Academic literature on the topic 'Measles genetic variability'

Of the 24 known measles genotypes, only D8 and B3 are responsible for outbreaks in the last years in Europe, Asia, and America. In this study the H gene of 92 strains circulating between 2015 and 2019 in Lombardy, Northern Italy, and 1273 H sequences available in GenBank were analyzed in order to evaluate the genetic variability and to assess the conservation of the immunodominant sites. Overall, in Lombardy we observed the presence of four different B3 and three different D8 clusters, each one of them including sequences derived from viruses found in both vaccinated and unvaccinated subjects. Worldwide, the residue 400 within the H protein, a position located within the main immune epitope, is mutated in all circulating strains that belong to the two globally endemic genotypes, B3 and D8. Our data demonstrate the usefulness of measles virus (MV) H gene sequencing. Indeed, the monitoring the H protein epitopes of circulating strains could be included in the measles laboratory surveillance activities in order to improve and optimize strategies for measles control, as countries go towards elimination phase.

Vaccines have revolutionized modern public health. The effectiveness of some vaccines is limited by the variation in response observed between individuals and across populations. There is compelling evidence that a significant proportion of this variability can be attributed to human genetic variation, especially for those vaccines administered in early life. Identifying and understanding the determinants of this variation could have a far-reaching influence upon future methods of vaccine design and deployment. In this review, we summarize the genetic studies that have been undertaken attempting to identify the genetic determinants of response heterogeneity for the vaccines against hepatitis B, measles and rubella. We offer a critical appraisal of these studies and make a series of suggestions about how modern genetic techniques, including genome-wide association studies, could be used to characterize the genetic architecture of vaccine response heterogeneity. We conclude by suggesting how the findings from such studies could be translated to improve vaccine effectiveness and target vaccination in a more cost-effective manner.

It has been reported that the phenomenon of biased hypermutation, associated with measles virus recovered from brain tissue, can be reproduced experimentally by propagation of measles virus in cultured neuroblastoma cells. The purpose of the research described in this thesis was to evaluate this claim and establish whether biased hypermutation could be induced in measles virus from an acute infection (ADD-MV), or in an RNA virus not implicated in neural disease such as human respiratory syncytial virus (huRS virus). Accordingly, an isolate of ADD-MV and huRS virus were passaged 10 times in two neuroblastoma cell lines (SK-N-SH and IMR-32), in human lung fibroblasts (MRC-5) and in monkey kidney epithelial (Vero) cells. The M gene sequence of huRS virus passaged 10 times in each cell line was compared with the M gene sequence of virus passaged once in the same cells. No base change was observed in the M gene of huRS virus as a result of passage in these cell lines. A 750 base pair fragment of the M gene of ADD-MV was sequenced. Comparison of the sequence obtained from virus passaged 10 times in Vero cells with that from virus passaged once did not reveal any base changes. The M gene sequence of virus passaged 10 times in SK-N-SH cells contained one base change, at position 222, a uracil to cytosine transition. Two base changes were observed in the M gene of virus passaged 10 times in MRC-5 cells, one at position 222, as described above, and one at position 217, a cytosine to guanine base change. No changes were observed in virus passaged 10 times in IMR-32 cells when compared to that passaged once. These data indicate that neither ADD-MV, nor huRS virus, underwent enhanced mutational events during propagation in cells of neural origin, contrary to observations reported previously by Wong, et al. (1989), which suggested that propagation in neuroblastoma cells induced biased hypermutation. To determine if biased hypermutation occurred at a frequency too low to be detected by cycle sequencing, the M gene of ADD-MV passaged 9 times in IMR-32 cells, generated by PCR, was cloned into the bacteriophage vector M13. Twenty-four clones were sequenced in full. In total 21 base changes were observed, however, no more than 5 changes were seen in any one clone, and there was no directional bias in the mutations observed. However, each clone contained a large deletion at the 3' region of the gene, ranging from 1008 to 622 bases. Thirteen of the clones also contained a 56 base insertion. This insertion was shown to have high identity with human mitochondrial transfer RNA (tRNA). The M gene of Yamagata-1 (SSPE) virus resembles that of acute measles, but contains a number of additional mutations, mainly uracil to cytosine transitions. Propagation of this virus in neuroblastoma cells resulted in additional mutations not observed in virus propagated in Vero cells (Wong et al., 1989). Yamagata-1 virus was passaged in the two neuroblastoma cell lines (SK-N-SH and IMR-32), both with and without propagation in human lung fibroblast cells prior to passage in the neuroblastoma cells. Three M gene clones from passage numbers one and five in both cell lines used for this experiment, were sequenced. Analysis of the M gene sequence of Yamagata-1 virus passaged in each cell line revealed that the virus stock contained a heterogeneous population of M gene mRNA's. Both mutated sequences and sequences identical to the M gene of ADD-MV were obtained. Comparisons of the M gene sequence from virus passaged 5 times in neuroblastoma cells with that passaged once revealed no biased hypermutational events. However, the sequence data determined from two clones appeared to be a chimera consisting of both ADD-MV M gene sequence and Yamagata-1 virus M gene sequence. The recombination point is at position 457, a G-A mutation which is the only base change common to both ADD-MV M gene and Yamagata-1 M gene sequence.

INTRODUZIONE. Il morbillo è una malattia esantematica estremamente contagiosa trasmissibile per via aerea causata da un virus della famiglia Paramyxoviridae, genere Morbillivirus. L’infezione spesso è causa di complicanze severe e decessi, è prevenibile con la vaccinazione e presenta i requisiti per l’eliminazione. L’Italia fa parte dei 12 paesi europei dove la trasmissione del morbillo è ancora endemica. Il Global Measles and Rubella Strategic Plan 2012 – 2020 ha fissato il goal di eliminazione del morbillo endemico nella Regione Europea dell’OMS. Per raggiungere tale obiettivo è necessario ottenere e mantenere coperture vaccinali elevate (>95%) e disporre di un sistema di sorveglianza sensibile e di qualità. La sorveglianza molecolare del morbillo è una componente chiave della verifica dell'eliminazione del morbillo endemico ed è uno strumento cruciale sia per stabilire eventuali link epidemiologici tra casi che si verificano nello stesso periodo in una determinata area geografica sia per identificare le possibili fonti di importazione. Con il progredire del programma di eliminazione, la diversità genetica dei ceppi di morbillo circolanti diminuisce. I continui viaggi aerei e la facilità di scambi tra i paesi favoriscono le importazioni in una determinata area geografica di varianti virali appartenenti allo stesso genotipo. In questo contesto, i tradizionali metodi di laboratorio non consentono di distinguere la trasmissione endemica da eventi di importazione delle stesse varianti virali. Con il progredire del programma di vaccinazione, inoltre, una quota sempre più alta di casi di morbillo si verifica in soggetti vaccinati. Le nuove e avanzate tecnologie devono quindi permette di ampliare le conoscenze su questi aspetti e consentire di identificare ceppi potenzialmente in grado di eludere la risposta immunitaria. SCOPO. Scopo del presente progetto di dottorato è quello di studiare e sorvegliare nel tempo l’epidemiologia molecolare del morbillo in vista dell’obiettivo di eliminazione, attraverso lo sviluppo e l’utilizzo di metodologie innovative molecolari e bioinformatiche. La ricerca pertanto si propone di combinare il metodo epidemiologico tradizionale con le tecniche molecolari e bioinformatiche che la nuova era offre. Un ulteriore obiettivo è stato quello di studiare casi confermati di morbillo in soggetti precedentemente vaccinati, al fine di valutare il fallimento vaccinale e identificare eventuali mutanti escape a livello del gene H. MATERIALI E METODI. Sono stati analizzati i campioni biologici provenienti da pazienti con diagnosi sospetta di morbillo raccolti nell’ambito della Sistema di Sorveglianza Integrata Morbillo e Rosolia della Regione Lombardia (rete MoRoNET), da marzo 2017 a luglio 2019. I campioni sono stati sottoposti a estrazione dell'RNA e a test di Real Time RT-PCR per l’identificazione del genoma del virus del morbillo. Tutti i campioni risultati positivi sono stati sottoposti a retrotrascrizione e a successiva amplificazione genica della regione N-450 del virus del morbillo mediante nested RT-PCR per la caratterizzazione genotipica. Campioni di interesse (N=50) sono stati sottoposti ad amplificazione del gene virale H mediante due emi-nested PCR e ad amplificazione dell’intero genoma attraverso l’utilizzo di specifiche coppie di primer per l’amplificazione di 10 frammenti parzialmente sovrapposti. Gli amplificati sono stati sequenziati e sono state analizzate filogeneticamente le sequenze N-450, N-450/H e l’intero genoma attraverso diversi programmi bioinformatici (ClustalX2, BioEdit, MEGA7) per le valutazioni filogenetiche. Lo studio dei casi vaccinati è stato condotto attraverso l’analisi dei dati sierologici ottenuti dal database del laboratorio di riferimento regionale; in aggiunta, è stata analizzata la sequenza amminoacidica (ottenuta tramite la conversione delle sequenze nucleotidiche grazie al programma BioEdit) della proteina H di 7 casi vaccinati e di 80 casi non vaccinati come gruppo controllo. I dati anagrafici, clinici ed epidemiologici relativi ai casi di morbillo analizzati nel presente lavoro sono stati ottenuti dal database della Regione Lombardia per le malattie infettive MAINF. RISULTATI. Complessivamente, tra marzo 2017 e luglio 2019 sono stati indagati 885 casi sospetti di morbillo segnalati a Milano e nelle aree limitrofe. Il 74.2% dei casi indagati è stato confermato in laboratorio. Il maggior numero di casi è stato confermato nel 2017 (50.4%). Le fasce d’età più colpite sono state quelle dei giovani tra i 15 e i 39 anni (63.0%) e degli adulti over-39 anni (21.5%), dato che si è confermato per tutti e tre gli anni esaminati. Il 92.1% dei casi confermati di morbillo non era vaccinato. La consultazione del database regionale MAINF ha permesso di classificare i casi confermati come sporadici (57.4%) o appartenenti a focolai (42.7%). Il 9.7% dei casi confermati dal nostro laboratorio si trovava fuori dal territorio in esame durante il periodo di incubazione della malattia. In questo modo è stato possibile definire l’importazione dell’infezione, sia dall’estero (44.3% dei casi importati) che da altre regioni italiane (55.7% dei casi importati). La caratterizzazione genotipica mediante sequenziamento della regione N-450 di Morbillivirus è stata completata con successo per il 95.3% dei casi. È stato possibile evidenziare la costante co-circolazione di due genotipi, il D8 (72.9%) e il B3 (26.1%). L’analisi intra-genotipica ha permesso di identificare complessivamente 16 varianti virali, di cui 8 già classificate dall’OMS e definite WHO named strain. In particolare, sono state identificate 5 WHO named strain D8 (Mv/Osaka.JPN/29.15, Mvs/London.GBR/21.16/2, MVi/Hulu-Langat.MYS/26.11, MVs/Gir-Somnath.IND/42.16, Mvs/Victoria.AUS/6.18) e 3 WHO named strain B3 (MV/Dublin.IRL/8.16, MVs/Saint-Denis.FRA/36.17, Mvs/Ljubljana.SVN.27.17). La D8-Osaka è stata la variante predominante nel 2017, la B3-Dublino nel 2018 e la D8-Gir Somnath nel 2019. Sono state inoltre identificate 69 sequenze non ancora classificate dall’OMS e momentaneamente denominate “no named strain”. Venti erano di genotipo D8 e 49 di genotipo B3. Alcune di queste sequenze hanno dato origine a eventi di trasmissione continua, altre a casi sporadici o a piccoli focolai familiari non propagandosi ulteriormente nella popolazione. È stata approfondita l’analisi molecolare di ceppi correlati a importanti eventi epidemici (focolai nosocomiali e familiari) e a casi notificati come sporadici avvenuti a breve distanza geografica e temporale dai focolai. È stato quindi sviluppato il sequenziamento e l’analisi filogenetica di una regione di più di 2000 nucleotidi, comprendente l’N-450 e tutto il gene H (N-450/H). Ciò ha reso possibile valutare la variabilità genetica (intra-variante) di ceppi 100% identici in N-450. L’analisi filogenetica di questa regione ha permesso di osservare che sequenze tra loro identiche nella sola regione N-450 formavano dei cluster e altre invece risultavano non strettamente correlate. In un secondo momento, gli stessi ceppi sono stati sottoposti a sequenziamento dell’intero genoma, la cui analisi filogenetica ha confermato i dati ottenuti in N-450/H, ma raggiungendo un maggior match con l’indagine epidemiologica e risultando più sensibile nel delineare le singole catene di trasmissione. È stato studiato il profilo sierologico di 33 casi confermati di morbillo con storia di vaccinazione documentata nel database regionale MAINF. Nel 18.2% dei casi, il riscontro di negatività in IgG in fase acuta (7-10 giorni dall’esordio del rash) ha suggerito una mancata risposta al vaccino (non-responder) e quindi un fallimento primario della vaccinazione. La gran parte dei casi vaccinati (81.8%) ha mostrato una risposta in IgG in fase acuta e pertanto inquadrabile come fallimento verosimilmente secondario. La capacità di trasmettere l’infezione da parte di soggetti con fallimento vaccinale è stata dimostrata nel 12% dei casi. Infine, i soggetti con fallimento primario avevano un’età mediana all’epoca dell’ultima dose più elevata rispetto a quelli con fallimento secondario (12 anni e 6 anni rispettivamente). Un ulteriore obiettivo del presente progetto è stato quello di analizzare 87 sequenze amminoacidiche della proteina H (target principale degli anticorpi neutralizzanti) identificate in soggetti vaccinati e non vaccinati. Di queste, 30, di cui 7 appartenenti a ceppi di casi vaccinati, presentavano sostituzioni in siti immunoepitopici. Le sostituzioni riscontrate sono state L247S, P247S, A400V, A192T e Q575K. Non sono invece state riscontrate mutazioni in siti funzionali della proteina, come per esempio residui di cisteina importanti per il mantenimento della struttura terziaria, o in siti di legame col recettore, responsabili del riconoscimento e dell’ingresso nella cellula ospite. CONCLUSIONI. L’andamento epidemiologico e genotipico dei casi di morbillo identificato nei 3 anni di studio a Milano e nelle aree limitrofe rispecchia quello riscontrato sul territorio nazionale. L’analisi intra-genotipica condotta ha permesso di identificare complessivamente 16 varianti virali, di cui 4 predominanti e un’elevata variabilità per entrambi i genotipi individuati (D8 e B3). Ciò conferma il pattern tipico nelle aree caratterizzate da un abbassamento delle coperture vaccinali e un aumento dei soggetti suscettibili alla malattia. Per dimostrare l’interruzione della circolazione all’interno del proprio territorio, i paesi devono essere in grado di distinguere le trasmissioni endemiche dai casi importati, in quanto la malattia può essere considerata eliminata solo in assenza di focolai endemici. La circolazione a livello mondiale di un ristretto numero di varianti virali limita l’informazione data dall’analisi filogenetica della regione N-450 e rende più complessa la ricostruzione delle rotte di trasmissione e la caratterizzazione dei focolai epidemici. Inoltre, non consente di ricostruire le catene di trasmissione e identificare i casi di importazione da fonti diverse. È pertanto importante mettere in atto nuove strategie metodologiche per ampliare la “finestra di sequenziamento”. L’analisi dell’intero genoma è risultata sensibile e in grado di ricostruire le catene di trasmissione e identificare i casi di importazione. Questa tecnica risulta tuttavia estremamente laboriosa e costosa e, nell’attuale contesto epidemiologico, non applicabile come metodica di routine. Potrebbe risultare invece la strategia ottimale per i paesi measles-free o che si stanno avvicinando all'eliminazione del morbillo, dove è necessario verificare l’introduzione solo di pochi ceppi virali. I risultati ottenuti nel corso dei 3 anni di dottorato sui ceppi coinvolti in importanti eventi epidemici a Milano e nelle aree limitrofe suggeriscono che l’analisi della regione N-450/H possa essere considerata una valida strategia di implementazione della sorveglianza molecolare in questa fase del programma di eliminazione. Per quanto riguarda lo studio dei casi confermati di morbillo in soggetti vaccinati, i risultati hanno dimostrato che la maggior parte dei fallimenti vaccinali non sono dovuti a una mancata risposta immunitaria (non-responder), ma piuttosto ad una perdita nel tempo della risposta immunologica vaccino-indotta. Tuttavia, la valutazione del titolo anticorpale è stata fatta durante la fase acuta della malattia e non si è potuto valutare quanti soggetti presentavano titoli anticorpali protettivi prima dell’infezione. Questa valutazione avrebbe contribuito a identificare i booster delle IgG in fase acuta causati dall’incontro col virus selvaggio. Il riscontro di una percentuale non trascurabile di casi di morbillo tra soggetti vaccinati che ha dato origine a focolai evidenzia la necessità di mantenere alta l’attenzione nel mettere in atto le misure di contenimento e diffusione della malattia anche in presenza di casi vaccinati. Ulteriori studi multidisciplinari devono essere condotti per confermare i risultati ottenuti e delineare opportuni piani risolutivi. L’analisi amminoacidica della proteina H ha permesso di identificare mutazioni in siti critici della proteina non osservate da studi precedenti. I dati ottenuti sono da un lato rassicuranti in quanto non mostrano la circolazione di mutanti escape, dall’altro documentano una variabilità della proteina H che impone la necessità di un monitoraggio costante. In conclusione, nel presente progetto di dottorato, sono state sviluppate e applicate metodologie innovative risultate utili per la corretta valutazione dello scenario epidemiologico attuale, caratterizzato dalla circolazione di ceppi endemici, dalla continua introduzione di varianti virali e da una quota non trascurabile di fallimenti vaccinali.
INTRODUCTION. Measles virus belongs to the morbillivirus genus of the family Paramyxoviridae. Infection with measles virus results in an extremely contagious exanthematic disease transmitted by air. It often causes severe complications and deaths, is preventable with vaccination and presents requirements for elimination. Italy is one of the 12 European countries where measles transmission is still endemic. The Global Measles and Rubella Strategic Plan 2012–2020 has set the goal of the elimination of endemic measles in the WHO European Region. To achieve this goal, high vaccination coverage must be obtained and maintained (>95%), and a sensitive and quality surveillance system must be ensured. Measles molecular surveillance is a key component to verify the endemic measles elimination, and a crucial tool both to establish any epidemiological link between cases occur in the same period and area, and to identify the importation sources. With the progress of the elimination program, the genetic diversity of circulating measles strains decreases. Continuous air travels and the ease world trade between countries facilitate imports into a given geographical area of viral variants belonging to the same genotype. In this context, traditional laboratory methods can not distinguish endemic transmission from import events of the same viral variant. In addition, as the vaccination program moves forward, an increasing proportion of measles cases occur in vaccinated individuals. New and advanced technologies must therefore allow us to broaden knowledge on measles in vaccinated people, and allow us to identify strains potentially capable of evading the immune response. AIM. The aim of the PhD project is to study and monitor in time the molecular epidemiology of measles in view of the elimination goal, through the develop and the use of innovative molecular and bioinformatic methodologies. Therefore, the research aims to combine the traditional epidemiological methods with the molecular and bioinformatic techniques of the new era. A further objective is to study measles confirmed cases in vaccinated people, in order to assess vaccination failure and to identify any escape mutant in the measles H gene. MATERIALS AND METHODS. From March 2017 to July 2019, biological specimens from patients with suspected measles were collected and analysed as part of the Measles and Rubella Integrated Surveillance System of the Lombardy Region (MoRoNET network). Viral RNA was extracted, and a Real Time RT-PCR was carried out for the measles genome identification. Retro-transcription was performed to all the measles positive samples, and a nested RT-PCR was conducted for the amplification of the N-450 region, in order to perform the genotyping. Samples of interest (N=50) have undergone two emi-nested PCR for the amplification of the H gene. Moreover, the amplification of the complete genome through specific couples of primers (which allow to obtain 10 overlapped fragments) has been conducted on the same samples. Amplicons were sequenced and a phylogenetic analysis was conducted on N-450 region, N-450/H region and on the whole genome, using the bioinformatic programs ClustalX2, BioEdit and MEGA7. Vaccinated measles cases were studied through analysis on serological data obtained from the regional referent laboratory database. In addition, the amino acid sequence of measles H protein (obtained from the conversion of the nucleotide sequences with BioEdit) was analysed in 7 vaccinated measles cases and 80 non-vaccinated measles cases, as control group. Personal, clinical and epidemiological data of measles cases analysed in this study were obtained from the Lombardy Region database of infectious diseases, MAINF. RESULTS. Overall, from March 2017 to July 2019, 885 suspected measles cases reported in Milan and surrounding areas were investigated. The 74.2% of measles cases was confirmed by laboratory investigations. The largest number of measles cases was confirmed in 2017 (50.4%). The age groups 15-39 years and over-39 years were the most affected over the all three years (63.0% and 42.7% of measles cases, respectively). The 92.1% of measles cases was unvaccinated. Regional database MAINF allowed to classify measles cases as sporadic (57.4%) or belonging to outbreaks (42.7%). The 9.7% of measles cases confirmed by our laboratory was outside the examined area during the incubation period of the disease. Genetic characterization was performed by the N-450 morbillivirus region sequencing and was completed with success in 95.3% of measles cases. The genotyping made possible to highlight the continuous co-circulation of two genotypes, D8 (72.9%) and B3 (26.1%). The intra-genotype analysis identified overall 16 viral variants, 8 of them already classified as WHO named strain. In particular, 5 WHO named strains D8 (Mv/Osaka.JPN/29.15, Mvs/London.GBR/21.16/2, MVi/Hulu-Langat.MYS/26.11, MVs/Gir-Somnath.IND/42.16, Mvs/Victoria.AUS/6.18) and 3 WHO named strains B3 (MV/Dublin.IRL/8.16, MVs/Saint-Denis.FRA/36.17, Mvs/Ljubljana.SVN.27.17) have been identified. The named strain D8-Osaka was the predominant genotype variant during the 2017. The named strain B3-Dublin was the predominant genotype variant during the 2018, and the named strain D8-Gir Somnath was the most frequently detected during the 2019. Moreover, 69 sequences not yet classified by the WHO have been identified and called “no-named strain”. Twenty no-named strains belonged to genotype D8 and 49 belonged to genotype B3. Some of them were responsible of continued transmission events, sporadic cases or small familiar outbreaks that did not further spread into the population. Molecular analysis has been deepened on strains correlated to nosocomial and familiar outbreaks and on strains correlated to sporadic cases occurred within short geographical and temporal distances from the outbreaks. It was therefore developed the sequencing and the phylogenetic analysis of a more than 2000 nucleotides region, which includes the N-450 region and the whole H gene (N-450/H). This made possible to evaluate the genetic variability of strains 100% identical in the N-450 region. Phylogenetic analysis of the N-450/H construct allowed to observe clusters within strains with the same N-450 region, whereas others 100% identical in N-450 were not strictly correlated. Subsequently, the whole genome sequencing was carried out on the same strains, and the phylogenetic analysis confirmed data obtained with N-450/H analysis. However, the whole genome sequencing analysis reached a greater match with the epidemiological investigation, and it resulted more sensitive outlining the single chains of transmission. The serological profile of 33 measles cases with a vaccination history was investigated. Negativity in IgG test during the acute phase of the disease (7-10 days from rash) was found in 18.2% of measles cases, suggesting a failure in vaccination response (non-responder) and therefore a primary vaccine failure. The majority of vaccinated measles cases (81.8%) showed an IgG response during the acute phase. Therefore, these cases could be placed in a secondary vaccine failure. Ability to transmit the infection to secondary cases was found in the 12% of vaccinated measles cases. In addition, the median age at the time of the last vaccine dose of measles cases with primary vaccine failure was higher than the one of measles cases with secondary vaccine failure (12 and 6 years old, respectively). Another goal of the present project was to analyse 87 aminoacidic sequences of measles H protein (first target of human neutralizing antibodies) identified in vaccinated and not vaccinated subjects. Of them, 30, of which 7 belonged to vaccinated measles cases, showed amino acid substitutions in antigenic epitopes. The substitutions were L247S, P247S, A400V, A192T, and Q575K. No mutations were found at functional sites of the protein, such as cysteine residues important for the maintenance of the tertiary protein structure, or on binding receptor sites, responsible for the recognition and the entry into the host cell. CONCLUSIONS. The epidemiological trend and genotypes of measles cases identified in Milan and the surrounding areas in the 3 years of study reflect what found on the national territory. The intra-genotyping analysis identified overall 16 viral variants, 4 of them predominant, and a high variability for both the D8 and B3 genotypes. This result confirms the typical pattern of areas characterized from a reduction of vaccination coverage and with an increment of susceptible subjects. To demonstrate the interruption of the virus circulation in the territory, countries must be able to distinguish endemic transmission form imported cases. Indeed, measles disease can be considered eliminated only in the absence of endemic outbreaks. The worldwide circulation of a small number of viral variants limits the information given by the phylogenetic analysis of the N-450 region, and makes more complex the reconstruction of transmission routes, as well as the outbreaks characterization. Furthermore, it is impossible to trace the chains of transmission and identify imported cases from different sources. It is therefore important to implement new methodologic strategies in order to extend the “window sequencing”. The whole genome analysis was sensitive and resulted able to trace the chains of transmission and identify the imported cases. This technique, however, is extremely laborious and expensive, and it is not applicable as routine tool in the actual epidemiological context. It could be instead the optimal strategy for measles-free countries, or for which ones is approaching the measles elimination, with only few cases to verify. The results obtained on strains involved in important epidemic events in Milan and surrounding areas during the 3-years PhD study suggest that the N-450/H analysis could be considered a good implementation strategy of the molecular surveillance in the actual elimination program phase. Regarding the study of measles cases in vaccinated subjects, it can be hypothesized that vaccination failure is not caused by an immunity system failure to vaccination (non-responder), but rather to a decline over the time of the immunity response vaccine-induced. However, the antibody evaluation was made during the acute phase of the disease, and it is not able to evaluate how many subjects had protective antibody titres before the infection. This evaluation would contribute to identified IgG boosters in acute phase caused by the wild-type measles strain exposure. The finding of an important proportion of vaccinated measles cases able to originate outbreaks highlight the necessity to maintain a high attention for the containment measures and for the spread disease control even in presence of vaccinated subjects. More multidisciplinary studies must be conducted to confirm the obtained results and to outline appropriate resolutive planes. Amino acid analysis of the measles H protein has led to identify mutations in critical protein sites observed for the first time. Data obtained are on the one hand reassuring because no escape mutant was found. On the other hand, data document a measles H protein variability which impose a constant monitoring. In conclusion, in this PhD project the innovative techniques developed and applied were found to be useful for the correct evaluation of the actual epidemiological scenario, which is characterized by the circulation of endemic measles strains, the continuous introduction of viral variants and a significant number of vaccine failures.

Genetic diversity comprises the total of genetic variability contained in a population and it represents the fundamental component of changes since it determines the microevolutionary potential of populations. There are several measures for quantifying the genetic diversity, most notably measures based on heterozygosity and measures based on allelic richness, i.e. the expected number of alleles in populations of same size. These measures differ in their theoretical background and, in consequence, they differ in their ecological and evolutionary interpretations. Therefore, in the present chapter these measures of genetic diversity were jointly analyzed, highlighting the changes expected as consequence of gene flow and genetic drift. To develop this analysis, computational simulations of extreme scenarios combining changes in the levels of gene flow and population size were performed.

Freshwater fish stocks are being exposed to increasing threats as a result of fisheries and aquaculture practices. Integrating genetic knowledge into fisheries and aquaculture management is becoming increasingly important in order to ensure the sustainability of species. So, I used SSR markers to evaluate the pattern of genetic variability in Silver Carp populations (175 samples) from five different sites of River Chenab, Pakistan. DNA was isolated and processed for analysis. There were no scoring errors related to large allele, no stuttering bands, and no null allele. The mean values of number of alleles, allelic richness, effective number of alleles, observed (Ho) and expected (He) heterozygosites, 1-Ho/He, inbreeding coefficient, pairwise population differentiation, and the gene flow provided data indicating loss of genetic diversity of silver carp in River Chenab (Pakistan). Reasons are overhunting, pollution, inbreeding, and poor control measures.

Developing new treatment options for individuals with ASD requires identifying the mechanisms that underlie core symptoms throughout the lifespan. Case-control designs are still very common but have well-recognized limitations; cutting-edge research combines large samples, prospective longitudinal designs, measures that can be back-translated to animal models, and advanced genetic techniques that identify common neuropathology. Insights from clinical studies have identified social attention as a putative early signature of autism emergence that may be modifiable with behavioural or pharmacological treatment, but evidence that reduced social attention has downstream effects on other social difficulties remains limited. Preclinical and genetic work has identified converging alterations in synaptic function, activity-dependent translation, oxidative stress, and immune dysfunction; some differences may be reversible in animal models, but successful human clinical trials remain rare. Moving forward requires greater integration of clinical and preclinical insights, and more focus on understanding variability across the spectrum.

<em>Abstract.</em>—The white sturgeon population <em>Acipenser transmontanus</em> in the Kootenai River was listed as endangered by the U.S. Fish and Wildlife Service (USFWS) in 1994 due to postglacial isolation and the virtual lack of recruitment since 1974. The Kootenai River White Sturgeon Conservation Aquaculture Program was initiated to preserve genetic variability, begin rebuilding natural age-class structure, and prevent extinction while measures are identified and implemented to restore natural recruitment. The program is part of a comprehensive recovery strategy detailed in the USFWS recovery plan for the Kootenai River population of white sturgeon. A breeding plan, including culture methods to minimize potential detrimental effects of conventional stocking programs, has been implemented to guide recovery, population management, and the systematic collection and spawning of wild adults before they are lost from the wild breeding population. Between 1990 and 2000, 33 families were produced from the mating of 51 wild white sturgeon broodstock. Genetic analysis indicated that five mitochondrial control region length variants represented in the wild white sturgeon population were represented in similar frequencies in the wild white sturgeon broodstock. A total of 2,702 hatchery-reared white sturgeon were released into the Kootenai River between 1992 and 1999. White sturgeon juveniles approved for release had no diagnostic disease symptoms and less than or equal to 10% prevalence of endemic pathogens. A total of 398 hatchery-reared fish were recaptured in the wild (14.7% of 2,702 stocked; single recapture events) during the 1993–1999 sampling period. The Kootenai River Conservation Aquaculture Program is currently meeting its objectives of reducing the threat of population extinction by providing frequent year classes from native broodstock, representing inherent within-population genetic diversity in its broodstock and progeny, and minimizing the introduction of disease into the recipient wild population.

The desire to enlarge the flight envelope of tactical aircraft and provide enhanced maneuvering capabilities has led to the use of forces and moments produced by the propulsion system to directly influence aircraft dynamics. This coupling between propulsion and flight dynamics is significant enough that traditional techniques for control system design and analysis are either conservative or inaccurate. An integrated approach is required in order to obtain an overall system that provides stability and performance with minimum pilot workload. The authors propose a solution that integrates existing techniques for linear robustness analysis, nonlinearity analysis, optimization, and robust identification into a software tool that facilitates the analysis of robustness of stability and dynamic performance of propulsion control systems and investigates the system’s ability to meet aggregate performance measures, specifically in the presence of fleet-wide component “variability.” The paper first discusses the motivation for the solution by identifying the variability problem in control system design. Following this, an overview of the proposed solution is presented with highlights of key elements, including a discussion on nonlinearity assessment techniques. The paper next describes the prototype version of the software tool and its initial analytical capabilities. Results of the prototype as applied against a nonlinear, engine control model using two different optimization routines, Genetic Algorithms and Particle Swarm Optimization, are presented to demonstrate the promising performance of both algorithms for finding the worst variability due to operating condition flight dynamics and aerothermal component degradation.