, and several were categorized as outliers to any of the established subtypes or as probable intersubtype recombinants. Some geographic clustering was evident, with subtypes A and B found in greater frequency in the western and eastern regions of the United States, respectively. Subtypes A, B, and C were found on more than one continent, and countries with more than two samples analyzed contained at least two subtypes. The broadest representation of subtypes was found in Munich, Germany, where three subtypes and one virus that was not classifiable by HMA were found. Thirteen samples were selected for DNA sequence determination over the same region of env used for HMA. Analysis of all available FIV env sequences from this and previous studies revealed the existence of recombinant viruses generated from subtype A/B, B/D, and A/C envelope gene sequences. Subtype A env sequences were less diverse than subtype B sequences, although both groups had well-supported clusters. Furthermore, the mutational pattern giving rise to diversification in the two subtypes differed, with the subtype A viruses showing half as many synonymous site mutations compared to subtype B yet showing similar levels of nonsynonymous site changes. These results are consistent with the hypothesis that FIV-B is an older virus group and is possibly more host adapted than FIV-A.

Piggy-back versus conventional technique in liver transplantation: report of a randomized trial.

Liver transplantation with preservation of the recipient vena cava (the "piggy-back" technique) has been proposed as an alternative to the traditional method. We performed a randomized study on 39 cirrhotic patients, 20 who underwent the piggy-back technique (group 1) and 19 the traditional method using venovenous bypass (group 2) to evaluate the feasibility and true advantages of the piggy-back technique compared to the traditional method. Two patients were switched to the conventional technique due to the presence of a caudate lobe embracing the vena cava in one patient and a caval lesion in the other. Statistically significant differences between the two groups were only found for the warm ischemia time (48.5 +/- 13 min for piggy-back vs 60 +/- 12 min for the conventional method) and for renal failure (zero cases in group 1 vs four cases in group 2). We therefore believe that liver transplantation with the piggy-back technique can easily be performed in almost all cases, and that only a few, specific situations, such as a very enlarged caudate lobe, do not justify its routine use.

Incubation time for feline immunodeficiency virus cultures.

In a recent report, Fiscus et al. (S. A. Fiscus, S. L. Welles, S. A. Spector, and J. L. Lathey, J. Clin. Microbiol. 33:246-247, 1995) have shown that qualitative human immunodeficiency virus cultures can be terminated at day 21 with minimal false-negative results. We have evaluated a large number of qualitative and quantitative feline immunodeficiency virus (FIV) isolations to determine how long FIV cultures should be incubated to obtain reasonably certain results. The rate at which FIV cultures became positive was influenced by whether the cats under study were naturally or experimentally infected, the duration of in vivo infection, and the number of infected peripheral blood mononuclear cells seeded. The results show that cultures for FIV isolation should be kept for 5 to 6 weeks.