Antonio Piralla

Cross-neutralization of four paramyxoviruses by a human monoclonal antibody

Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel β-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine.

Correlation of Rhinovirus Load in the Respiratory Tract and Clinical Symptoms in Hospitalized Immunocompetent and Immunocompromised Patients

While human rhinoviruses (HRVs) are well accepted as a major cause of common cold syndromes (rhinitis), their role in the etiology of lower respiratory tract infections is still controversial, and their detection in asymptomatic patients is relatively common. The HRV pathogenic role in four groups of hospitalized patients (pediatric immunocompetent and immunocompromised patients, and adult immunocompetent and immunocompromised patients) was investigated by quantifying HRV load in nasopharyngeal aspirates or bronchoalveolar lavage samples by real‐time reverse transcription PCR (RT‐PCR). Real‐time RT‐PCR was performed in duplicate on all respiratory samples resulting positive by qualitative RT‐PCR. In addition, molecular typing allowed detection of all known HRV species (A, B, and C). In immunocompetent pediatric patients HRVs were mostly associated with lower respiratory tract infections (in the absence of other viral agents) and wheezing, when viral load was ≥106 RNA copies/ml. In young immunocompromised patients (stem cell transplantation recipients), an inverse correlation between HRV persistence over time and time at which the infection occurred after transplantation was observed, whereas in adult immunocompromised patients (lung transplant recipients) HRVs could be detected at a medium–low level (<105 RNA copies/ml) in bronchoalveolar lavage samples taken routinely from asymptomatic patients. In conclusion, when detected at high viral load, HRVs may cause severe upper and lower respiratory tract infections, whereas when detected at a medium–low viral load, an event more frequent in immunocompromised subjects, they may represent only bystander viruses. J. Med. Virol. 81:1498–1507, 2009.

Human respiratory coronavirus HKU1 versus other coronavirus infections in Italian hospitalised patients

Abstract Background Human respiratory coronavirus (hCoV) HKU1 infections were reported for the first time in 2005 in Hong Kong. Objective To investigate epidemiological, clinical, and diagnostic features of HKU1 infections. Study design Longitudinal, prospective study from November 2005 through May 2006 in a hospitalised patient population. Results Overall, 48/426 (11.3%) patients were found to be infected by hCoV acute respiratory tract infections (ARTI). Of these, 10 (19.2%) were caused by HKU1 (6 single infections and 4 coinfections) during the period January–May 2006. Diagnosis was made by using RT-PCR for all four hCoVs, and in parallel, in-house developed group-specific monoclonal antibodies (MAbs) for HKU1 and 229E. HKU1-specific MAb was able to retrospectively identify 8 of 10 HKU1 strains detected by RT-PCR. Phylogenetic analysis showed that four HKU1 strains were genotype A and six genotype B. In HKU1-infected patients, the predominant clinical symptom was rhinorrhea (nine patients). Within group II hCoV, HKU1-infected patients had a significantly lower rate of lower ARTI compared to OC43-infected patients. Conclusion HKU1 hCoV strains circulated in northern Italy during the winter–spring season 2005–2006. Both HKU1 genotypes were detected. HKU1-specific MAb may contribute to the rapid diagnosis of HKU1 infections currently performed by RT-PCR.

Comparative evaluation of eight commercial human cytomegalovirus IgG avidity assays

BACKGROUND The interpretation of a positive IgM antibody result to human cytomegalovirus (HCMV) in a pregnant woman is of major importance for the correct management of the pregnancy. Determination of HCMV-specific IgG avidity is considered an useful approach for distinguishing IgM antibody due to primary HCMV infection from IgM antibody elicited during non-primary infection. OBJECTIVE Comparative evaluation of eight commercial HCMV IgG avidity assays currently available in Europe. STUDY DESIGN A panel of 198 sequential samples collected from 65 pregnant women at 0-90, 91-180, and >180 days after the onset of primary HCMV infection was retrospectively tested by Abbott, BioMérieux, Bio-Rad, DiaSorin, Diesse, Euroimmun, Radim, and Technogenetics HCMV IgG avidity assays according to the manufacturers instructions. RESULTS None of the 198 samples tested yielded identical scores by the kits under evaluation. The Euroimmun and Radim assays showed the best correlation with expected results in terms of low (0-90 days), intermediate (90-180 days) and high (>180 days) avidity results, respectively. The best accuracy in diagnosing a recent (<90 days after the onset) or non-recent (>180 days after the onset) primary HCMV infection was shown by Radim followed by Euroimmun and Diesse. The best correlation with a well established in-house developed HCMV IgG avidity assay was shown by Radim. CONCLUSIONS HCMV IgG avidity kits need to be improved and standardized. In the meantime, highly specific IgM assays are preferable for screening purposes in pregnant women.

Naturally occurring mutations to HCV protease inhibitors in treatment-naïve patients

BackgroundProtease inhibitors (PIs) to treat hepatitis C (HCV) virus infection have been approved and others are under development.ResultsThe aims of this study were to illustrate natural polymorphisms in the HCV protease and measure the frequency of PI resistance mutations in different HCV genotypes from PI-naïve patients.Direct sequencing of HCV NS3/4A protease was performed in 156 HCV patients naïve to PIs who were infected with genotype 1a (n = 31), 1b (n = 39), 2 (n = 30), 3 (n = 33) and 4 (n = 23).Amino acid (aa) substitutions associated with HCV PI resistance were found in 17/156 (10.8%) sequences. Mutations V36L, T54S, V55A/I, and Q80K/L were observed in 29% of patients with genotype 1a, and V55F, Q80L/N and M175L in 10% of patients with genotype 1b. The mutation V158M was found in 3% of patients with genotype 2, D168Q was present in 100% of patients with genotype 3 and D168E was observed in 13% of patients with genotype 4. In addition, multiple aa polymorphisms not associated with PI resistance were detected in patients with genotypes 1a, 1b and 4.ConclusionsAlthough major PI resistance mutations were not detected, other resistance mutations conferring low level resistance to PIs together with a number of natural polymorphisms were observed in proteases of PI naïve HCV patients. A more extensive analysis is needed to better evaluate the impact of baseline resistance and compensatory mutations in the efficacy of HCV PI treatment.

Human rhinovirus and human respiratory enterovirus (EV68 and EV104) infections in hospitalized patients in Italy, 2008-2009.

The epidemiology of picornavirus infections along with associated risk factors for lower respiratory tract infections (LRTI) and duration of virus shedding were investigated in 985 hospitalized patients in the period October 2008-September 2009. One-third of patients were human rhinovirus (HRV)-positive. Of 336 HRV-associated episodes, 153 (45.5%) were sustained by HRV-A, 31 (9.2%) by HRV-B, and 93 (27.7%) by HRV-C, while 7 episodes showed multiple HRV types and 52 were sustained by undefined HRV species. Independent risk factors for LRTI included high viral load and age less than 5 years. Twenty (2.1%) patients were enterovirus (EV)-positive (12 had EV-68, 7 EV-104, and 1 E-13 infection). Half of the EV-positive patients had a LRTI and were younger with respect to patients with upper RTI (median 18 months versus 37 years; P < 0.001). HRVs are often the cause of LRTI in children less than 5 years, frequently in association with a high viral load.

Phylogenetic Patterns of Human Respiratory Picornavirus Species, Including the Newly Identified Group C Rhinoviruses, during a 1-Year Surveillance of a Hospitalized Patient Population in Italy

ABSTRACT Human rhinovirus species C (HRV-C) was the second most common HRV species detected in hospitalized patients in Italy with acute respiratory disease during a 1-year surveillance period. Sequencing of the picornavirus VP4/VP2 region allowed molecular typing of HRV-A and HRV-B and provisional typing of HRV-C.

Phylogenetic characterization of enterovirus 68 strains in patients with respiratory syndromes in Italy.

Enterovirus 68 (EV‐D68) was associated with mild to severe respiratory infections. In the last 4 years, circulation of different EV‐D68 strains has been documented worldwide. In this study, the phylogenetic characterization of nine EV‐D68 strains identified in patients in the 2010–2012 period and 12 additional EV‐D68 Italian strains previously identified in 2008 in Italy was described. From January 2010 to December 2012, a total of 889 respiratory specimens from 588 patients stayed or visited at the Fondazione IRCCS Policlinico San Matteo were positive for HRV or HEV. Extracted nucleic acids were amplified by one‐step RT‐PCR with primer specific for VP1 region of EV‐D68 and purified positive PCR products were directly sequenced. Overall, 9/3736 (0.24%) patients were EV‐D68 positive. Of these, 7/9 (77.8%) were pediatric and two (22.2%) were adults. Five out of seven (71.4%) pediatric patients had lower respiratory tract infection with oxygen saturation <94%. Four cases were detected from August through October 2010, while five other cases from September through December 2012. The Italian EV‐D68 strains in 2008 belonged to clade A (n = 5) and clade C (n = 7). In 2010 all the Italian strains belonged to clade A (n = 4) and in 2012, four Italian strains belonged to clade B and one to clade A. In conclusion, we provide additional evidence supporting a role of EV‐D68 in severe respiratory infection in pediatric patients. In addition, all the three EV‐D68 clades circulating worldwide were identified in Italy in a 5‐year period of time. J. Med. Virol. 86:1590–1593, 2014.

Molecular epidemiology of primary human cytomegalovirus infection in pregnant women and their families

The source of human cytomegalovirus (HCMV) infection was investigated in 29 pregnant women with primary HCMV infection by comparing DNA sequences of UL146, UL144 and a portion of UL55 gene of HCMV strains circulating within each family. Thirteen families were identified in which the pregnant woman, the husband and/or a child were shedding HCMV. In three of these families, both the woman and the husband suffered from a concomitant primary HCMV infection. Phylogenetic analysis of UL146, UL144, and UL55 genes indicated that strains circulating within each family were identical, whereas strains from different families appeared to be distinct. However, identical UL146, UL144, and UL55 DNA sequences were observed sporadically among unrelated strains. A child rather than the husband was the virus source for the great majority of pregnant women. No association was observed between UL144 polymorphisms and intrauterine transmission. J. Med. Virol. 80:1415–1425, 2008.

Characteristics and Their Clinical Relevance of Respiratory Syncytial Virus Types and Genotypes Circulating in Northern Italy in Five Consecutive Winter Seasons

In order to investigate the genetic diversity and patterns of the co-circulating genotypes of respiratory syncytial virus (RSV) and their possible relationships with the severity of RSV infection, we studied all of the RSV-positive nasopharyngeal samples collected from children during five consecutive winters (2009–2010, 2010–2011, 2011–2012, 2012–2013 and 2013–2014). The RSVs were detected using the respiratory virus panel fast assay and single-tube RT-PCR, their nucleotides were sequenced, and they were tested for positive selection. Of the 165 positive samples, 131 (79.4%) carried RSV-A and 34 (20.6%) RSV-B; both groups co-circulated in all of the study periods, with RSV-A predominating in all the seasons except for winter 2010–2011, which had a predominance of RSV-B. Phylogenetic analysis of the RSV-A sequences identified genotypes NA1 and ON1, the second replacing the first during the last two years of the study period. The RSV-B belonged to genotypes BA9 and BA10. BA9 was detected in all the years of the study whereas BA only desultorily. Comparison of the subjects infected by RSV-A and RSV-B types did not reveal any significant differences, but the children infected by genotype A/NA1 more frequently had lower respiratory tract infections (p<0.0001) and required hospitalisation (p = 0.007) more often than those infected by genotype A/ON1. These findings show that RSV has complex patterns of circulation characterised by the periodical replacement of the predominant genotypes, and indicate that the circulation and pathogenic role of the different RSV strains should be investigated as each may have a different impact on the host. A knowledge of the correlations between types, genotypes and disease severity may also be important in order to be able to include the more virulent strains in future vaccines.