A recent view about encephalomyocarditis virus circulating in compartmentalised animal population in Northern Italy

Abstract

Encephalomyocarditis virus (Picornaviridae, Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis and reproductive disorders in various mammals, especially in swine. Despite the disease occurred endemically in pig farms since 1997, the recent increase of death experimented in Northern Italy prompted to furtherly investigate the evolution of the virus and the actual spread of the infection. Italian EMC viruses, collected between 2013 and 2019, showed an overall antigenic stability. The in-house ELISA Monoclonal Antibodies based, able to reveal changes in seven different antigenic sites, showed only sporadic and occasional mutations in considered samples and the subsequent phylogenetic analysis confirmed antigenic panel's remarks. All the isolates could be classified within a unique lineage, which comprise other European strains and confirm that the viruses currently circulating in Italy developed from a unique common ancestor. Despite the demonstrated stability of virus, some putative newly emerged variants were detected through antigenic profile analysis and phylogenesis. Finally, the serosurvey proved that spread of EMCV is greater than the diffusion of fatal infections would suggest, due to subclinical circulation of EMCV. It demonstrated an increase in the proportion of seropositive farms, if compared with previous data with no remarkable differences between farms with and without clinical evidence of disease.

Figure 1

Geographic origin of EMCV recent isolates. The map shows the exact location of samples’ collection. The main box is an overall picture of the sampling area involving Northern and Central Italy; two boxes display magnifications of affected area. The major study area, big square, included almost all of the Po Valley, primarily Lombardy, Emilia-Romagna and Veneto, even if analysis also considered some samples from Piedmont (Cuneo, in the south-western border of the box) and Friuli Venezia-Giulia (near to Pordenone, in the north-eastern border of the box). Affected Central Italy area, tiny square, was smaller and included a zone between south of Tuscany (close to Grosseto) and Umbria (province of Perugia). Each red dot represents one village where sampling took place. Two or more farms could be located in the same village and indicated by a unique icon. Pictures were generated on-line on https://it.batchgeo.com/.

Figure 2

Workflow. The diagram shows the pattern of activities and experiments carried out, from sampling to obtainment of data. In dark cells, all the operations performed to get the results, presented in lighter cells.

Figure 3

Characterization of antigenic profile of recent isolates. Graphical representation of reactivity of MAbs against recent EMCV isolates considered in the analysis. The upper rows of the tables indicate MAbs features: names, epitopes’ characteristics, areas and eventual involvement in viral neutralization. Letters from A to R, on the left, indicate the groups generated clustering analysed samples accordingly to homogeneity in induce antibody reactivity, and the size of each group is indicated alongside. Colours of squares denote reactivity level: black, high; grey medium; white, null. The picture revealed an overall homogeneity of reactivity of MAbs listed in area 1, 2 and 7 and a discrete, but occasional and unfixed, variability of the other MAbs. Maintenance of high reactivity of 3A3 II (completely black column) and loss of reactivity of 4H2 (almost completely white column) are evident in the picture. Even changes in epitopes of area 2 of samples clustered in Group C, with consequent loss of reactivity by specific MAbs, are clearly noticeable.

Figure 4

Maximum Likelihood phylogenetic tree of EMCV strains. Phylogenetic analysis of EMCV strains based on sequences of gene coding for 1D (VP1) capsidic protein. (A) analysis included recent EMCV isolates and strains collected in databases, respectively listed in Supplementary Table S1 and S2. Resulting tree is obtained by Maximum Likelihood method with 1000 bootstrap (see text). Sequences, all belonging to EMCV-1, clustered into 6 linages (LIN A – LIN G, sequences of LIN F were not included in the analysis), accordingly to Vyshemirskii and colleagues. (B) magnification of LIN B, including all the samples considered by the analysis. Brackets indicate closer subgroups: (I) strains isolated from wild hosts in the recovery centre for wild animals near to Grosseto (Tuscany); (II) strains isolated from primates in the natural park “Natura Viva” in province of Verona (Veneto); (III) strains isolated in 2015 in Emilia-Romagna and clustered in Group C of Antigenic Panel; (IV) strains isolated from samples originated from an unique village in province of Brescia (Lombardy), showing higher phylogenetic distance. Asterisks sign strains originated from consecutive samplings in three farms; * farm located in Province of Cremona (Lombardy) with samples very closely clustering; ** farm located in province of Verona (Veneto) and distributed in various groups; *** farm located in province of Brescia (Lombardy) with evidence of strains clustering together. Bootstrap values are indicated over the branches, when lower of 0.7 they are omitted.

Figure 5

Serosurvey of pig herds in Northern Italy. Graphs compare seroprevalence percentage obtained during serosurvey of swine farms of Lombardy and Emilia-Romagna in 2016 with data of 2010 and 2000. Farms were considered as positive (black) if percentage of seropositive sera was higher than 10% and negative with lower percentage of seropositivity. Negatives include farms with seroprevalence equal to 0 (white) and with seroprevalence lower than 10% (dotted). (A) total of farms considered.(B) herds divided between Fattening and Breeding. Only 2016 and 2010 data considered. Differences between data were statistically validated with one-sided Fisher’s exact test and p value obtained for every couples compared was lower than 0.001. *, in 2000, serosurvey divided farms by seroprevalence percentage in four groups: neg, < 5%, 5–15%, > 15%. All farms with seroprevalence lower than 15% were considered as “negative with seroprevalence” (dotted).