Academic literature on the topic 'Measles N-450 sequence'

A genetic analysis of circulating measles virus (MeV) provides strong evidence of an interruption in endemic measles and supports the elimination status of this disease. This study investigated 219 MeVs isolated between 2015 and 2020. Based on the 450 nucleotide sequences of the nucleoprotein gene (N-450), three genotypes of the H1, D8 and B3 with 8, 18 and 6 different N-450 sequences, respectively, were identified. The H1 genotype virus has not circulated in Taiwan since 2017, and the D8 and B3 genotype MeVs became dominant between 2018 and 2019. Different D8 genotype variants were imported from neighboring countries, and the majority of MeV variants were detected only for a short period. However, MVs/Gir Somnath.IND/42.16[D8], a named strain designated by the World Health Organization (WHO), was detected over 2 years. To explore whether the endemic transmission of measles has been underestimated, another sequence window of the hypervariable, noncoding regions between the matrix (M) and fusion (F) genes (MF-NCR) was introduced to clarify the transmission chain. From the chronological sequence analysis of MeVs with N-450 and MF-NCR sequence windows, no endemic MeV variants lasted over 4 weeks, providing strong evidence to support the contention that Taiwan has reached the status for measles elimination.

Virologic surveillance is a critical component of measles management. One of the criteria for verification of elimination of endemic measles is genetic analysis of wild-type viruses to demonstrate lack of an indigenous genotype. Measles is yet to be eliminated in China, and genotype H1 has been detected continuously since virologic surveillance was initiated in 1993. Virologic surveillance has been very active in China, providing a unique opportunity to conduct a detailed study of the evolution of a single, endemic genotype over a timespan of nearly two decades. Phylogenetic analysis performed on the 450 nt coding sequence for the C-terminal 150 amino acids of the nucleoprotein (N-450), fusion (F) gene and haemagglutinin (H) gene confirmed the continued circulation of genotype H1 viruses for 19 years. No evidence of selective pressure for the H protein was found. The substitution rates ranged from 0.75×10−3 substitutions site−1 year−1 for H to 1.65×10−3 substitutions site−1 year−1 for N-450. The time of most recent common ancestor (TMRCA) for genotype H1 was estimated as approximately 1985 (95 % highest probability density, 1979–1989). Finally, the overall diversity of measles sequences from China decreased from 2005 to 2012, coincident with a substantial decrease in measles cases. The results suggest that detailed evolutionary analyses should facilitate the documentation of eventual measles elimination in China. Moreover, the molecular approaches used in this study can be applied in other countries approaching measles elimination.

We report the co-circulation of a new lineage of measles virus (MV) and an Edmonston-like (Ed-like) genotype of MV in China during 1995–7. Sequence analysis of 25 strains was performed on a 282 nucleotides (nt) region of the nucleoprotein (N) gene, a 450-nt region of the haemagglutinin (H) gene and a 152-nt region of the matrix (M) gene by direct sequencing of RT-PCR amplicons obtained from clinical specimens. The entire H gene was sequenced from two strains. The results showed that 24/25 Chinese strains belonged to a new genogroup and were distinct from the vaccine strains used in China and the UK, and also from MV strains previously described in Europe, Africa and the USA. The remaining strain was Ed-like. Two strains of the new genotype (IV) and one of the Ed-like genotype were also detected in the UK in 1996.

Abstract Background The long-term clinical course of patients with an enterovirus central nervous system infection (ECI) is poorly understood. Methods We performed a nationwide population-based cohort study of all Danish patients with ECI diagnosed 1997–2016 (n = 1745) and a comparison cohort from the general population individually matched on date of birth and sex (n = 17 450). Outcomes were categorized into mortality and risk of cancer and likely measures of neurological sequelae: neuropsychiatric morbidities, educational landmarks, use of hospital services, employment, receipt of disability pension, income, number of sick leave days, and nursing home residency. Results Mortality in the first year was higher among patients with ECI (mortality rate ratio [MRR] = 10.0; 95% confidence interval [CI], 4.17–24.1), but thereafter mortality was not higher (MMR = 0.94; 95% CI, 0.47–1.86). Long-term outcomes for patients with ECI were not inferior to those of the comparison cohort for risk of cancer, epilepsy, mental and behavioral disorders, dementia, depression, school start, school marks, high school education, use of hospital services, employment, receipt of disability pension, income, days of sick leave, or nursing home residency. Conclusions Diagnosis of an ECI had no substantial impact on long-term survival, health, or social/educational functioning.

Three genotypes (B3, D8, and H1) of the measles virus (MeV) have recently caused global outbreaks. In Korea, four measles outbreaks were reported during 2018–2019 and most patients were infants and health care workers in their 20s and 30s. To investigate the genetic characteristics and molecular epidemiology of the outbreaks, we analyzed the sequence of MeVs by targeting the N-450, MF-NCR, and/or H gene regions. Considering their phylogenetic relationships, besides the N-450 and MF-NCR sequences that are commonly used for genotyping MeVs, the MF-NCR-H sequence was related to the dynamics for identifying the transmission of MeVs. Phylogenetic clustering patterns reconstructed from the MF-NCR-H sequence set revealed that genotype D8 caused three of the four outbreaks, while B3 seemed to have induced the fourth outbreak. These results suggest that the MF-NCR-H sequence is useful for rapid confirmation of measles outbreaks and to identify the epidemiological routes of MeVs.

ABSTRACT Along with the implementation of measles case-based surveillance, measles vaccine-associated rash illness (VARI) cases were detected in China. To better understand the characteristics of VARI, 101 VARI cases confirmed by measles virus genotyping in 2011 to 2018 were analyzed in this study. With the decrease in measles incidence, the detection rate of VARI cases increased among the cases confirmed by genotyping. Compared with genotype H1 wild-type measles, VARI occurred throughout the year, without obvious seasonal distribution. Infants and children of ages 8 to 23 months were the main population of VARI. VARI mainly occurred within 14 days after measles vaccination. The number of VARI cases peaked on the 8th day after measles vaccination, which was later than that of genotype H1 wild-type measles cases with a measles vaccination history. VARI presents clinical symptoms similar to those of measles. The frequencies of the “3Cs” (cough, coryza, and conjunctivitis), Koplik spots, and complications in VARI cases were significantly lower than those in wild-type measles cases. In total, 94.06% of sequences from VARI cases were identical to measles vaccine strain S191 in the C-terminal 450-nucleotide sequence of the nucleoprotein (N-450) gene. A few substitutions were found in N-450 sequences of the VARI cases. The confirmation of VARI has become an emerging issue in the process of measles elimination. Rapid confirmation of VARI is critical for measles surveillance and will help to determine the response measures for measles, especially in measles preelimination and elimination settings. The suspected measles cases with measles-containing vaccine (MCV) vaccination were recommended to be tested by the laboratory to identify wild-type measles or VARI.

Abstract Background Measles caused by measles virus (MeV) is a highly contagious viral disease which has also been associated with complications including pneumonia, myocarditis, encephalitis, and subacute sclerosing panencephalitis. The current study isolated 33 strains belonging to 2 groups, outbreak and sporadic strains, in 13 cities of Shandong province, China from 2013 to 2019. Comparison of genetic characterization among 15 outbreak strains and 18 sporadic strains was performed by analyzing nucleotide sequences of the C-terminal region of N protein gene (N-450). Results All 33 stains belonged to genotype H1. The outbreak strains and sporadic strains distributed crossly in phylogenetic tree. Sequences alignment revealed some interesting G to A transversion which changed the amino acids on genomic sites 1317, 1422, and 1543. The nucleotide and amino acid similarities among outbreak isolates were 98–100% (0–10 nucleotide variations) and 97.7–100%, respectively; They were 97.3–100% and 96.6–100%, respectively for sporadic isolates. Evolutionary genetics analysis revealed that the mean evolution rates of outbreak and sporadic isolates were 1.26 N 10− 3 and 1.48 N 10− 3 substitutions per site per year separately, which were similar with corresponding data before 2012. Local transmission analysis suggested that there were three transmission chains in this study, two of them originated from Japan. Outbreak cases and sporadic cases emerged alternatively and were reciprocal causation on the transmission chains. Conclusions Our study investigated the phylogeny and evolutional genetics of MeV during a 7-year surveillance, and compared epidemic and genetic characteristics of outbreak strains and sporadic strains. These results underscore the importance of evolutionary study alongside with sporadic cases in discovering and tracing possible outbreaks, especially in the stage of measles elimination.

Abstract Foodborne viruses such as hepatitis E virus (HEV) pose an increasing risk to public health and to confidence in Irish food. Hepatitis E has been acknowledged as a significant pathogen of likely zoonotic transmission, with pork products and shellfish being implicated as potential sources. The European Food Safety Authority has recommended that systematic strain typing of viruses in humans, animals, and food commodities is needed to improve understanding of etiological agents and foodborne transmission pathways, in particular for HEV. The dominant autochthonous genotype of HEV in Europe is genotype 3, thought to be associated with consumption of contaminated food, specifically pork products. However, little is known about the epidemiology of HEV in Ireland. In 2016, HEV became a notifiable disease in Ireland. Following this, as part of the Department of Agriculture, Food and the Marine-funded FoVIRA study, the molecular epidemiology of HEV in Irish clinical samples has been characterized for the first time. HEV RNA-positive clinical specimens from 2016 were genetically characterized (n = 14). A 450 nucleotide fragment of the ORF2 region of the HEV genome was amplified, with contiguous sequence assembly performed using DNA Lasergene v14. Sequences were aligned with ClustalW implemented in Bioedit v7.1.9 and compared to reference strains from GenBank. A maximum likelihood phylogenetic tree was constructed using the Hasegawa–Kishino–Yano model and a discrete gamma distribution to model evolutionary rate distances between sites. Evolutionary analyses were conducted in MEGA7. Statistical support was provided by bootstrapping with 1,000 replicates. Fourteen strains belonged to genotype 3 and were classified as the following subtypes: 3c (n = 7), 3e (n = 4), 3f (n = 1), 3 untyped (n = 1), and 1 untyped. Phylogenetic analysis showed the formation of two distinct clusters of genotype 3:3abchij and 3efg, with strong bootstrap support. A genotype 1 was detected and found to be associated with travel. Data generated from this research will contribute to a risk exposure assessment and will be used to identify potential control points and risk mitigation measures for viral foodborne pathogens. This study will provide a unique opportunity to build national capability in the area of food testing within Irish public laboratories.

Background: Measles transmissions due to case importations challenge public health systems globally and herd immunities in all countries. In 2019, an imported measles case and its subsequently outbreak was found in the Zhoushan Islands. Here, the process of epidemiological investigation and virus tracing were summarized to provide references for the prevention and control of measles in the future.Materials and methods: The data on the demographic, epidemiological, and clinical manifestation of measles cases in this outbreak were collected. The 450 bp fragments of the measles virus (MeV) N gene were amplified and sequenced. The genome of the first imported case was further isolated. Then, the maximum-likelihood and time-scaled phylogenetic analysis was conducted.Results: A total of 28 measles cases were confirmed. Their onsets were between March 13 and May 18, 2019. The first patient was from the Ukraine. He was confirmed at the Fever Clinic in Zhoushan hospital on March 15, 2019 and at the same time, another patient had visited the hospital due to another illness and 10 days later, this second case had onset (March 25, 2019). The epidemic curve shows sustained community transmission. The majority of the following cases (19/26) were clustered on the Donggang street which was close to where the second case worked. The 22 measles virus strains successfully isolated from this outbreak all belonged to the D8.2a sub-cluster and clustered with the KY120864/MVs/GirSomnath.IND/42.16/[D8] which was the predominant genotype in the Ukraine during 2018-2019. The analysis of the complete D8 genotype genome pointed to the fact that this prevailing strain originated from India in 2015 and its substitution rate was estimated as 6.91 × 10−4 (5.64–7.98 × 10−4) nucleotide substitutions/site/year.Conclusion: This outbreak was caused by an imported case from the Ukraine. There was a possible nosocomial infection between the first case and the second case. Then, the second case played an important role in the spread of virus due to her occupation. The molecular phylogenetic analysis could help to track the origin of the virus. Increasing and maintaining the high level of vaccination coverage (≥95%) and an efficient response to imported cases are essential to prevent and control the recurrence and outbreak of measles virus.

Introduction Nun habe ich Euch genug geschrieben, diesen Brief wenn sei [sic] lesen würden, dann würde ich den Genickschuß bekommen.Now I have written you enough, this letter if they would read it, I would get the neck shot. (M., all translations from German sources and quotations by the author) When the German soldier Otto M. wrote these lines from Russia to his family on 3 September 1943 during the Second World War, he knew that his war letter would not be subject to the National Socialist censorship apparatus. The letter contains, inter alia, detailed information about the course of the war on the front, troop locations, and warnings about the Nazi regime. M., as he wrote in the letter, smuggled it past the censorship via a “comrade”. As a German soldier, M. was a member of the Volksgemeinschaft—a National Socialist concept that drew a “racist and anti-Semitic borderline” (Wildt 48)—and was thus not socially excluded due to his status. Nevertheless, in the sentence quoted above, M. anticipates possible future consequences of his deviant actions, which would be carried out by “them”—potentially leading to his violent death. This article investigates how social and societal exclusion is brought forth by everyday media practices such as writing letters. After an introduction to the thesis under discussion, I will briefly outline the linguistic research on National Socialism that underlies the approach presented. In the second section, the key concepts of agency and dispositif applied in this work are discussed. This is followed by two sections in which infrastructural and interactional practices of exclusion are analysed. The article closes with some concluding remarks. During the Second World War, Wehrmacht soldiers and their relatives could not write and receive letters that were not potentially subject to controls. Therefore, the blunt openness with which M. anticipated the brutal sanctions of behavioural deviations in the correspondence quoted above was an exception in the everyday practice of war letter communication. This article will thus pursue the following thesis: private communication in war letters was subject to specific discourse conditions under National Socialism, and this brought forth excluding agency, which has two intertwined readings. Firstly, “excluding” is to be understood as an attribute of “agency” in the sense of an acting entity that either is included and potentially excludes or is excluded due to its ascribed agency. For example, German soldiers who actively participated in patriotic service were included in the Volksgemeinschaft. By contrast, Jews or Communists, to name but a few groups that, from the perspective of racist Nazi ideology, did not contribute to the community, were excluded from it. Such excluding agencies are based on specific practices of dispositional arrangement, which I refer to as infrastructural exclusion of agency. Secondly, excluding agency describes a linguistic practice that developed under National Socialism and has an equally stabilising effect on it. Excluding agency means that agents, and hence protagonists, are excluded by means of linguistic mitigation and omission. This second reading emphasises practices of linguistic construction of agency in interaction, which is described as interactional exclusion of agency. In either sense, exclusion is inextricably tied to the notion of agency, which is illustrated in this article by using data from field post letters of the Second World War. Social exclusion, along with its most extreme manifestations under fascism, is both legitimised and carried out predominantly through discursive practices. This includes for the public domain, on the one hand, executive language use such as in laws, decrees, orders, court hearings, and verdicts, and on the other hand, texts such as ideological writings, speeches, radio addresses, folk literature, etc. Linguistic research on National Socialism and its mechanisms of inclusion and exclusion has long focussed on the power of a regulated public use of language that seemed to be shaped by a few protagonists, most notably Hitler and Goebbels (Schlosser; Scholl). More recent works, however, are increasingly devoted to the differentiation of heterogeneous communities of practice, which were primarily established through discursive practices and are manifested accordingly in texts of that time (Horan, Practice). Contrary to a justifiably criticised “exculpation of the speakers” (Sauer 975) by linguistic research, which focusses on language but not on situated, interactional language use, such a perspective is increasingly interested in “discourse in National Socialism, with a particular emphasis on language use in context as a shared, communicative phenomenon” (Horan, Letter 45). To understand the phenomenon of social and societal exclusion, which was constitutive for National Socialism, it is also necessary to analyse those discursive practices of inclusion and exclusion through which the speakers co-constitute everyday life. I will do this by relating the discourse conditions, based on Foucault’s concept of dispositif (Confessions 194), to the agency of the correspondents of war letters, i.e. field post letters. On Agency and Dispositif Agency and dispositif are key concepts for the analysis of social exclusion, because they can be applied to analyse the situated practices of exclusion both in terms of the different capacities for action of various agents, i.e. acting entities, and the inevitably asymmetrical arrangement within which actions are performed. Let me first, very briefly, outline some linguistic conceptions of agency. While Ahearn states that “agency refers to the socioculturally mediated capacity to act” (28) and thus conceives agency as a potential, Duranti understands agency “as the property of those entities (i) that have some degree of control over their own behavior, (ii) whose actions in the world affect other entities’ (and sometimes their own), and (iii) whose actions are the object of evaluation (e.g. in terms of their responsibility for a given outcome)” (453). Deppermann considers agency to be a means of social and situational positioning: “‘agency’ is to capture properties of the subject as agent, that is, its role with respect to the events in which it is involved” (429–30). This is done by linguistic attribution. Following Duranti, this analysis is based on the understanding that agency is established by the ascription of action to an entity which is thereby made or considered accountable for the action. This allows a practice-theoretical reference to Garfinkel’s concept of accountability and identifies agentive practices as “visibly-rational-and-reportable-for-all-practical purposes” (7). The writing of letters in wartime is one such reflexive discursive practice through which agents constitute social reality by means of ascribing agency. The concept of semantic roles (Fillmore; von Polenz), offers another, distinctly linguistic access to agency. By semantic roles, agency in situated interaction is established syntactically and semantically. Put simply, a distinction is made between an Agent, as someone who performs an action, and a Patient, as someone to whom an action occurs (von Polenz 170; semantic roles such as Agent, Patient, Experiencer, etc. are capitalised by convention). Using linguistic data from war letters, this concept is discussed in more detail below. In the following, “field post” is considered as dispositif, by which Foucault means a thoroughly heterogeneous ensemble consisting of discourses, institutions, architectural forms, regulatory decisions, laws, administrative measures, scientific statements, philosophical, moral and philanthropic propositions – in short, the said as much as the unsaid. Such are the elements of the apparatus [dispositif]. The apparatus [dispositif] itself is the system of relations that can be established between these elements. (Foucault, Confessions 194) The English translation of the French “dispositif” as “apparatus” encourages an understanding of dispositif as a rather rigid structure. In contrast, the field post service of the Second World War will be used here to show how such dispositifs enable practices of exclusion or restrict access to practices of inclusion, while these characteristics themselves are in turn established by practices or, as Foucault calls them, procedures (Foucault, Discourse). An important and potentially enlightening notion related to dispositif is that of agencement, which in turn is borrowed from Deleuze and Guattari and was further developed in particular in actor-network theory (Çalışkan and Callon; Gherardi). What Çalışkan and Callon state about markets serves as a general description of agencement, which can be defined as an “arrangement of heterogeneous constituents that deploys the following: rules and conventions; technical devices; metrological systems; logistical infrastructures; texts, discourses and narratives …; technical and scientific knowledge (including social scientific methods), as well as the competencies and skills embodied in living beings” (3). This resembles Foucault’s concept of dispositif (Foucault, Confessions; see above), which “denotes a heterogeneous ensemble of discursive and nondiscursive elements with neither an originary subject not [sic] a determinant causality” (Coté 384). Considered morphosemantically, agencement expresses an important interrelation: in that it is derived from both the French agencer (to construct; to arrange) and agence (agency; cf. Hardie and MacKenzie 58) and is concretised and nominalised by the suffix -ment, agencement elegantly integrates structure and action according to Giddens’s ‘duality of structure’. While this tying aspect certainly contributes to a better understanding of dispositional arrangements and should therefore be considered, agencement, as applied in actor-network theory, emphasises above all “the fact that agencies and arrangements are not separate” (Çalışkan and Callon) and is, moreover, often employed to ascribe agency to material objects, things, media, etc. This approach has proven to be very fruitful for analyses of socio-technical arrangements in actor-network theory and practice theory (Çalışkan and Callon; Gherardi). However, within the presented discourse-oriented study on letter writing and field post in National Socialism, a clear analytical differentiation between agency and arrangement, precisely in order to point out their interrelation, is essential to analyse practices of exclusion. This is why I prefer dispositif to agencement as the analytical concept here. Infrastructural Exclusion of Agency in Field Post Letters In the Second World War, writing letters between the “homeland” and the “frontline” was a fundamental everyday media practice with an estimated total of 30 to 40 billion letters in Germany (Kilian 97). War letters were known as field post (Feldpost), which was processed by the field post service. The dispositif “field post” was, in opposition to the traditional postal service, subject to specific conditions regarding charges, transport, and above all censorship. No transportation costs arose for field post letters up to a weight of 250 grams. Letters could only be sent by or to soldiers with a field post number that encoded the addresses of the field post offices. Only soldiers who were deployed outside the Reich’s borders received a field post number (Kilian 114). Thus, the soldiers were socially included as interactants due to their military status. The entire organisation of the field post was geared towards enabling members of the Volksgemeinschaft to communicatively shape, maintain, and continue their social relationships during the war (Bergerson et al.). Applying Foucault, the dispositif “field post” establishes selection and exclusion mechanisms in which “procedures of exclusion” (Discourse 52) become manifest, two of which are to be related to the field post: “exclusion from discourse” and “scarcity of speaking subjects” (Spitzmüller and Warnke 73). Firstly, “procedures of exclusion ensure that only certain statements can be made in discourse” (Spitzmüller and Warnke 73). This exclusion procedure ought to be implemented by controlling and, ultimately, censoring field post letters. Reviews were carried out by censorship offices (Feldpostprüfstellen), which were military units independent of the field post offices responsible for delivery. Censorship initially focussed on military information. However, “in the course of the war, censorship shifted from a control measure aimed at defence towards a political-ideological review” (Kilian 101). Critical remarks could be legally prosecuted and punished with prison, penitentiary, or death (Kilian 99). Hence, it is assumed that self-censorship played a role not only for public media, such as newspapers, but also for writing private letters (Dodd). As the introductory quotation from Otto M. shows, writers who spread undesirable information in their letters anticipated the harshest consequences. In this respect, randomised censorship—although only a very small proportion of the high volume of mail was actually opened by censors (Kilian)—established a permanent disposition of control that resulted in a potentially discourse-excluding social stratification of private communication. Secondly, the dispositif “field post” was inherently exclusive and excluding, as those who did not belong to the Volksgemeinschaft could not use the service and thus could not acquire agentive capacity. The “scarcity of speaking subjects” (Spitzmüller and Warnke 73) was achieved by restricting participation in the field post system to members of the Volksgemeinschaft. Since agency is based on the most basic prerequisite, namely the ability to act linguistically at all, the mere possibility of exercising agency was infrastructurally restricted by the field post system. Excluding people from “agency-through-language” means excluding them from an “agency of an existential sort” (Duranti 455), which is described here, regarding the field post system, as infrastructural exclusion of agency. Interactional Exclusion of Agency in Field Post Letters In this section, I will elaborate how agency is brought forth interactionally through linguistic means on the basis of data from a field post corpus that was compiled in the project “Linguistic Social History 1933 to 1945” (Kämper). The aim of the project is an actor-based description of discursive practices and patterns at the time of National Socialism, which takes into account the fact that society in the years 1933 to 1945 consisted of heterogeneous communities of practice (Horan, Practice). Letter communication is considered to be an interaction that is characterised by mediated indexicality, accountability, reflexivity, sequentiality, and reciprocity (Dang-Anh) and is performed as situated social practice (Barton and Hall). The corpus of field letters examined here provides access to the everyday communication of members of the ‘integrated society’, i.e. those who were neither high-ranking members of the Nazi apparatus nor exposed to the repressions of the fascist dictatorship. The corpus consists of about 3,500 letters and about 2.5 million tokens. The data were obtained by digitising letter editions using OCR scans and in cooperation with the field post archive of the Museum for Communication Berlin (cf. sources below). We combine qualitative and quantitative methods, the latter providing heuristic indicators for in-depth hermeneutical analysis (Felder; Teubert). We apply corpus linguistic methods such as keyword, collocation and concordance analysis to the digitised full texts in order to analyse the data intersubjectively by means of corpus-based hermeneutic discourse analysis (Dang-Anh and Scholl). However, the selected excerpts of the corpus do not comprise larger data sets or complete sequences, but isolated fragments. Nevertheless, they illustrate the linguistic (non-)constitution of agency and thus distinctively exemplify exclusionary practices in field post letter writing. From a linguistic point of view, the exclusion of actors from action is achieved syntactically and semantically by deagentivisation (Bernárdez; von Polenz 186), as will be shown below. The following lines were written by Albert N. to his sister Johanna S. and are dated 25 June 1941, shortly after the beginning of the German Wehrmacht’s military campaign in Russia (Russlandfeldzug) a few days earlier. Vor den russ. Gefangenen bekommt man einen Ekel, d.h. viele Gefangene werden nicht gemacht.One gets disgusted by the Russian prisoners, i.e. many prisoners are not made. (N.) In the first part of the utterance, “mitigation of agency” (Duranti 465) is carried out using the impersonal pronoun “man” (“one”) which does not specify its referent. Instead, by means of deagentivisation, the scope of the utterance is generalised to an indefinite in‑group of speakers, whereby the use of the impersonal pronoun implies that the proposition is valid or generally accepted. Moreover, the use of “one” generalises the emotional expression “disgust”, thus suggesting that the aversive emotion is a self-evident affect experienced by everyone who can be subsumed under “one”. In particular, this includes the author, who is implicitly displayed as primarily perceiving the emotion in question. This reveals a fundamental practice of inclusion and exclusion, the separating distinction between “us”/“we” and “them”/“the others” (Wodak). In terms of semantic roles, the inclusive and generalised formal Experiencer “one” is opposed to the Causative “Russian prisoner” in an exclusionary manner, implicitly indicating the prisoners as the cause of disgust. The subsequent utterance is introduced by “i.e.”, which marks the causal link between the two phrases. The wording “many prisoners are not made” strongly suggests that it refers to homicides, i.e. executions carried out at the beginning of the military campaign in Russia by German troops (Reddemann 222). The depiction of a quasi-universal disgust in the first part establishes a “negative characterization of the out-group” (Wodak 33) which, in the expressed causal relation with the second phrase, seems to morally legitimise or at least somehow justify the implied killings. The passive form entirely omits an acting entity. Here, deagentivisation obscures the agency of the perpetrators. However, this is not the only line between acting and non-acting entities the author draws. The omission of an agent, even the impersonal “one”, in the second part, and the fact that there is no talk of self-experienceable emotions, but war crimes are hinted at in a passive sentence, suggest the exclusion of oneself as a joint agent of the indicated actions. As further data from the corpus indicate, war crimes are usually not ascribed to the writer or his own unit as the agents but are usually attributed to “others” or not at all. Was Du von Juden schreibst, ist uns schon länger bekannt. Sie werden im Osten angesiedelt.What you write about Jews is already known to us for some time. They are being settled in the East. (G.) In this excerpt from a letter, which Ernst G. wrote to his wife on 22 February 1942, knowledge about the situation of the Jews in the war zone is discussed. The passage appears quite isolated with its cotext in the letter revolving around quite different, trivial, everyday topics. Apparently, G. refers in his utterance to an earlier letter from his wife, which has not been preserved and is therefore not part of the corpus. “Jews” are those about whom the two agents, the soldier and his wife, write, whereas “us” refers to the soldiers at the front. In the second part, agency is again obscured by deagentivisation. While “they” anaphorically refers to “Jews” as Patients, the agents of their alleged resettlement remain unnamed in this “agent-less passive construction” (Duranti 466). Jews are depicted here as objects being handled—without any agency of their own. The persecution of the Jews and the executions carried out on the Russian front (Reddemann 222), including those of Jews, are euphemistically played down here as “settlements”. “Trivialization” and “denial” are two common discursive practices of exclusion (Wodak 134) and emerge here, as interactional exclusion of agency, in one of their most severe manifestations. Conclusion Social and societal exclusion, as has been shown, are predominantly legitimised as well as constituted, maintained, and perpetuated by discursive practices. Field post letters can be analysed both in terms of the infrastructure—which is itself constituted by infrastructuring practices and is thus not rigid but dynamic—that underlies excluding letter-writing practices in times of war, and the extent to which linguistic excluding practices are performed in the letters. It has been shown that agency, which is established by the ascription of action to an entity, is a central concept for the analysis of practices of exclusion. While I propose the division into infrastructural and interactional exclusion of agency, it must be pointed out that this can only be an analytical distinction and both bundles of practices, that of infrastructuring and that of interacting, are intertwined and are to be thought of in relation to each other. Bringing together the two concepts of agency and dispositif, despite the fact that they are of quite different origins, allows an analysis of exclusionary practices, which I hope does justice to the relation of interaction and infrastructure. By definition, exclusion occurs against the background of an asymmetrical arrangement within which exclusionary practices are carried out. Thus, dispositif is understood as an arranged but flexible condition, wherein agency, as a discursively ascribed or infrastructurally arranged property, unfolds. Social and societal exclusion, which were constitutive for National Socialism, were accomplished not only in public media but also in field post letters. Writing letters was a fundamental everyday media practice and the field post was a central social medium during the National Socialist era. However, exclusion occurred on different infrastructural and interactional levels. As shown, it was possible to be excluded by agency, which means exclusion by societal status and role. People could linguistically perform an excluding agency by constituting a division between “us” and “them”. Also, specific discourses were excluded by the potential control and censorship of communication by the authorities, and those who did not suppress agency, for example by self-censoring, feared prosecution. Moreover, the purely linguistic practices of exclusion not only constituted or legitimised the occasionally fatal demarcations drawn under National Socialism, but also concealed and trivialised them. As discussed, it was the perpetrators whose agency was excluded in war letters, which led to a mitigation of their actions. In addition, social actors were depreciated and ostracised through deagentivisation, mitigation and omission of agency. In extreme cases of social exclusion, linguistic deagentivisation even prepared or resulted in the revocation of the right to exist of entire social groups. The German soldier Otto M. feared fatal punishment because he did not communicatively act according to the social stratification of the then regime towards a Volksgemeinschaft in a field post letter. This demonstrates how thin the line is between inclusion and exclusion in a fascist dictatorship. I hope to have shown that the notion of excluding agency can provide an approach to identifying and analytically understanding such inclusion and exclusion practices in everyday interactions in media as dispositional arrangements. However, more research needs to be done on the vast yet unresearched sources of everyday communication in the National Socialist era, in particular by applying digital means to discourse analysis (Dang-Anh and Scholl). Sources G., Ernst. “Field post letter: Ernst to his wife Irene. 22 Feb. 1942.” Sei tausendmal gegrüßt: Briefwechsel Irene und Ernst Guicking 1937–1945. Ed. Jürgen Kleindienst. Berlin: JKL Publikationen, 2001. Reihe Zeitgut Spezial 1. M., Otto. 3 Sep. 1943. 3.2002.7163. Museum for Communication, Berlin. Otto M. to his family. 16 Sep. 2020 <https://briefsammlung.de/feldpost-zweiter-weltkrieg/brief.html?action=detail&what=letter&id=1175>. N., Albert. “Field post letter: Albert N. to his sister Johanna S. 25 June 1941.” Zwischen Front und Heimat: Der Briefwechsel des münsterischen Ehepaares Agnes und Albert Neuhaus 1940–1944. Ed. Karl Reddemann. Münster: Regensberg, 1996. 222–23. References Ahearn, Laura M. “Agency and Language.” Handbook of Pragmatics. Eds. Jan-Ola Östman and Jef Verschueren. Amsterdam: John Benjamins Publishing Company, 2010. 28–48. Barton, David, and Nigel Hall. Letter Writing as a Social Practice. Amsterdam: John Benjamins Publishing Company, 2000. Bergerson, Andrew Stuart, Laura Fahnenbruck, and Christine Hartig. “Working on the Relationship.” Private Life and Privacy in Nazi Germany. Eds. Elizabeth Harvey et al. Vol. 65. Cambridge: Cambridge UP, 2019. 256–79. Bernárdez, Enrique. “A Partial Synergetic Model of Deagentivisation.” Journal of Quantitative Linguistics 4.1–3 (1997): 53–66. Çalışkan, Koray, and Michel Callon. “Economization, Part 2: A Research Programme for the Study of Markets.” Economy and Society 39.1 (2010): 1–32. Coté, Mark. “What Is a Media Dispositif? Compositions with Bifo.” Journal of Communication Inquiry 35.4 (2011): 378–86. Dang-Anh, Mark, and Stefan Scholl. “Digital Discourse Analysis of Language Use under National Socialism: Methodological Reflections and Applications.” Writing the Digital History of Nazi Germany. Eds. Frederike Buda and Julia Timpe. Boston, Berlin: De Gruyter, forthcoming. Dang-Anh, Mark. Protest twittern: Eine medienlinguistische Untersuchung von Straßenprotesten. Bielefeld: Transcript, 2019. Locating Media/Situierte Medien 22. 22 Sep. 2020 <https://doi.org/10.14361/9783839448366>. Deleuze, Gilles, and Félix Guattari. A Thousand Plateaus: Capitalism and Schizophrenia. Trans. Brian Massumi. Minneapolis: University of Minnesota Press, 1987. Deppermann, Arnulf. “Unpacking Parental Violence in Narratives: Agency, Guilt, and Pedagogy in Narratives about Traumatic Interpersonal Experiences.” Applied Linguistics 41.3 (2020): 428–51. Dodd, W.J. National Socialism and German Discourse. Cham: Springer International Publishing, 2018. Duranti, Alessandro. “Agency in Language.” A Companion to Linguistic Anthropology. Ed. Alessandro Duranti. Malden, Mass.: Blackwell, 2004. 451–73. Felder, Ekkehard. “Lexik und Grammatik der Agonalität in der linguistischen Diskursanalyse.” Diskurs – Interdisziplinär. Eds. Heidrun Kämper and Ingo H. Warnke. Berlin: De Gruyter, 2015. 87–121. Fillmore, Charles J. “The Case for Case.” Universals in Linguistic Theory. Eds. Emmon Bach and Robert T. Harms. London: Holt, Rinehart and Winston, 1968. 1–88. Foucault, Michel. “The Confessions of Flesh.” Power/Knowledge: Selected Interviews and Other Writings, 1972–1977. Ed. Michel Foucault. New York: Vintage Books, 1980. 194–228. ———. “The Order of Discourse.” Untying the Text: A Post-Structuralist Reader. Ed. Robert J.C. Young. London: Routledge & Kegan Paul, 1981. 51–78. Garfinkel, Harold, ed. Studies in Ethnomethodology. Cambridge: Polity Press, 1967. Gherardi, Silvia. “To Start Practice Theorizing Anew: The Contribution of the Concepts of Agencement and Formativeness.” Organization 23.5 (2016): 680–98. Giddens, Anthony. Central Problems in Social Theory. London: Macmillan Education UK, 1979. Hardie, Iain, and Donald MacKenzie. “Assembling an Economic Actor: The Agencement of a Hedge Fund.” The Sociological Review 55.1 (2007): 57–80. Horan, Geraldine. “‘Er zog sich die ‚neue Sprache‘ des ‚Dritten Reiches‘ über wie ein Kleidungsstück‘: Communities of Practice and Performativity in National Socialist Discourse.” Linguistik online 30.1 (2007): 57–80. 22 Sep. 2020 <https://doi.org/10.13092/lo.30.549>. ———. “‘Lieber Guter Onkel Hitler’: A Linguistic Analysis of the Letter as a National Socialist Text-Type and a Re-Evaluation of the ‘Sprache im/des Nationalsozialismus’ Debate.” New Literary and Linguistic Perspectives on the German Language, National Socialism, and the Shoah. Eds. Peter Davies and Andrea Hammel. Rochester, NY: Camden House, 2014. 45–58. Kämper, Heidrun. “Sprachliche Sozialgeschichte 1933 bis 1945 – Ein Projektkonzept.” Sprachliche Sozialgeschichte des Nationalsozialismus. Eds. Heidrun Kämper and Britt-Marie Schuster. Bremen: Hempen Verlag, 2018. 9–25. Kilian, Katrin Anja. “Das Medium Feldpost als Gegenstand interdisziplinärer Forschung: Archivlage, Forschungsstand und Aufbereitung der Quelle aus dem Zweiten Weltkrieg.” Dissertation. Technische Universität Berlin, 2001. 22 Sep. 2020 <https://doi.org/10.14279/depositonce-322>. Reddemann, Karl, ed. Zwischen Front und Heimat: Der Briefwechsel des münsterischen Ehepaares Agnes und Albert Neuhaus 1940–1944. Münster: Regensberg, 1996. Sauer, Christoph. “1933–1945.” Handbuch Sprache und Politik: In 3 Bänden. Eds. Thomas Niehr, Jörg Kilian, and Martin Wengeler. Bremen: Hempen Verlag, 2017. 975–98. Schlosser, Horst Dieter. Sprache unterm Hakenkreuz: Eine andere Geschichte des Nationalsozialismus. Köln: Böhlau, 2013. Scholl, Stefan. “Für eine Sprach- und Kommunikationsgeschichte des Nationalsozialismus: Ein Programmatischer Forschungsüberblick.” Archiv für Sozialgeschichte 59 (2019): 409–44. Spitzmüller, Jürgen, and Ingo H. Warnke. Diskurslinguistik: Eine Einführung in Theorien und Methoden der transtextuellen Sprachanalyse. Berlin, New York: De Gruyter, 2011. Teubert, Wolfgang. “Corpus Linguistics: An Alternative.” Semen 27 (2009): 1–25. Von Polenz, Peter. Deutsche Satzsemantik: Grundbegriffe des Zwischen-den-Zeilen-Lesens. Berlin: De Gruyter, 1985. Wildt, Michael. “Volksgemeinschaft: A Modern Perspective on National Socialist Society.” Visions of Community in Nazi Germany. Eds. Martina Steber and Bernhard Gotto. Oxford: Oxford UP, 2014. 43–59. Wodak, Ruth. “Discourse and Politics: The Rhetoric of Exclusion.” The Haider Phenomenon in Austria. Eds. Ruth Wodak and Anton Pelinka. New Brunswick, NJ: Transaction Publishers, 2002. 33–60.

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.