Molecular characterization of emerging Echovirus 11 (E11) shed light on the recombinant origin of a variant associated with severe hepatitis in neonates.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. Due to the limited data available, the World Health Organization (WHO) considers public health risk to the general population to be low. The present study investigated the genetic variation and molecular evolution of E11 genomes collected from May to December 2023. Whole genome sequencing (WGS) was performed for 16 E11 strains. Phylogenetic analysis on WG showed how all Italian strains belonged to genogroup D5, similarly to other E11 strains recently reported in France and Germany all together aggregated into separate clusters. A cluster-specific recombination pattern was also identified using phylogenetic analysis of different genome regions. Echovirus 6 was identified as the major recombinant virus in 3Cpro and 3Dpol regions. The molecular clock analysis revealed that the recombination event probably occurred in June 2018 (95% HPD interval: Jan 2016-Jan 2020). Shannon entropy analyses, within P1 region, showed how 11 amino acids exhibited relatively high entropy. Five of them were exposed on the canyon region which is responsible for receptor binding with the neonatal Fc receptor. The present study showed the recombinant origin of a new lineage of E11 associated with severe neonatal infections.

Dynamics of viral DNA shedding and culture viral DNA positivity in different clinical samples collected during the 2022 mpox outbreak in Lombardy, Italy.

BACKGROUND: Mpox virus (MPXV) has recently spread outside of sub-Saharan Africa. This large multicentre study was conducted in Lombardy, the most densely populated Italian region accounting for more than 40% of Italian cases. The present study aims to: i) evaluate the presence and the shedding duration of MPXV DNA in different body compartments correlating the MPXV viability with the time to onset of symptoms; ii) provide evidence of MPXV persistence in different body compartment as a source of infection and iii) characterize the MPXV evolution by whole genome sequencing (WGS) during the outbreak occurred in Italy. MATERIAL AND METHODS: The study included 353 patients with a laboratory-confirmed diagnosis of MPXV infection screened in several clinical specimens in the period May 24th - September 1st, 2022. Viral isolation was attempted from different biological matrices and complete genome sequencing was performed for 61 MPXV strains. RESULTS: MPXV DNA detection was more frequent in the skin (94.4%) with the longest median time of viral clearance (16 days). The actively-replicating virus in cell culture was obtained for 123/377 (32.6%) samples with a significant higher viral quantity on isolation positive samples (20 vs 31, p < 0.001). The phylogenetic analysis highlighted the high genetic identity of the MPXV strains collected, both globally and within the Lombardy region. CONCLUSION: Skin lesion is gold standard material and the high viral load and the actively-replicating virus observed in genital sites confirms that sexual contact plays a key role in the viral transmission.

Increased circulation of echovirus 11 in the general population and hospital patients as elicited by the non-polio enterovirus laboratory-based sentinel surveillance in northern Italy, 2023.

OBJECTIVES: Following the alert of echovirus 11 (E-11) infection in neonates in EU/EEA Member States, we conducted an investigation of E-11 circulation by gathering data from community and hospital surveillance of enterovirus (EV) in northern Italy from 01 August 2021 to 30 June 2023. METHODS: Virological results of EVs were obtained from the regional sentinel surveillance database for influenza-like illness (ILI) in outpatients, and from the laboratory database of ten hospitals for inpatients with either respiratory or neurological symptoms. Molecular characterization of EVs was performed by sequence analysis of the VP1 gene. RESULTS: In our ILI series, the rate of EV-positive specimens showed an upward trend from the end of May 2023, culminating at the end of June, coinciding with an increase in EV-positive hospital cases. The E-11 identified belonged to the D5 genogroup and the majority (83%) were closely associated with the novel E-11 variant, first identified in severe neonatal infections in France since 2022. E-11 was identified sporadically in community cases until February 2023, when it was also found in hospitalized cases with a range of clinical manifestations. All E-11 cases were children, with 14 out of 24 cases identified through hospital surveillance. Of these cases, 60% were neonates, and 71% had severe clinical manifestations. CONCLUSION: Baseline epidemiological data collected since 2021 through EV laboratory-based surveillance have rapidly tracked the E-11 variant since November 2022, alongside its transmission during the late spring of 2023.

Mpox Virus in the Pharynx of Men Having Sex with Men: A Case Series.

The recent Mpox virus (MPV) outbreak in Europe and North America, primarily among men who have sex with men (MSM), raised concerns about various transmission sources. We examined patients with Mpox from an urban STI center in Lombardy, Italy, between May and August 2022. Demographic, transmission, and clinical data were collected using a standardized form. Initial and subsequent tests were conducted using the RealStar Orthopoxvirus PCR Kit 1.0 (Altona Diagnostics, Hamburg, Germany) for skin lesions and oropharyngeal swabs. A total of 15 patients were recruited, all MSM, with 40% being HIV-positive. Almost all reported recent unprotected sexual activity. Oropharyngeal symptoms were observed in a minority, and oral cavity lesions were present in 20% of cases. MPV DNA was detected in skin lesions of 93% of patients and in oropharyngeal swabs of 87%. Skin samples exhibited a higher viral load than pharyngeal samples, with the latter persisting longer. Prospective follow-up of 11 individuals revealed an average pharyngeal persistence of 5.3 days beyond skin lesion clearance, reaching up to 80 days in an immunosuppressed case. Our findings indicate that MPV replication can persist in the pharynx asymptomatically and for an extended period.

An overview of SARS-CoV-2 variants circulating in the 2020-2022 period in Lombardy.

Since the beginning of the pandemic, SARS-CoV-2 has shown genetic variability. All the variants that have sustained pandemic waves have shown several mutations, especially in the Spike protein that could affect viral pathogenesis. A total of 15,729 respiratory samples, collected between December 2020 and August 2022, have been included in this study. We report the circulation of SARS-CoV-2 variants in the Lombardy region, Italy, in a 2-year study period. Alpha, Delta, and Omicron variants became predominant causing the majority of cases whereas Beta or Gamma variants mostly caused local outbreaks. Next-generation sequencing revealed several mutations and few deletions in all of the main variants. For example, 147 mutations were observed in the Spike protein of Omicron sublineages; 20% of these mutations occurred in the receptor-binding domain region.

Preliminary results on an autochthonous dengue outbreak in Lombardy Region, Italy, August 2023.

In August 2023, six locally acquired dengue virus 1 infections were detected in Lodi province, Lombardy Region, in northern Italy, where the vector Aedes albopictus is present. Four cases were hospitalised, none died. The viruses clustered with Peruvian and Brazilian strains collected between 2021 and 2023. This preliminary report highlights the importance of continued integrated surveillance of imported vector-borne virus infections and the potential for tropical disease outbreaks in highly populated regions of northern Italy where competent vectors are present.

Asymptomatic infection with clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) in carnivore pets, Italy, April 2023.

In April 2023, an outbreak of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses carrying the T271A mammalian adaptive mutation in the PB2 protein was detected in a backyard poultry farm in Italy. Five domestic dogs and one cat living on the premises had seroconverted in the absence of clinical signs. Virological and serological monitoring of individuals exposed to the virus proved the absence of human transmission, however, asymptomatic influenza A(H5N1) infections in mammalian pets may have important public health implications.

Fulminant echovirus 11 hepatitis in male non-identical twins in northern Italy, April 2023.

Echovirus 11 (E11) has recently been associated with a series of nine neonatal cases of severe hepatitis in France. Here, we present severe hepatitis caused by E11 in a pair of twins. In one of the neonates, the clinical picture evolved to fulminant hepatitis. The E11 genome showed 99% nucleotide identity with E11 strains reported in the cases in France. Rapid genome characterisation using next generation sequencing is essential to identify new and more pathogenetic variants.

Autoantibodies neutralizing type I IFNs underlie West Nile virus encephalitis in ∼40% of patients.

Mosquito-borne West Nile virus (WNV) infection is benign in most individuals but can cause encephalitis in <1% of infected individuals. We show that ∼35% of patients hospitalized for WNV disease (WNVD) in six independent cohorts from the EU and USA carry auto-Abs neutralizing IFN-α and/or -ω. The prevalence of these antibodies is highest in patients with encephalitis (∼40%), and that in individuals with silent WNV infection is as low as that in the general population. The odds ratios for WNVD in individuals with these auto-Abs relative to those without them in the general population range from 19.0 (95% CI 15.0-24.0, P value <10-15) for auto-Abs neutralizing only 100 pg/ml IFN-α and/or IFN-ω to 127.4 (CI 87.1-186.4, P value <10-15) for auto-Abs neutralizing both IFN-α and IFN-ω at a concentration of 10 ng/ml. These antibodies block the protective effect of IFN-α in Vero cells infected with WNV in vitro. Auto-Abs neutralizing IFN-α and/or IFN-ω underlie ∼40% of cases of WNV encephalitis.

Characterization of immune response against monkeypox virus in cohorts of infected patients, historic and newly vaccinated subjects.

Monkeypox virus (MPXV) is a zoonotic disease endemic in the rainforest countries of Central and West Africa. Understanding the immune response in zoonosis is fundamental to prevent and contrast viral spreading. MPXV is a close relative of Variola (smallpox) virus and vaccination with vaccinia virus gives approximatively 85% of protection against MPXV. With the emergence of the recent MPXV outbreak, JYNNEOS vaccine has been proposed to individuals at high-risk of exposure. Comparative data on MPXV immune response in vaccinated or infected subjects are still limited. Here we set-up an immunofluorescence method for the evaluation of humoral response elicited by natural infection and healthy vaccinated subjects, including historically smallpox-vaccinated individuals and newly vaccinated subjects. Neutralization assay was also included, and in vaccinated subjects, cell-mediated response was evaluated. We observed that the natural infection produces a strong immune response that can control the disease. In naïve subjects, a second dose boosts the serological response to levels similar to those of the MPXV patients. Last, smallpox-vaccinated controls retain a degree of protection, even after years from vaccination, most visible in the t-cellular response.

The WEST Study: A Retrospective and Multicentric Study on the Impact of Steroid Therapy in West Nile Encephalitis.

Background: The use of steroid therapy in potentially life-threatening neuroinvasive forms of West Nile infection (WNND) is controversial. The aim of this study is to assess the efficacy of steroid therapy in reducing intrahospital mortality, length of stay, and neurological sequelae at discharge. Methods: This was a multicenter, retrospective, observational study conducted in 5 hospitals in Northern Italy, headed by the Fondazione IRCSS Policlinico San Matteo (Pavia). We extracted all patient data with WNND diagnoses, comparing patients who received steroid treatment with patients who did not receive steroid treatment between January 2014 and January 2022. Comparisons between the 2 groups were performed using chi-square tests for categorical variables and Mann-Whitney tests for non-normal continuous data, and a generalized linear model for the binomial family was carried out. Results: Data from 65 WNND patients were extracted. Among these patients, 33 (50.7%) received steroid therapy at any point during their hospitalization. Receiving steroid therapy did not significantly reduce intrahospital mortality (odds ratio [OR], 1.70; 95% CI, 0.3-13.8; P = .89) or neurological sequelae at discharge (OR, 0.53; 95% CI, 0.16-1.76; P = .47). Conclusions: Steroid treatment is currently used on a single-case basis in severe WNND. More prospective data are needed to demonstrate a protective effect on mortality and neurological sequelae.

Characteristics and outcomes of vaccinated and nonvaccinated patients hospitalized in a single Italian hub for COVID-19 during the Delta and Omicron waves in Northern Italy.

OBJECTIVE: We compared the characteristics and outcomes of vaccinated and nonvaccinated patients hospitalized with COVID-19. DESIGN: We analyzed patients hospitalized in a COVID hub during three one-month periods: (i) October 15, 2020-November 15, 2020 (prevaccination peak); (ii) October 15, 2021-November 15, 2021 (Delta wave); (iii) December 15, 2021-January 15, 2022 (Omicron wave). To define the epidemiologic context, SARS-CoV-2 infection in healthcare workers was analyzed. RESULTS: SARS-CoV-2 infection incidence in healthcare workers was 146 cases per 1000 persons in 2020 (prevaccination) and 67 in 2021 (postvaccination, when the Omicron variant caused most infections). There were 420 hospitalized patients in the prevaccination period, 51 during the Delta wave (52.1% vaccinated) and 165 during the Omicron wave (52.9% vaccinated). During the Delta wave, a significantly higher number of nonvaccinated (29.2%) than vaccinated patients (3.7%) were admitted to the intensive care unit (ICU) (p = 0.019). Nonvaccinated patients were younger and had a lower rate of concomitant medical conditions (53.2% vs 83.7%; p < 0.001) during the Omicron wave when 80% of patients admitted to ICU and all those who died were still infected by the Delta variant. CONCLUSIONS: Vaccine effectiveness in fragile individuals appears to be lower because of a faster immunity decline. However, the Omicron variant seems to cause less severe COVID-19.

Human Herpes Virus 7-related encephalopathy in children.

BACKGROUND: Primary HHV7 infection is almost ubiquitous, and it can present as exanthema subitem. Little is known on the clinical relevance of HHV7 neuroinvasion in immunocompetent children. METHODS: We describe 12 patients (median age 9.45 years, 50% males) with acute encephalopathy and active HHV7 infection. In all patients, HHV7-DNA was detected on cerebrospinal fluid (CSF) by RT-PCR. RESULTS: 7/12 patients had meningoencephalitis (two with ADEM and one with MOG antibody-associated CIS); 5/12 showed acute neuropsychiatric symptoms. EEG showed anomalies exclusively in patients with meningoencephalitis. Six patients had RMN anomalies. CSF HHV7 copies ranged between 20 and 3,500 copies/mL (median 66 copies/mL) and mean HHV7 CSF/blood ratio was 0.75. Outcome was favorable in all children, although 3/12 had minor neurobehavioral sequelae. Mean follow-up period of 5.2 months. CONCLUSIONS: HHV7 can determine neuroinvasion in immunocompetent children, leading to acute encephalopathy. Blood-brain barrier damage and high CSF/blood viral copies ratio correlated with a more severe presentation. We speculate on the importance of immune-mediated mechanisms in provoking clinical features.

Comparative analysis of SARS-CoV-2 quasispecies in the upper and lower respiratory tract shows an ongoing evolution in the spike cleavage site.

Studies are needed to better understand the genomic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe viral quasispecies population of upper and lower respiratory tract by next-generation sequencing in patients admitted to intensive care unit. A deep sequencing of the S gene of SARS-CoV-2 from 109 clinical specimens, sampled from the upper respiratory tract (URT) and lower respiratory tract (LRT) of 77 patients was performed. A higher incidence of non-synonymous mutations and indels was observed in the LRT among minority variants. This might be explained by the ability of the virus to invade cells without interacting with ACE2 (e.g. exploiting macrophage phagocytosis). Minority variants are highly concentrated around the gene portion encoding for the Spike cleavage site, with a higher incidence in the URT; four mutations are highly recurring among samples and were found associated with the URT. Interestingly, 55.8% of minority variants detected in this locus were T>G and G>T transversions. Results from this study evidenced the presence of selective pressure and suggest that an evolutionary process is still ongoing in one of the crucial sites of spike protein associated with the spillover to humans.

SARS-CoV-2 infections in pediatric patients: A comparison of three pandemic waves.

The pediatric population seems to be at a lower risk of developing severe clinical symptoms of COVID-19. However, the clinical and epidemiological characteristics of COVID-19 in children are yet to be fully clarified. This retrospective observational study aimed to evaluate the frequency of pediatric laboratory-confirmed COVID-19 patients from February 2020 to April 2021. A total of 740 (5.1% of total) pediatric COVID-19 cases were observed during the study period. The peak of pediatric cases was observed in November 2020, with 239 cases. During the first wave of pandemics, the frequency of pediatric cases was 0.89% (49/5877 cases), ranging from 0.6% in February 2020 to 1.3% in April 2020. On the contrary, after the beginning of the second wave, the frequency of pediatric cases raised from 5.3% in September 2020 to 9.4%in February 2021, with an overall frequency of 8.2% (690/8416 cases). A different rate of SARS-CoV-2 circulation was observed among the pediatric population between the pandemic waves. During the second wave, two peaks of cases were observed. The last peak was associated with the spread of a more transmissive SARS-CoV-2 strain (VOC 202012/01).

Spread of multiple SARS-CoV-2 lineages April-August 2020 anticipated the second pandemic wave in Lombardy (Italy).

During the early phase of the pandemic (20 February-4 April 2020), we have investigated the temporal and geographical evolution of the virus in Lombardy showing the circulation of at least seven lineages distributed differently in the Region. In the present study, the molecular epidemiology of SARS-CoV-2 was monitored in a period between two pandemic waves in order to track the circulation of new variants (April-August 2020). A great majority of SARS-CoV-2 strains (70.8%) belonged to lineages B, B.1, B.1.1 and B.1.1.1, and five strains belonging to four lineages were already reported in Italy (B.1.1.148, B.1.1.162, B.1.1.71, and B.1.425). In addition, 21 SARS-CoV-2 strains belonged to six lineages not previously observed in Italy were detected. No variants of concern were observed. A total of 152/1274 (11.3%) amino acid changes were observed among spike gene sequences and only 26/152 (17.1%) occurred in the receptor-binding domain region of the spike protein. Results of this study are indicative of ongoing transmission throughout the lockdown period, rather than re-introduction of novel lineages past lockdown. The use of molecular epidemiology in Italy should be promoted in order to provide additional understanding of the transmission of the disease and to have major effect on controlling the spread of disease.

The early phase of the COVID-19 epidemic in Lombardy, Italy.

BACKGROUND: In the night of February 20, 2020, the first epidemic of the novel coronavirus disease (COVID-19) outside Asia was uncovered by the identification of its first patient in Lombardy region, Italy. In the following weeks, Lombardy experienced a sudden increase in the number of ascertained infections and strict measures were imposed to contain the epidemic spread. METHODS: We analyzed official records of cases occurred in Lombardy to characterize the epidemiology of SARS-CoV-2 during the early phase of the outbreak. A line list of laboratory-confirmed cases was set up and later retrospectively consolidated, using standardized interviews to ascertained cases and their close contacts. We provide estimates of the serial interval, of the basic reproduction number, and of the temporal variation of the net reproduction number of SARS-CoV-2. RESULTS: Epidemiological investigations detected over 500 cases (median age: 69, IQR: 57-78) before the first COVID-19 diagnosed patient (February 20, 2020), and suggested that SARS-CoV-2 was already circulating in at least 222 out of 1506 (14.7%) municipalities with sustained transmission across all the Lombardy provinces. We estimated the mean serial interval to be 6.6 days (95% CrI, 0.7-19). Our estimates of the basic reproduction number range from 2.6 in Pavia (95% CI, 2.1-3.2) to 3.3 in Milan (95% CI, 2.9-3.8). A decreasing trend in the net reproduction number was observed following the detection of the first case. CONCLUSIONS: At the time of first case notification, COVID-19 was already widespread in the entire Lombardy region. This may explain the large number of critical cases experienced by this region in a very short timeframe. The slight decrease of the reproduction number observed in the early days after February 20, 2020 might be due to increased population awareness and early interventions implemented before the regional lockdown imposed on March 8, 2020.

SARS-CoV-2 vaccine breakthrough infections with the alpha variant are asymptomatic or mildly symptomatic among health care workers.

Vaccine breakthrough SARS-CoV-2 infection has been monitored in 3720 healthcare workers receiving 2 doses of BNT162b2. SARS-CoV-2 infection is detected in 33 subjects, with a 100-day cumulative incidence of 0.93%. Vaccine protection against acquisition of SARS-CoV-2 infection is 83% (95%CI: 58-93%) in the overall population and 93% (95%CI: 69-99%) in SARS-CoV-2-experienced subjects, when compared with a non-vaccinated control group from the same Institution, in which SARS-CoV-2 infection occurs in 20/346 subjects (100-day cumulative incidence: 5.78%). The infection is symptomatic in 16 (48%) vaccinated subjects vs 17 (85%) controls (p = 0.01). All analyzed patients, in whom the amount of viral RNA was sufficient for genome sequencing, results infected by the alpha variant. Antibody and T-cell responses are not reduced in subjects with breakthrough infection. Evidence of virus transmission, determined by contact tracing, is observed in two (6.1%) cases. This real-world data support the protective effect of BNT162b2 vaccine. A triple antigenic exposure, such as two-dose vaccine schedule in experienced subjects, may confer a higher protection.

Incidence of SARS-CoV-2 infection in health care workers from Northern Italy based on antibody status: immune protection from secondary infection- A retrospective observational case-controlled study.

OBJECTIVE: The protection from SARS-CoV-2 infection induced by SARS-CoV-2 anti-S1 and anti-S2 IgG antibody positivity resulting from natural infection was evaluated. METHODS: The frequency of SARS-CoV-2 infection (as determined by virus RNA detection) was evaluated in a group of 1,460 seropositive and a control group of 8,150 seronegative healthcare workers in three Centres of Northern Italy in the period June-November 2020. Neutralizing serum titers were analyzed in seropositive subjects with or without secondary SARS-CoV-2 infection. RESULTS: During the 6-month survey, 1.78% seropositive subjects developed secondary SARS-CoV-2 infection while 6.63% seronegative controls developed primary infection (odds ratio: 0.26; 95% confidence interval: 0.17-0.38). Secondary infection was associated with low or absent serum neutralizing titer (p<0.01) and was mildly symptomatic in 45.8% cases vs 71.4% symptomatic primary infections (odds ratio: 0.34; 95% confidence interval: 0.16-0.78). CONCLUSIONS: Immunity from natural infection appears protective from secondary infection; therefore, vaccination of seronegative subjects might be prioritized.