Structure and dynamics of bio-hydrogels investigated by neutron and X-ray scattering techniques

Hyaluronic acid (HYA) is a natural polysaccharide, belonging to the family of glycosaminoglycans, characterized by the repetition of a disaccharide unit of glucuronic acid and N-acetylglucosamine. HYA is present throughout the animal kingdom, from the extracellular matrices in which most tissues differentiate, to the vitreous of the human eye and the synovial joint fluid. Thanks to its ability to form viscous solutions in water, HYA is widely used to treat inflammatory and degenerative joint diseases, a group of pathologies with a high impact in society since they contribute heavily to the rise of health costs and they affect life quality. Beside HYA, we investigated a chemically modified form of hyaluronic acid, HYADD, obtained derivatizing the polysaccharide backbone with hexadecylic (C-16) side chains, through amide bonds, with a 1-3 mol-% degree of substitution of repeating units. Even if the modification is very small, it alters dramatically some macroscopic properties such as the elastic behavior and the rheological response. The resulting system is a relatively stable hydrogel at polymer concentrations higher than 0.3% (weight of polymer/total volume), whereas native hyaluronic acid forms highly viscous solutions only at concentrations ten times higher. HYADD, however, does not differ from HYA in other relevant features such as charge density along the chain and swelling ability, as well as in its interaction behavior with metabolites and cellular material in the synovial matrix. On the basis of the beneficial effects noticed in recent tests performed on animal models of osteoarthritis, HYADD is expected to have a positive clinical effect in joint mobility and function. The aim of this PhD thesis is to compare the natural gel and the chemically modified one focusing on microscopic structural and dynamic properties such as the structure and organization of the polymer chains, the dynamics of the polymer and the self-diffusivity of water in the gels. We have performed several experiments using X-ray and neutron scattering to probe gels at different polymer concentrations and temperatures. From the structural point of view we found that HYADD has a structure more compact than HYA owing to the hexadecylic branches added along the chain. The characteristic lengths derived from neutron and X-ray experiments (~ 150 Å) are in fair agreement with those resulting from dynamics light scattering measurements. The small angle diffraction patterns show, in addition, the presence of some degree of order in the organization of the natural saccharide chain in the most concentrated gels. The dynamics of HYA and HYADD shows some small differences in the 100-1000 ps timescale (mostly in the low-Q region) that can be related to the presence of the chemical modification. On the other hand the diffusive dynamics of gel water is similar to that of bulk water for both HYA and HYADD gels at least up to 10% gel concentration. In the frame of a collaboration with the group of Prof. Santi at the Department of Pharmacy of Parma University, we contributed to the characterization of a novel platform called Patch-non Patch®, developed for transdermal drug delivery. In particular, using quasielastic neutron scattering we investigated the diffusivity of a model drug (lidocaine) through the patch. The obtained data indicate that the diffusion of lidocaine is triggered by that of hydration water present in the patch. In the framework of a CNR PhD grant, I spent 18 months at the Institut Laue Langevin in Grenoble, where I joined the group of Italian and French researchers in charge of the backscattering spectrometer IN13. Under the supervision of Dr. Francesca Natali, I took part to the group activities, contributing to the technical developments and to the assistance to users during their experiments.L'acido ialuronico (HYA) è un polisaccaride naturale, appartenente alla famiglia dei glicosaminoglicani, caratterizzato dalla ripetizione di un' unità disaccaridica composta da acido glucuronico ed N-acetilglucosammina. Lo HYA è presente in tutto il regno animale, dalle matrici extracellulari in cui molti tessuti si differenziano, all'umor vitreo dell'occhio umano, al fluido sinoviale in corrispondenza delle giunture. Grazie alla sua abilità di formare soluzioni viscose in acqua, lo HYA è ampiamente impiegato nei trattamenti delle malattie infiammatorie e degenerative delle articolazioni, un gruppo di patologie con elevato impatto sociale in quanto fortemente legate all'aumento dei costi sanitari ed alla qualità della vita. Dell'acido ialuronico abbiamo studiato anche una forma chimicamente modificata, lo HYADD, ottenuta derivatizzando, attraverso legami ammidici, la catena principale del polisaccaride con catene laterali esadeciliche (C-16) con un grado di sostituzione dell'1-3% delle unità ripetitive. La modifica, pur essendo piccola, altera drammaticamente alcune delle proprietà macroscopiche come il comportamento elastico e la riposta reologica del polimero. Con lo HYADD si ottiene un idrogel relativamente stabile a concentrazioni polimeriche superiori allo 0.3% (peso polimero/ volume totale), mentre l'acido ialuronico naturale forma soluzioni viscose a concentrazioni dieci volte superiori. Lo HYADD, tuttavia, non differisce dallo HYA in altre caratteristiche importanti quali la densità di carica lungo la catena, l'abilità di 'swelling' o il comportamento d'interazione con metaboliti e materiale cellulare nella matrice sinoviale. Sulla base degli effetti benefici notati in recenti test condotti su modelli animali di osteoartrite, si pensa che lo HYADD possa avere effetti clinici positivi nella mobilità e nella funzione delle articolazioni. Lo scopo principale di questa tesi è quello di confrontare il gel naturale e quello chimicamente modificato sulla base di proprietà strutturali quali l'organizzazione delle catene polimeriche e di proprietà dinamiche come la micro diffusività dell'acqua nel gel. Per studiare i due sistemi polimerici in diverse condizioni di temperatura e concentrazione, abbiamo condotto diversi esperimenti di scattering di raggi X e neutroni. Dal punto di vista strutturale abbiamo scoperto che lo HYADD ha una struttura più compatta dello HYA, dovuta ai bracci laterali presenti lungo la catena principale. Le lunghezze caratteristiche (~150 Å) derivate dagli esperimenti di neutroni e raggi X, sono in buon accordo con quelle risultanti dalle misure di scattering dinamico della luce. Sui gels più concentrati, inoltre, i patterns di diffrazione a basso angolo mostrano la presenza di qualche grado d'ordine nell'organizzazione della catena saccaridica di HYA. La dinamica delle catene di HYA e HYADD mostra alcune piccole differenze sulla scala temporale tra i 100 e i 1000 ps (principalmente nella regione a bassi Q), che può essere collegata alla presenza della modifica chimica. La dinamica diffusiva dell'acqua nel gel invece risulta simile a quella dell'acqua bulk sia per lo HYA che per lo HYADD, almeno fino ad una concentrazione del 10%. Nel contesto di una collaborazione col gruppo della Prof.ssa Santi del Dipartimento di Farmacia dell'Università di Parma, abbiamo contribuito alla caratterizzazione di una nuova piattaforma chiamata Patch-non Patch®, sviluppata per il trasporto transdermico del farmaco. In particolare, abbiamo studiato la diffusività di un farmaco modello (lidocaina) attraverso il cerotto con lo scattering quasi-elastico di neutroni. I dati ottenuti indicano che la diffusione della lidocaina è triggerata da quella dell'acqua di idratazione presente nel cerotto. Grazie al finanziamento del CNR, ho potuto trascorrere 18 mesi all' Institut Laue Langevin a Grenoble, ospite del gruppo di ricercatori italo-francesi responsabili dello spettrometro a retrodiffusione IN13. Sotto la supervisione della Dott.ssa Francesca Natali, ho partecipato alle attività del gruppo, contribuendo ad esempio agli sviluppi tecnici e all'assistenza degli utilizzatori durante gli esperimenti

Genetic diversification patterns in swine influenza A virus (H1N2) in vaccinated and nonvaccinated animals

Influenza A viruses (IAVs) are characterized by having a segmented genome, low proofreading polymerases, and a wide host range. Consequently, IAVs are constantly evolving in nature causing a threat to animal and human health. In 2009 a new human pandemic IAV strain arose in Mexico because of a reassortment between two strains previously circulating in pigs; Eurasian "avian-like" (EA) swine H1N1 and "human-like" H1N2, highlighting the importance of swine as adaptation host of avian to human IAVs. Nowadays, although of limited use, a trivalent vaccine, which include in its formulation H1N1, H3N2, and, H1N2 swine IAV (SIAV) subtypes, is one of the most applied strategies to reduce SIAV circulation in farms. Protection provided by vaccines is not complete, allowing virus circulation, potentially favoring viral evolution. The evolutionary dynamics of SIAV quasispecies were studied in samples collected at different times from 8 vaccinated and 8 nonvaccinated pigs, challenged with H1N2 SIAV. In total, 32 SIAV genomes were sequenced by next-generation sequencing, and subsequent variant-calling genomic analysis was carried out. Herein, a total of 364 de novo single nucleotide variants (SNV) were found along all genetic segments in both experimental groups. The nonsynonymous substitutions proportion found was greater in vaccinated animals suggesting that H1N2 SIAV was under positive selection in this scenario. The impact of each substitution with an allele frequency greater than 5% was hypothesized according to previous literature, particularly in the surface glycoproteins hemagglutinin and neuraminidase. The H1N2 SIAV quasispecies evolution capacity was evidenced, observing different evolutionary trends in vaccinated and nonvaccinated animals

Evolution of Swine Influenza Virus H3N2 in Vaccinated and Nonvaccinated Pigs after Previous Natural H1N1 Infection

Swine influenza viruses (SIV) produce a highly contagious and worldwide distributed disease that can cause important economic losses to the pig industry. Currently, this virus is endemic in farms and, although used limitedly, trivalent vaccine application is the most extended strategy to control SIV. The presence of pre-existing immunity against SIV may modulate the evolutionary dynamic of this virus. To better understand these dynamics, the viral variants generated in vaccinated and nonvaccinated H3N2 challenged pigs after recovery from a natural A(H1N1) pdm09 infection were determined and analyzed. In total, seventeen whole SIV genomes were determined, 6 from vaccinated, and 10 from nonvaccinated animals and their inoculum, by NGS. Herein, 214 de novo substitutions were found along all SIV segments, 44 of them being nonsynonymous ones with an allele frequency greater than 5%. Nonsynonymous substitutions were not found in NP; meanwhile, many of these were allocated in PB2, PB1, and NS1 proteins. Regarding HA and NA proteins, higher nucleotide diversity, proportionally more nonsynonymous substitutions with an allele frequency greater than 5%, and different domain allocations of mutants, were observed in vaccinated animals, indicating different evolutionary dynamics. This study highlights the rapid adaptability of SIV in different environments.info:eu-repo/semantics/publishedVersio

Vaccination against swine influenza in pigs causes different drift evolutionary patterns upon swine influenza virus experimental infection and reduces the likelihood of genomic reassortments

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios.This research was funded by grants AGL2016–75280-R from Ministerio de Ciencia, Innovación y Universidades from the Spanish government. ÁL-V has a pre-doctoral fellowship FPI 2017, Ministerio de Ciencia, Innovación y Universidades from the Spanish government.The authors would like to thank Laura Baioni for the sequencing libraries preparation. The authors also want to thank the IRTA-CReSA animal facilities staff for their work during the in vivo experiment. Lastly, we would like to thank Marta Pérez and all the people from CReSA who were involved in the experiment during the COVID-19 lockdowns.info:eu-repo/semantics/publishedVersio

Arbovirus Screening in Mosquitoes in Emilia-Romagna (Italy, 2021) and Isolation of Tahyna Virus

Several viruses can be transmitted by mosquitoes. We searched some of these viruses in 20,778 mosquitoes, collected in 95 traps on the plains of Emilia-Romagna (North of Italy) in 2021. We detected West Nile virus (WNV) and Usutu virus (USUV) in pools of Culex (Cx.) pipiens. In addition, we detected two insect-specific flaviviruses in three pools of Aedes (Ae.) caspius and in two of Ae. vexans. Tahyna virus (TAHV) was detected in six pools, three of Ae. caspius and three of Cx. pipiens, and one isolated strain was obtained from one of the Ae. caspius pools. Moreover, we detected TAHV in pools of several mosquito species (Ae. caspius, Ae. vexans, Ae. albopictus, Anopheles maculipennis s.l.) collected in the previous year of surveillance. Our data indicate Ae. caspius as the species most infected with TAHV in the surveyed area. Together with the likely plasticity of the cycle, we reported strong genome stability of the TAHV, probably linked to a successful adaptation of the virus to its ecological niche. Interestingly, in six pools of Cx. pipiens we detected two associated viruses among USUV, WNV, TAHV and all the three viruses in two pools. This result allows us to assume the presence of particular conditions that prompt the circulation of arboviruses, creating the conditions for viral hot spots. While no human diseases related to Tahyna virus were reported in Italy, its detection over the years suggests that it is worth investigating this virus as a potential cause of disease in humans in order to assess its health burden. IMPORTANCE We reported in this work the detection of three Arboviruses (Arthropod-borne viruses) in mosquitoes collected in Emilia-Romagna in 2021. In addition to West Nile and Usutu viruses, which were reported from more than 10 years in the study area, we detected and isolated Tahyna virus (TAHV). We also reported detections of TAHV obtained in previous years of surveillance in different species of mosquitoes. TAHV is the potential causative agent of summer influenza-like diseases and also of meningitis. Even if human cases of disease referable to this virus are not reported in Italy, its relevant presence in mosquitoes suggests investigating the possibility they could

Filogenetska analiza HA i Na gena virusa influence svinja u Srbiji od 2016 do 2018 godine

Pigs are very important for the epidemiology of influenza A viruses, being commonly infected with the lineages of most adapted H1N1, H3N2, H1N2 swine subtypes. Epidemiological complexity of swine influenza is increasing by a periodic spillover of human or avian viruses in the pig population when genetic shifts can occur. The objectives of this research were to determine the presence of the influenza A virus in nasal and tracheobronchial swabs and lung tissue samples of ill and dead pigs on commercial farms, to determine circulating subtypes and characterize them through the phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes. A total of 255 samples collected from 13 farms were analyzed by means of real-time RT-PCR. The genome of influenza A virus was detected in 24 samples, which represented a 61.5% prevalence at the farms level (influenza A virus was confirmed in 8 out of 13 farms included in this study). Based on HA and NA gene sequences of 8 viruses, the circulation of H1N1 and H3N2 subtypes of influenza A viruses were determined. In addition, one farm exhibited a time separated circulation of H1N1 and H3N2 virus subtypes. Using Influenza Research Database, our viruses of the H1 subtype were classified into 1C.2.1 and 1A.3.3.2. clade. Based on the nucleotide sequences of HA genes, three viruses of the H1N1 subtype belong to the H1N1pdm09 lineage, and the other four to Eurasian “avian-like” H1avN1 lineage; while based on NA genes sequences, these seven viruses belong to Eurasian “avian-like” H1avN1 lineage. Both HA and NA genes of the virus of the H3N2 subtype belonged to the A/swine/ Gent/1/1984-like H3N2 lineage.Svinje su veoma važne u epidemiologiji infl uenca A virusa, jer je većina zapata širom sveta zaražena nekim od tri podtipa (H1N1, H1N2 ili H3N2). Pored toga, one su prijemčive i za ljudske i ptičije infl eunca A viruse, usled čega u njihovom organizmu može doći do genetskog reasortiranja i stvaranja genotipski i fenotipski novih virusa. Cilj ovog istraživanja je bio da se analizom nosnih i traeo-bronhijalnih briseva i pluća poreklom od bolesnih i ugilnulih svinja ispita prisustvo infl uenca A virusa, da se odrede cirkulišući podtipovi i fi logenetski okarakterišu kroz analizu sekvenci HA i NA gena. Tokom ovog istraživanja sa 13 farmi je sakupljeno i metodom realtime RT-PCR pregledano je 255 uzoraka. Genom infl uenca A virusa je utvrđen u 24 uzorka. Prevalencija virusa na nivou farmi iznosila je 61.5%. Kompletno je sekvenciran genom 8 virusa koji su poticali sa sedam farmi. Na osnovu sekvenci HA i NA gena potvrđena je cirkulacija H1N1 i H3N2 podtipova. Na jednoj farmi ustanovlejna je cirkulacija oba podtipa, ali u različitim periodima ispitivanja. Na osnovu sekvence HA gena, sedam virusa H1 podtipa su grupisani u 1C.2.1 i 1A.3.3.2 genske grupe i H1N1pdm09 i evroazijsku “avian-like” H1avN1 linije. Na osnovu sekvence neuraminidaza gena ovih sedam H1N1 virusa su grupisani u evroazijsku “avian-like” H1avN1 liniju. Virus H3N2 podtipa na osnovu sekvenci HA i NA gena pripada liniji A/Swine/ Gent/1/1984 - “like” liniji

Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool.We thank Jaime Maldonado and HIPRA (Spain) for the A/Swine/Spain/ 54008/2004 (H3N2) strain, Edward J. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) IAV strain, T. M. Chambers and the University of Kentucky for the A/Equine/OH/1/03 (H3N8) IAV strain, and Hon Ip and the U.S. Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbor Seal/New Hampshire/179629/2011 (H3N8) IAV strains. We thank Sergio López, David Solanes, Francisco X. Abad, Jordi Alberola, Jaume Martorell, and Eduard J. Cunilleras for help in providing different samples and during the experimental infections, as well as the personnel in Cat3 laboratories and the animal house. We thank Adolfo García-Sastre for providing materials and for support as the principal investigator of the NIAID-funded Center for Research in Influenza Pathogenesis (HHSN266200700010C). The research leading to these results received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), the Research Infrastructures Action under grant FP7-228393 (a NADIR project), and projects AGL2010-22200-C02-01 and AGL2007-60274 of the Spanish Ministry of Science and Innovation

Identification and Characterization of Swine Influenza Virus H1N1 Variants Generated in Vaccinated and Nonvaccinated, Challenged Pigs

Influenza viruses represent a continuous threat to both animal and human health. The 2009 H1N1 A influenza pandemic highlighted the importance of a swine host in the adaptation of influenza viruses to humans. Nowadays, one of the most extended strategies used to control swine influenza viruses (SIVs) is the trivalent vaccine application, whose formulation contains the most frequently circulating SIV subtypes H1N1, H1N2, and H3N2. These vaccines do not provide full protection against the virus, allowing its replication, evolution, and adaptation. To better understand the main mechanisms that shape viral evolution, here, the SIV intra-host diversity was analyzed in samples collected from both vaccinated and nonvaccinated animals challenged with the H1N1 influenza A virus. Twenty-eight whole SIV genomes were obtained by next-generation sequencing, and differences in nucleotide variants between groups were established. Substitutions were allocated along all influenza genetic segments, while the most relevant nonsynonymous substitutions were allocated in the NS1 protein on samples collected from vaccinated animals, suggesting that SIV is continuously evolving despite vaccine application. Moreover, new viral variants were found in both vaccinated and nonvaccinated pigs, showing relevant substitutions in the HA, NA, and NP proteins, which may increase viral fitness under field conditions.info:eu-repo/semantics/publishedVersio

Reconstructing the recent West Nile virus lineage 2 epidemic in Europe and Italy using discrete and continuous phylogeography

West Nile virus lineage 2 (WNV-2) was mainly confined to sub-Saharan Africa until the early 2000s, when it was identified for the first time in Central Europe causing outbreaks of human and animal infection. The aim of this study was to reconstruct the origin and dispersion of WNV-2 in Central Europe and Italy on a phylodynamic and phylogeographical basis. To this aim, discrete and continuous space phylogeographical models were applied to a total of 33 newly characterised full-length viral genomes obtained from mosquitoes, birds and humans in Northern Italy in the years 2013-2015 aligned with 64 complete sequences isolated mainly in Europe. The European isolates segregated into two highly significant clades: a small one including three sequences and a large clade including the majority of isolates obtained in Central Europe since 2004. Discrete phylogeographical analysis showed that the most probable location of the root of the largest European clade was in Hungary a mean 12.78 years ago. The European clade bifurcated into two highly supported subclades: one including most of the Central/East European isolates and the other encompassing all of the isolates obtained in Greece. The continuous space phylogeographical analysis of the Italian clade showed that WNV-2 entered Italy in about 2008, probably by crossing the Adriatic sea and reaching a central area of the Po Valley. The epidemic then spread simultaneously eastward, to reach the region of the Po delta in 2013, and westward to the border area between Lombardy and Piedmont in 2014; later, the western strain changed direction southward, and reached the central area of the Po valley once again in 2015. Over a period of about seven years, the virus spread all over an area of northern Italy by following the Po river and its main tributaries

Evolution of Swine Influenza Virus H3N2 in Vaccinated and Nonvaccinated Pigs after Previous Natural H1N1 Infection

Swine influenza viruses (SIV) produce a highly contagious and worldwide distributed disease that can cause important economic losses to the pig industry. Currently, this virus is endemic in farms and, although used limitedly, trivalent vaccine application is the most extended strategy to control SIV. The presence of pre-existing immunity against SIV may modulate the evolutionary dynamic of this virus. To better understand these dynamics, the viral variants generated in vaccinated and nonvaccinated H3N2 challenged pigs after recovery from a natural A(H1N1) pdm09 infection were determined and analyzed. In total, seventeen whole SIV genomes were determined, 6 from vaccinated, and 10 from nonvaccinated animals and their inoculum, by NGS. Herein, 214 de novo substitutions were found along all SIV segments, 44 of them being nonsynonymous ones with an allele frequency greater than 5%. Nonsynonymous substitutions were not found in NP; meanwhile, many of these were allocated in PB2, PB1, and NS1 proteins. Regarding HA and NA proteins, higher nucleotide diversity, proportionally more nonsynonymous substitutions with an allele frequency greater than 5%, and different domain allocations of mutants, were observed in vaccinated animals, indicating different evolutionary dynamics. This study highlights the rapid adaptability of SIV in different environments