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Enterovirus (EV) and parechovirus (HPeV) are common viruses in the neonatal period, with similar seasonality and symptomatology. They also are the main causes of aseptic meningitis in newborns and children under 1 year of age. We compared the clinical signs, laboratory data, brain, and neurodevelopmental outcome of 10 infants with HPeV and 8 with EV meningitis. In patients with EV meningitis, serum C-reactive protein (CRP) values were significantly higher than those of patients with HPeV infection. Procalcitonin values were low in both groups. White blood cell (WBC) and lymphocyte values were significantly higher in EV patients. None of the infants had a brain lesion on cerebral ultrasound neither negative neurological outcome. Based solely on symptoms, it is not possible to distinguish HPeV from EV infection. C-reactive protein, WBC, and lymphocyte values might allow the physician to assume EV infection. The gold standard test for diagnosis remains real-time polymerase chain reaction on cerebral spinal fluid., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Enteroviruses (EVs) are ubiquitous viruses that circulate worldwide, causing sporadic or epidemic infections, typically during the summer and fall. They cause a broad spectrum of illnesses, ranging from an unspecified febrile clinical presentation to a severe illness. EVs are recognized to be the most frequent etiological agents of aseptic meningitis in children. However, as the infection is usually mild and self-limiting, it remains underestimated, and the epidemiology of EVs is poorly understood. To date, no vaccine or effective therapy for all types of enteroviruses is available, and EVs constitute a public health concern. Here, we investigated the molecular epidemiology of EV strains circulating in the Lazio region over a 10-year time span (2012-2023) by using a sequence-typing approach and phylogenetic analysis. The epidemiological trend of EV infection has undergone changes during the SARS-CoV-2 pandemic (2020-2021), which resulted in a modification in terms of the number of diagnosed cases and seasonality. From 2022, the circulation of EVs showed a behavior typical of the pre-pandemic period, although changes in predominantly circulating strains have been noted. Both epidemic and sporadic circulation events have been characterized in the Lazio region. Further analyses are needed to better characterize any strain with higher potential pathogenic power and to identify possible recombinant strains.

Since spring 2022, the global epidemiology of the monkeypox virus (MPXV) has changed. The unprecedented increase of human clade II MPXV cases worldwide heightened concerns about this emerging zoonotic disease. We analysed the positivity rates, viral loads, infectiousness, and persistence of MPXV DNA for up to 4 months in several biological samples from 89 MPXV-confirmed cases. Our data showed that viral loads and positivity rates were higher during the first two weeks of symptoms for all sample types. Amongst no-skin-samples, respiratory specimens showed higher MPXV DNA levels and median time until viral clearance, suggesting their usefulness in supporting MPXV diagnosis, investigating asymptomatic patients, and monitoring viral shedding. Infectious virus was cultured from respiratory samples, semen, and stools, with high viral loads and collected within the first 10 days. Notably, only one saliva and one semen were found positive for viral DNA after 71 and 31 days from symptoms, respectively. The focus on bloodstream samples showed the best testing sensitivity in plasma, reporting the overall highest MPXV DNA detection rate and viral loads during the 3-week follow-up as compared to serum and whole-blood. The data here presented can be useful for MPXV diagnostics and a better understanding of the potential alternative routes of its onward transmission., (© 2024. The Author(s).)

The recent multi-country outbreak of the zoonotic monkeypox virus (MPXV) infection in humans without an epidemiological link with endemic areas has raised concerns about the route of transmission. Since the infection spread largely among men who have sex with men who, in most cases, presented primary lesions of the genital and oral mucosa, sexual transmission has been proposed. In the present study, we retrospectively evaluated specimens of vesicular lesions collected from the skin and genital tract of 35 patients (23 positive and 12 negative) presenting at our Institute for monkeypox (mpox) diagnosis by using a novel molecular syndromic vesicular virus panel (VVP) assay. All MPXV-positive samples but one was confirmed; however, the viral syndromic analysis revealed that 8.6% of them were coinfected with one or more viruses, and 17% had at least a virus different from the MPXV. The percentage of coinfections increased to more than 25% when nonviral pathogens, such as gonorrhea and syphilis, were also considered. These results show the usefulness of syndromic diagnosis in cases where MPXV is suspected (and vice versa) and at the same time highlight that the broader screening of sexually transmitted infections in the population with high-risk sexual behavior is critical to ensure a complete etiology and appropriate treatment.

During the 2022-outbreak, peculiar clinical presentations of Mpox have been described, some of which can make the diagnosis of the disease extremely challenging. Here we report a case series of fourteen patients with Mpox pharynogotonsillar involvement (PTI) seen at National Institute for Infectious Diseases, "Lazzaro Spallanzani", in Rome, Italy from May to September 2022. All included patients were men who have sex with men (median age 38 years) reporting unprotected sex within three weeks from symptoms onset. Seven out of fourteen patients needed hospitalization due to uncontrolled pain, reduced airspace and difficulty swallowing, of whom five were effectively treated with tecovirimat or cidofovir. The remaining two patients were treated with symptomatic drugs. The typical Mpox muco-cutaneous manifestations were not observed simultaneously with PTI in three patients, two of whom developed the lesions after several days, while one never manifested them. Polymerase Chain Reaction (PCR) for Mpox virus was positive in oropharyngeal swab, saliva and serum. Although PTI occurs in only a small percentage of Mpox cases, its diagnosis is of utmost importance. In fact, this localization, if not identified, could lead to serious complications in the absence of early antiviral treatment and to missed diagnosis with an increased risk of disease transmission., Competing Interests: Declaration of Competing Interest The authors declare that no conflicting financial interests or other competing relationships exist., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Since the beginning of the COVID-19 pandemic, large-scale genomic sequencing has immediately pointed out that SARS-CoV-2 has rapidly mutated during the course of the pandemic, resulting in the emergence of variants with a public health impact. In this context, strictly monitoring the circulating strains via NGS has proven to be crucial for the early identification of new emerging variants and the study of the genomic evolution and transmission of SARS-CoV-2. Following national and international guidelines, the Lazio region has created a sequencing laboratory network (WGSnet-Lazio) that works in synergy with the reference center for epidemiological surveillance (SERESMI) to monitor the circulation of SARS-CoV-2. Sequencing was carried out with the aims of characterizing outbreak transmission dynamics, performing the genomic analysis of viruses infecting specific categories of patients (i.e., immune-depressed, travelers, and people with severe symptoms) and randomly monitoring variant circulation. Here we report data emerging from sequencing activities carried out by WGSnet-Lazio (from February 2020 to October 2022) linked with epidemiological data to correlate the circulation of variants with the clinical and demographic characteristics of patients. The model of the sequencing network developed in the Lazio region proved to be a useful tool for SARS-CoV-2 surveillance and to support public health measures for epidemic containment.

We report two cases of SARS-CoV-2 recombinant variant XE detected in nasopharyngeal swabs (NPS) of hospitalized patients with no evident epidemiological link in Lazio, Central Italy. Whole-Genome Sequencing (WGS) performed on an Ion Torrent GSS5 platform according to Italian flash surveys showed genomes corresponding to the PANGOLIN unclassified lineage and the Nextclade XE clade. Further analyses were then carried out to investigate more deeply the genetic characteristics of these XE-like sequences. When phylogenetic trees, by using IQ-TREE, were built splitting the genome into two regions according to the putative XE recombination site, the upstream and downstream regions were seen to be clustered near BA.1 and BA.2 sequences, respectively. However, our XE-like sequences clustered separately, with a significant bootstrap, from the classified European and Italian XE strains, although the recombination site between BA.1 and BA.2 was identified at the nucleotide site 11556 by RDP4 software, consistent with the putative XE breakpoint. These findings show the risk of the introduction of novel recombinant variants of SARS-CoV-2 and the existence of XE-like strains, phylogenetically separated, that could make their exact taxonomy difficult. It follows the need for continued SARS-CoV-2 surveillance by WGS.

Background: The aim of this study was to characterize the genome of a recombinant Enterovirus associated with severe and fatal nosocomial infection; it was typed as Echovirus 11 (E-11) according to the VP1 gene. Enterovirus infection is generally asymptomatic and self-limited, but occasionally it may progress to a more severe clinical manifestation, as in the case described here. Recombination plays a crucial role in the evolution of Enteroviruses (EVs) and has been recognized as the main driving force behind the emergence of epidemic strains associated with severe infection. Therefore, it is of utmost importance to monitor the circulation of recombinant strains for surveillance purposes., Methods: Enterovirus-RNA was detected in the serum and liver biopsy of patients involved in the nosocomial cluster by commercial One-Step qRT-PCR method and the Enterovirus strains were isolated in vitro. The EVs typing was determined by analyzing the partial-length of the 5'UTR and VP1 sequences with the web-based open-access Enterovirus Genotyping Tool Version 0.1. The amplicons targeting 5'UTR, VP1 and overlapping fragments of the entire genome were sequenced with the Sanger method. Phylogenetic analysis was performed comparing the VP1 and the full-genome sequences of our strains against an appropriate reference set of Enterovirus prototypes of the Picornaviridae genera and species retrieved from the Enterovirus Genotyping Tool. Recombination analysis was performed using RDP4 software., Results: The Neighbor-Joining tree of the VP1 gene revealed that the 4 patients were infected with an identical molecular variant of Echovirus 11 (E-11). While the phylogenetic and the RDP4 analysis of the full-genome sequences provided evidence that it was a chimeric strain between an E-11 and a Coxsackievirus B (CV-B)., Conclusions: The chimeric structure of the E-11 genome might have contributed to the severe infection and epidemic feature of the strain, but further biological characterizations are needed. The evidence reported in this study, highlights the limit of typing techniques based on the VP1 gene, as they fail to identify the emergence of recombinant strains with potentially more pathogenic or epidemic properties, thus providing only partial information on the epidemiology and pathogenesis of Enteroviruses., (© 2022. The Author(s).)

A descriptive analysis of common respiratory pathogens (CRPs) detected in nasopharyngeal swabs (NPSs) from hospitalized patients with influenza-like illness during the fall seasons of the past three years, 2019-2021, in the Lazio region, Italy, was conducted to assess whether or not CRP circulation changed because of COVID-19 during the fall season. The results observed in a total of 633 NPSs subjected to molecular diagnosis for CRPs by multiplex PCR assay during the autumn seasons (exactly from week 41 to week 50) were compared with each other. In 2019, in 144 NPSs, the more represented CRPs were rhinovirus/enterovirus (7.6%) and influenza A/B (4.2%). In 2020, 55 (21.6%) out of 255 NPSs resulted positive for SARS-CoV-2 and, except for one case of Legionella pneumophila , the CRPs detected were exclusively rhinovirus/enterovirus (4.7%). In 2021, among 234 NPSs, 25.6% resulted positive for SARS-CoV-2, 14.5% for respiratory syncytial virus (RSV), and 12.8% for rhinovirus/enterovirus. Compared with 2019, in 2020, CRP circulation was severely limited to a few cases; in 2021, instead, infections by RSV (detected also among adults), rhinovirus/enterovirus, and other respiratory pathogens were observed again, while influenza was practically absent. The comparison of the CRPs detected in the NPSs depicts a different circulation in the Lazio region during the last three fall seasons. CRP monitoring has a direct impact on the prevention and control strategies of respiratory infectious diseases, such as the non-pharmacological interventions implemented in response to the COVID-19 pandemic. Future studies should investigate the impact of specific interventions on the spread of respiratory infections.

Purpose: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid., Methods: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital., Results: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with C t values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction., Conclusion: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation., (© 2021. The Author(s).)

Background: Limited data are available about the predictors and outcomes associated with prolonged SARS-CoV-2 RNA shedding (VS)., Methods: A retrospective study including COVID-19 patients admitted to an Italian hospital between March 1 and July 1, 2020. Predictors of viral clearance (VC) and prolonged VS from the upper respiratory tract were assessed by Poisson regression and logistic regression analyses. The causal relation between VS and clinical outcomes was evaluated through an inverse probability weighted Cox model., Results: The study included 536 subjects. The median duration of VS from symptoms onset was 18 days. The estimated 30-day probability of VC was 70.2%. Patients with comorbidities, lymphopenia at hospital admission, or moderate/severe respiratory disease had a lower chance of VC. The development of moderate/severe respiratory failure, delayed hospital admission after symptoms onset, baseline comorbidities, or D-dimer >1000ng/mL at admission independently predicted prolonged VS. The achievement of VC doubled the chance of clinical recovery and reduced the probability of death/mechanical ventilation., Conclusions: Respiratory disease severity, comorbidities, delayed hospital admission and inflammatory markers negatively predicted VC, which resulted to be associated with better clinical outcomes. These findings highlight the importance of prompt hospitalization of symptomatic patients, especially where signs of severity or comorbidities are present., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We report phylogenetic and mutational analysis by NGS of six SARS-CoV-2 strains from patients flying from Bangladesh to Italy (July 2020). Data suggest that no further circulation of such imported strains occurred in Italy, stating the efficacy of early screening at the point of entry and supporting the importance of molecular epidemiology in monitoring the efficacy of control measures., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rueca, Di Caro, Gruber, Messina, Giombini, Valli, Lalle, Lanini, Vairo, Capobianchi and Bartolini.)

In Italy, the first SARS-CoV-2 infections were diagnosed in Rome, Lazio region, at the end of January 2020, but sustained transmission occurred later, since the end of February. From 1 February to 12 April 2020, 17,164 nasopharyngeal swabs were tested by real time PCR for the presence of SARS-CoV-2 at the Laboratory of Virology of National Institute for Infectious Diseases "Lazzaro Spallanzani" (INMI) in Rome. In the same period, coincident with the winter peak of influenza and other respiratory illnesses, 847 samples were analyzed by multiplex PCR assay for the presence of common respiratory pathogens. In our study the time trend of SARS-CoV-2 and that of other respiratory pathogens in the same observation period were analysed. Overall, results obtained suggest that the spread of the pandemic SARS-CoV-2 virus did not substantially affect the time trend of other respiratory infections in our region, highlighting no significant difference in rates of SARS-CoV-2 infection in patients with or without other respiratory pathogens. Therefore, in the present scenario of COVID-19 pandemic, differential diagnosis resulting positive for common respiratory pathogen(s) should not exclude testing of SARS-CoV-2., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The 2013-2016 Ebola virus disease (EVD) outbreak showed a lack of diagnostic point-of-care methods. Currently, EBOV diagnosis relies on quantitative reverse-transcription-PCR (RT- qPCR), highly specific and sensitive, but requiring skilled personnel and well-equipped laboratories. In field settings, these factors and others, such as samples' time of collection and transportation, determine a prolonged turnaround-time to final results. In outbreak scenarios, a rapid and transportable method could eliminate issues of cohorting suspected and actual EVD patients for lack of diagnostic certainty. The aim of this study was the field evaluation of the new fast, easy-to-use and reliable RT-qPCR assay and platform for EBOV detection, developed in the framework of the EbolaMoDRAD project by CLONIT S.r.l. and STMicroelectronics S.r.l., Study Design: We evaluated its performance during the outbreak and in further studies in the EVD laboratory at the Princess Christian Maternity Hospital (PCMH) in Freetown (Sierra Leone) run by Emergency NGO and the Italian National Institute for Infectious Diseases (INMI). The assay was tested on residual aliquots of clinical specimens from EBOV-positive or -negative patients (n=116, EVD prevalence 37%)., Results and Conclusion: Overall, the test was very easy-to-use and the instrument was robust and reliable in field-settings. The sensitivity of the assay was 100% and the specificity was 98.63% (95%CI: 96.34-100.92%). The positive and negative predictive values were 97.73 (95%CI:94.77-100.68%) and 100%, respectively. The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, especially in resource-limited settings., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In Italy in 2016, acute flaccid myelitis developed in a woman who had received a hematopoietic stem cell transplant. Enterovirus D68 viral genome was detected in respiratory and cerebrospinal fluid samples, and the viral protein 1 sequence clustered with lineage B3. Immunocompromised adults may be at risk for enterovirus D68-associated neurologic complications.

Here, we report a patient who developed diplopia secondary to a right cranial nerve III and IV palsy, as well as fever and headache. Cerebrospinal fluid analysis (CSF) showed high varicella-zoster virus (VZV)-DNA viral load (>300,000,000 copies/ml). VZV antibodies in CSF was ≥1:16. Diagnosis of neurological reactivation of VZV infection was made without the presence of characteristic vesicular rash. Quantitative real-time PCR for VZV and intrathecal dosage of VZV IgM and IgG should be performed in cases suspected for viral encephalitis and also in all patients with not otherwise attributable cranial nerve lesions.

In 2009 an influenza A epidemic caused by a swine origin H1N1strain, unusual in human hosts, has been described. The present research is aimed to perform the first phylogenetic investigation on the influenza virus A (H1N1) strains circulating in Montenegro, from December 1, 2009, when the first case of death due to H1N1 was confirmed, and the epidemic began causing a total of four fatalities. The phylogenetic analysis of the strains circulating showed the absence of a pure Montenegrin cluster, suggesting the occurrence of multiple re-introductions in that population from different areas till as far as the early 2010. The time to most recent common ancestor (TMRCA) for the complete dataset has been dated in early 2008, pre-dating the first Montenegrin identification of H1N1 infection. These data suggest that virus was spreading undetected, may be as a consequence of unidentified infections in returning travelers. Anyhow, the estimated TMRCA of Montenegrin strains is fully consistent to that found in different areas. Compatibly with the time coverage of the study period here analyzed, molecular dynamic of Montenegrin strains follows similar trend as in other countries. J. Med. Virol. 88:1905-1913, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

In March/April 2009, a new pandemic influenza A virus (A(H1N1)pdm09) emerged and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century. Influenza virus may be present in the infected host as a mixture of variants, referred to as quasi-species, on which natural and immune-driven selection operates. Since hemagglutinin (HA) and non-structural 1 (NS1) proteins are relevant in respect of adaptive and innate immune responses, the present study was aimed at establishing the intra-host genetic heterogeneity of HA and NS1 genes, applying ultra-deep pyrosequencing (UDPS) to nasopharyngeal swabs (NPS) from patients with confirmed influenza A(H1N1)pdm09 infection. The intra-patient nucleotide diversity of HA was significantly higher than that of NS1 (median (IQR): 37.9 (32.8-42.3) X 10(-4) vs 30.6 (27.4-33.6) X 10(-4) substitutions/site, p = 0.024); no significant correlation for nucleotide diversity of NS1 and HA was observed (r = 0.319, p = 0.29). Furthermore, a strong inverse correlation between nucleotide diversity of NS1 and viral load was observed (r = - 0.74, p = 0.004). For both HA and NS1, the variants appeared scattered along the genes, thus indicating no privileged mutation site. Known polymorphisms, S203T (HA) and I123V (NS1), were observed as dominant variants (>98%) in almost all patients; three HA and two NS1 further variants were observed at frequency >40%; a number of additional variants were detected at frequency <6% (minority variants), of which three HA and four NS1 variants were novel. In few patients multiple variants were observed at HA residues 203 and 222. According to the FLUSURVER tool, some of these variants may affect immune recognition and host range; however, these inferences are based on H5N1, and their extension to A(H1N1)pdm09 requires caution. More studies are necessary to address the significance of the composite nature of influenza virus quasi-species within infected patients.

In vitro models of HCV infection have allowed for the clarifying of molecules and mechanisms involved in the main steps of virus cell-entry. HCV entry and neutralization appear to be closely related. Neutralizing antibodies inhibit the E2-CD81 binding, therefore CD81 is considered to be a major target of immune response. The tight-junction proteins are also implicated in E2-binding to CD81 and successive steps of virus entry, in cooperation with several co-receptors, whose involvement has still to be elucidated. Increasing evidence has emphasized the importance of cell-to-cell HCV-transmission in chronic infection. This route for infection could favour virus-escape from host-neutralization though its CD81-dependency is still debated. The main reasons which have delayed our understanding of HCV-infection are here critically reviewed, as are the challenges faced by investigators in the field. A deeper insight into the different pathways involved could help to elucidate some crucial features of HCV infection mechanisms and disclose important implications in its pathogenesis, which could help in suggesting new targets for successful immune-prophylactic/therapeutic strategies.

During the 2009 pandemic the Virology Laboratory of L. Spallanzani, Rome, Italy, adopted a real-time RT-PCR developed by the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia to diagnose pandemic influenza A/H1N1 (H1N1pdm). A new multiplex real-time RT-PCR distributed by Astra Diagnostics, coupled with the extraction system developed and commercialized by Siemens Healthcare Diagnostics (referred to as the RealStar system), was tested for the ability to detect and type influenza A in clinical samples, with particular emphasis on influenza A-positive samples untyped by the CDC method. Seventy-six nasopharyngeal swabs, resulting by the CDC method H1N1pdm (n=7), H3N2 (n=3), and not subtyped (n=66), were re-analysed with the RealStar system. All H3N2 and H1N1pdm-positive samples were correctly identified; among the untyped samples, the RealStar system detected 24/66 (36.4%) H1N1pdm and 1/66 (1.5%) seasonal influenza A. In conclusion, the RealStar system confirmed the results of all the influenza A-positive samples subtyped by the CDC method, and was able to type 37.9% of samples untyped by the CDC method. However, 62.1% of samples, detected as influenza A-positive but not subtyped by the CDC method, were found to be negative by the RealStar system. Further investigation is needed to explain this latter, unexpected, finding., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: The first influenza pandemic of the 21th century was ignited by a new strain of influenza A virus (A/H1N1pdm). Specific patient groups, including those with comorbidities, pregnant women, young children, older and immunocompromised patients, are at increased risk for serious influenza-related disease. This study was aimed at investigating the influence of clinical presentation, antiviral treatment and possible drug resistance-associated mutations, on the extent and duration of viral shedding in patients infected with A/H1N1pdm., Methods: An observational study was performed, based on retrospective review of clinical and laboratory records of patients who were hospitalized for A/H1N1pdm infection at the National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy, between April 24 and December 31, 2009. Among 119 hospitalized patients, 39 were selected for a post hoc analysis, based on the availability of serial nasopharyngeal swabs samples and related information., Results: Eleven out of the 39 study patients (28.2%) presented with pneumonia; 29 (74.4%) received antiviral treatment. Patients with pneumonia were significantly older than patients without pneumonia. The mean values of viral RNA concentration were not significantly increased in patients with pneumonia, but a significant increase in the duration of viral shedding was observed as compared to patients without pneumonia. In patients receiving antivirals, the viral RNA concentration was significantly reduced in comparison to untreated patients at days 4-5 after symptom onset, while the overall duration of viral shedding was only marginally affected. A significant correlation between duration of viral shedding and time elapsed between symptom onset and therapy start was observed, with a significant reduction of days of viral shedding when therapy was initiated within 2 days of symptoms appearance. No known drug resistance mutations were detected in patients with prolonged viral shedding., Conclusions: Our results show that severe respiratory illness is associated with delayed virus clearance in patients with A/H1N1pdm infection. Antivirals caused an early reduction of viral load, but only marginally affected the overall duration of shedding. Prolonged shedding was not associated with the emergence of strains carrying known drug-resistance mutations.

Molecular testing of 270 consecutive nasopharyngeal swab samples taken in May and June 2009 and 274 samples from patients hospitalized between July and December 2009 showed similar findings of respiratory viruses, with influenza A pandemic virus H1N1/09 being the most represented, followed by human parainfluenza virus type 3 and rhinoviruses. Statistical analyses suggested virus cocirculation in the absence of viral interference.

Influenza A( H1N1)v has spread rapidly in all parts of the globe in 2009 as a true pandemic, although fortunately a clinically mild one. The relevant evolutionary steps for the new virus to adapt to human populations occurred very early during the pandemic, before the end of April. Of the several resulting clades or clusters, clade 7 appeared later and proved more successful, substituting all other early clades before the bulk of the worldwide infections occurred.

Virus cell-to-cell spread has been reported for many different viruses and may contribute to pathogenesis of viral disease. The role played by cell-to-cell contact in hepatitis C virus (HCV) transmission was studied in vitro by cell co-cultivation experiments. A human lymphoblastoid B-cell line, infected persistently with HCV in vitro (TO.FE(HCV)), was used as HCV donor [Serafino et al., 2003]; recipient cells were the human hepatoma HepG2 cell line. Both cell types were co-cultured for 48 hr to allow the cell-to-cell contacts. The hepatoma HepG2 cells are not permissive to free-virus infection, but they were infected successfully using TO.FE(HCV) cells as source of virus. The kinetics of viral RNA synthesis and the percentage of infected cells were compared in cell-mediated-and cell-free-viral infection. After co-cultivation, a consistent proportion of hepatoma cells replicated HCV and stably expressed viral antigens. Virus produced was infectious as demonstrated by the ability to reinfect fresh B-cells. This cell model shows that permissiveness to HCV infection can be achieved in vitro in non-permissive hepatoma cells by direct cell-to-cell contacts with infected human B-cells. This mechanism of virus spread may also play a pathogenic role in vivo., (Copyright 2005 Wiley-Liss, Inc.)

Objectives: The aim of this study was to investigate transthyretin (prealbumin) effects on Abeta25-35-induced cytotoxicity., Design and Methods: In view of the well-recognized literature data demonstrating that Abeta25-35 fibrillar aggregates cause in vitro cytotoxicity to human red blood cells and apoptotic changes to SK-N-BE neuroblastoma cells in cultures (ultrastructural evidence), we tested transthyretin effects on these two experimental models., Results: Incubation of Abeta25-35 with transthyretin (at transthyretin concentrations equal to CSF physiological levels) demonstrated both inhibition of red blood cells lysis and neutralization of SK-N-BE neuroblastoma cells ultrastructural apoptotic changes. Moreover, transthyretin was shown to be able to inhibit the formation of fibrillar macroaggregates of Abeta25-35., Conclusions: The findings imply that experimental systems investigating Abeta-induced cytotoxicity consider the protective interaction of transthyretin with Abeta; an interaction to be considered also in vivo in view of the fact that transthyretin immunoreactivity has been previously demonstrated in amyloid plaques of brains from Alzheimer's disease patients.

Background: Fibrillar aggregates of amyloid beta 25-35 (Abeta(25-35)) form rapidly in vitro able to lyse human red blood cells (RBCs). Human sera, albumin, and apolipoprotein E (ApoE) each limit fibrillation and cytotoxicity. Potentially, these substances protect neurons from Abeta(1-40/42) aggregates. Transferrin (TF) is investigated in this study., Methods: The Mattson red blood cells model was employed to determine whether co-incubation of transferrin and Abeta(25-35) prevented lysis. The formation of fibrillar Abeta(25-35) in the presence of transferrin was investigated using Congo red staining and spectrophotometric studies., Results: We found that incubation of 20 muM Abeta(25-35) with physiologic levels of transferrin prevented red blood cells lysis and the formation of macro-aggregates., Conclusions: These in vitro results suggest that transferrin may limit fibrillar beta amyloid formation in vivo and cytotoxicity.

Iacovacci et al. [(1997a) Research in Virology 148:147-151] described that the euploid diploid cells, of the normal human bone marrow-derived lymphoblastoid B-cell line TO.FE., are susceptible to hepatitis C virus (HCV) infection and support long term virus production. Transmission electron microscopy described some steps of HCV replication cycle in this in vitro infected cellular system [Serafino et al. (1997) Research in Virology 148:153-159]. In the present study, in order to identify the intracellular sites involved in HCV replication, the ultrastructural changes associated with infection in TO.FE. cells were correlated with the subcellular localisation of structural and nonstructural viral proteins. Transmission electron microscopy and confocal microscopy data indicate that these viral proteins appeared located in the Golgi apparatus and endoplasmic reticulum, suggesting an active involvement of these compartments in viral assembly and morphogenesis. Furthermore, transmission and scanning electron microscopic observations on cultures infected chronically support the hypothesis that these cellular compartments may serve as starting sites of the morphological changes associated to viral infection and replication, leading to cell-cell fusion, syncytia formation, and finally lysis of infected cells and virus release., (Copyright 2003 Wiley-Liss, Inc.)

Inhibition of the lysis of human red blood cells (RBCs) exposed to amyloid peptide Abeta25-35 is an model for screening natural and synthetic substances potentially protective against amyloid damage. In this system, human serum and a component, namely apolipoprotein E (apoE), completely prevent RBC lysis. This report demonstrates that albumin, another serum component, is 8-fold more protective: a concentration of 12.5 microg/ml protects RBCs against 20 microM-Abeta25-35, and prevents the formation of fibrillar Abeta25-35 aggregates stainable by Congo Red. The biological relevance of these findings is suggested by the following: (1) a large fraction ( approximately 90%) of circulating Abeta1-42 is bound to albumin; (2) albumin immunoreactivity is present in brain amyloid plaques; and (3) incubation of Abeta with albumin rapidly decreases detectable levels of free Abeta suggesting epitope masking. The results add new and important functional consequences to the amyloid-albumin relationship and imply that experimental systems investigating Abeta cytotoxicity should consider the protective interaction of albumin.

Mattson et al. (9) demonstrated lysis of human red blood cells (RBC) exposed to amyloid peptide Abeta(25-35), a new experimental model for amyloid-beta toxicity. Lysis resulted from poreformation in the RBC membranes and was completely prevented by concurrent exposure to Congo red We demonstrate that human serum, purified ApoE from human plasma, and recombinant isoforms of ApoE neutralize the Abeta(25-35) cytotoxicity: the E2 and E4 isoforms were marginally more effective than E3. Second, we demonstrate that Abeta(25-35) forms fibrils in the reaction mixtures using electron-microscopy. Together these results suggest that the RBC model might be useful in preliminary identification of natural and synthetic substances able to protect against amyloid-beta cytotoxic effects due to fibrillar Abeta(25-35). Such compounds would be candidate molecules for testing in neuronal systems.

In this preliminary report, we provide evidence that the human B-lymphoblastoid cell line (LCL) CE, bone-marrow-derived, previously reported to be permissive to hepatitis C virus, is also permissive to HIV1 infection. HIV1 genomes were detectable in cell supernatants, virus RNA transcripts and proviral DNAs in cell extracts at different times post-infection. Therefore, we propose this LCL cell line as a tool for exploring the mutual interactions of the two viruses in double-infected cells.

We previously demonstrated that the human lymphoblastoid B-cell line (LCL) TOFE, derived from normal human bone marrow, is permissive to HCV infection. In this report we developed an in vitro HCV adsorption-inhibition assay based on TOFE cells, to reveal the presence of neutralizing antibodies in sera from acutely infected patients.

In order to directly ascertain the presence of HCV virus infection in livers of patients with HCV chronic hepatitis, we investigated, by transmission electron microscopy (TEM), liver biopsies from 2 adults and 4 children for the presence of virus-like particles (VLPs). The plasmas of these HCV-positive patients were HCV-RNA-positive, with high ALT values. In liver tissue samples examined, we were able to detect plus and minus strands of HCV RNA by strand-specific RT-PCR. Aggregates or single VLPs of about 45 nm in diameter were detectable in variable amounts in endoplasmic cisternae and in hepatocyte cytoplasms of infected patients. These results emphasize the relevance of performing TEM assays to confirm the diagnosis of HCV infection.

In this study, we analysed by transmission electron microscopy (TEM), sequential details of morphological modifications that accompanied viral morphogenesis in the lymphoblastoid cell line (LCL) TOFE infected in vitro with hepatitis C virus (HCV). As previously reported, we observed virus-like particles (VLPs) in cytoplasmic vesicles mainly located in the perinuclear region of infected cells. In this area, the Golgi apparatus and the endoplasmic reticulum (ER) appeared hyperplastic, remarkably enriched in vesicles and lysosomal structures. Furthermore, only in this perinuclear region, cytopathic-effect(CPE)-like changes seemed to originate, consisting in enlarged cytoplasmic vacuoles filled with degenerative amorphous material containing VLPs. Finally, the complete filling-up of the cytoplasm with these degenerative vacuoles, in addition to cellular lysis displayed by some cells, appeared as the possible terminal pattern of the infectious process. Our data suggest that in vitro HCV-infected TOFE cells undergo typical CPE-like changes that may be connected with virus replication.

The molecular features of hepatitis C virus (HCV) replication in human fetal hepatocytes (HFHs) were addressed in this study. Using a competitive reverse-transcription polymerase chain reaction (RT-PCR) assay for the quantitation of HCV-RNA molecules, the highest level of viral replication was detected 30 days' postinfection. At this time point, viral particles of 41 to 45 nm in diameter accumulated in the cell cytoplasm. Their density in cell extracts and culture medium was distributed between heavy (1.180-1.360 g/cm3) and light fractions (1.105-1.050 g/cm3) of a sucrose gradient, while, in the serum inoculum, they had a positive fraction at 1.180 g/cm3. In infected HFHs, minus-strand HCV RNA was observed in fractions displaying a sedimentation coefficient of 28 S to 18 S, while plus-strand HCV RNA showed a peak restricted to the 21 S fraction; the HCV RNA of serum inoculum had a sedimentation coefficient of 38 to 40 S, which revealed the presence of HCV RNA of unique positive polarity. The 21 S RNA fraction of cell extracts was resistant to 20 minutes of RNase I digestion, while the same incubation time totally inactivated a comparable amount of HCV RNA purified from the serum inoculum, revealing the presence of completely and/or partially double-stranded HCV-RNA molecules in the infected cells. Detection in HFHs of replicative forms and replicative intermediates suggests that the dynamic profile of HCV replication in these cells is similar to that described in other flaviviruses.

The susceptibility of Vero cells and derivative cell clones to hepatitis C virus (HCV) infection was assayed by qualitative and quantitative polymerase chain reaction (PCR)-based methods. Cell extracts from Vero cells inoculated with HCV were tested for the presence of both positive and negative strands of HCV RNA; in parallel, cell-free HCV genomes were assayed in culture supernatant fluids. Quantitation of genomic HCV RNA molecules in infected cells by competitive reverse transcription PCR (cRT-PCR) indicated that HCV replication was more efficient in a derivative clone (named clone 10) than in parental Vero cells or other clones under study. Analysis of HCV-binding to cell receptors, performed by cRT-PCR quantitation of viral particles adsorbed to the cell surface, demonstrated a 10-fold higher virus-binding level of clone 10 than that of parental Vero cells. The results shown here indicate that the Vero clone 10 may constitute an efficient model system for analysing early events in HCV infection as well as a source of virus for diagnostic and biotechnological applications.

Hepatitis C virus (HCV)-infected TOFE cells, a human bone marrow-derived B-cell line, were studied by transmission electron microscopy (TEM). We reported the presence of virus-like particles, (VLPs) having a diameter of 45 nm on average, within the cytoplasm of HCV-infected cells and their absence in uninfected cells. The VLPs were mostly seen in dilated cisternae of endoplasmic reticulum (ER) and consisted of an electron-dense core and a membrane envelope with surface projections. In the HCV-infected TOFE cells, examination by TEM revealed tubular structures similar to those observed in liver cells of chimpanzees and humans infected by the virus. In some instances, the outer membrane of the particles was connected to the ER membrane, indicating possible virus budding into the cisternae. Rarely, coated vesicles containing a particle were seen. In most of the infected cells, enlarged cytoplasmic vacuoles filled with degenerative amorphous material were observed. The data suggest a flavivirus-like pattern of HCV morphogenesis in infected non-hepatic cells and identify lymphoblastoid TOFE cells as a valuable system for the study of the sequential steps of the infection process and the morphological effects produced by HCV infection.

The ability of hepatitis C virus (HCV) to replicate in two B-cell lines, CE and TOFE, derived from bone marrow of healthy subjects was compared using qualitative and quantitative molecular methods. The presence of intracellular negative-stranded HCV RNA (replicative intermediate) was investigated by nested polymerase chain reaction (PCR) in the infected cultures at different times after infection. The amounts of positive-stranded HCV RNA (genomic RNA copies) synthesized and released from cells one week after in vitro infection were determined by competitive PCR after reverse transcription of viral RNA for the 5' viral untranslated region. In both cell lines, HCV RNA replication took place, but the TOFE cell line appeared to be a more efficient virus producer than the CE cell line. The TOFE cell line could be a valuable and reliable tool for basic and clinical HCV studies.