Small Ruminant lentiviruses are heterogeneous group of virus able to infect goat and sheep. Four different genotypes were classified, and several subtypes were recognized in each group. This heterogeneity can influence both the host interaction and the diagnostic test results. In vivo and in vitro properties were investigated for different SRLV genotypes and recent studies demonstrated how small changes in the genetic sequences can interfere with the cell tropism during the viral replicative cycle. In the same way, given the antigenic diversity, diagnostic tests can fail in the identification of seropositive animals when they are based on heterologous antigens. In 2012 we conducted serological screening on 581 goats in Aosta Valley, finding a very high prevalence (49.57%). The 75% of the flocks showed a high reactivity against SRLV-A antigens, associated to ovine hosts. Molecular characterization confirmed that the SRLV-A8 subgroup were specifically associated to the Valdostana breed. In this report a viral strain was isolated and further characterised. A three years old Valdostana goat was selected based on specific immunoresponse and genetic sequence characterization (SRLV-A8). During regular slaughter, target tissues were collected and tissue explants were obtained from the mammary gland and lymph nodes, lung, mediastinal lymph nodes, synovial membrane, choroid plexus, and spleen. Cultures were maintained over five passages and weekly examined (CPE and RT activity). RT activity was detected only in spleen culture with a decreasing signal over time, suggesting a scarce susceptibility of the spleen fibroblasts selected during tissue explantation. A foetal caprine lung cell line, known to be permissive to SRLV, was also unsuccessful when used in co-culture. No CPE was observed in any culture. We then infect blood derived macrophage (BDM) obtained from SRLV free herd using the supernatant of the first passage spleen explant, resulting in the highest RT activity after 6 dpi. Supernatant was collected, centrifuged for eliminating the cells debris and concentrated using Amicon-15 100 kDa centrifugal filter tubes. Total RNA was extracted from the concentrated supernatant and reverse transcribed as double stranded cDNA. Nextera XT protocol was applied and MiSeq platform was used to fully sequence the virus on a 2x250 V2 Nano flowcell. A total of 165,156 reads were obtained and assembled with Ray. The analysis of the Gag sequence confirmed the subtype definition as well as the Pol gene sequence. Interestingly, the assembly procedure resulted in two different consensus sequences, showing differences along Tat and Env codifying sequences. Env protein is known to play a role in cell receptor and co-receptor binding. In particular, the hyper variable region HV2 was recognized as an important receptor binding site as well as a linear neutralizing epitope: in one of the two consensus sequence the HV2 region was absent, and the deletion was confirmed by the high coverage (1600X against 700X) and by Sanger sequencing. As previously demonstrated, mutation of the HV2 region may lead to a restricted tissue tropims and a marker of low pathogenic potential of A8 subtype in goat. The second env variant, harbouring a complete HV2 motif was not selected during in vitro propagation suggesting no advantages in terms of tissue tropism. Further studies are planned to verify this hypothesis including entry assays using viral pseudotypes using both env variant.

SMALL RUMINANT LENTIVIRUS A8 IN VALDOSTANA GOATS

Barbara Colitti;Margherita Profiti;Maria Teresa Capucchio;Luigi Bertolotti;Sergio Rosati
2017-01-01

Abstract

Small Ruminant lentiviruses are heterogeneous group of virus able to infect goat and sheep. Four different genotypes were classified, and several subtypes were recognized in each group. This heterogeneity can influence both the host interaction and the diagnostic test results. In vivo and in vitro properties were investigated for different SRLV genotypes and recent studies demonstrated how small changes in the genetic sequences can interfere with the cell tropism during the viral replicative cycle. In the same way, given the antigenic diversity, diagnostic tests can fail in the identification of seropositive animals when they are based on heterologous antigens. In 2012 we conducted serological screening on 581 goats in Aosta Valley, finding a very high prevalence (49.57%). The 75% of the flocks showed a high reactivity against SRLV-A antigens, associated to ovine hosts. Molecular characterization confirmed that the SRLV-A8 subgroup were specifically associated to the Valdostana breed. In this report a viral strain was isolated and further characterised. A three years old Valdostana goat was selected based on specific immunoresponse and genetic sequence characterization (SRLV-A8). During regular slaughter, target tissues were collected and tissue explants were obtained from the mammary gland and lymph nodes, lung, mediastinal lymph nodes, synovial membrane, choroid plexus, and spleen. Cultures were maintained over five passages and weekly examined (CPE and RT activity). RT activity was detected only in spleen culture with a decreasing signal over time, suggesting a scarce susceptibility of the spleen fibroblasts selected during tissue explantation. A foetal caprine lung cell line, known to be permissive to SRLV, was also unsuccessful when used in co-culture. No CPE was observed in any culture. We then infect blood derived macrophage (BDM) obtained from SRLV free herd using the supernatant of the first passage spleen explant, resulting in the highest RT activity after 6 dpi. Supernatant was collected, centrifuged for eliminating the cells debris and concentrated using Amicon-15 100 kDa centrifugal filter tubes. Total RNA was extracted from the concentrated supernatant and reverse transcribed as double stranded cDNA. Nextera XT protocol was applied and MiSeq platform was used to fully sequence the virus on a 2x250 V2 Nano flowcell. A total of 165,156 reads were obtained and assembled with Ray. The analysis of the Gag sequence confirmed the subtype definition as well as the Pol gene sequence. Interestingly, the assembly procedure resulted in two different consensus sequences, showing differences along Tat and Env codifying sequences. Env protein is known to play a role in cell receptor and co-receptor binding. In particular, the hyper variable region HV2 was recognized as an important receptor binding site as well as a linear neutralizing epitope: in one of the two consensus sequence the HV2 region was absent, and the deletion was confirmed by the high coverage (1600X against 700X) and by Sanger sequencing. As previously demonstrated, mutation of the HV2 region may lead to a restricted tissue tropims and a marker of low pathogenic potential of A8 subtype in goat. The second env variant, harbouring a complete HV2 motif was not selected during in vitro propagation suggesting no advantages in terms of tissue tropism. Further studies are planned to verify this hypothesis including entry assays using viral pseudotypes using both env variant.
2017
LXXI Convegno SISVet
Napoli - Italia
28-30 Giugno 2017
Sisvet - Abstract Book -Comunicazioni Orali
Società Italiana Scienze Veterinarie - SISVet
191
191
9788890909245
Barbara Colitti, Elisabetta Coradduzza, Margherita Profiti, Maria Teresa Capucchio, Marco Ragionieri, Giantonella Puggioni, Luigi Bertolotti, Sergio Rosati
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1682602
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