Sheep retroviruses can be used to map the selective preferences of early farmers and trace livestock movements across Europe.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of the host germline transmitted vertically from generation to generation. It is hypothesized that some ERVs are used by the host as restriction factors to block the infection of pathogenic retroviruses. Indeed, some ERVs efficiently interfere with the replication of related exogenous retroviruses. However, data suggesting that these mechanisms have influenced the coevolution of endogenous and/or exogenous retroviruses and their hosts have been more difficult to obtain. Sheep are an interesting model system to study retrovirus-host coevolution because of the coexistence in this animal species of two exogenous (i.e., horizontally transmitted) oncogenic retroviruses, Jaagsiekte sheep retrovirus and Enzootic nasal tumor virus, with highly related and biologically active endogenous retroviruses (enJSRVs). Here, we isolated and characterized the evolutionary history and molecular virology of 27 enJSRV proviruses. enJSRVs have been integrating in the host genome for the last 5–7 million y. Two enJSRV proviruses (enJS56A1 and enJSRV-20), which entered the host genome within the last 3 million y (before and during speciation within the genus Ovis), acquired in two temporally distinct events a defective Gag polyprotein resulting in a transdominant phenotype able to block late replication steps of related exogenous retroviruses. Both transdominant proviruses became fixed in the host genome before or around sheep domestication (∼ 9,000 y ago). Interestingly, a provirus escaping the transdominant enJSRVs has emerged very recently, most likely within the last 200 y. Thus, we determined sequentially distinct events during evolution that are indicative of an evolutionary antagonism between endogenous and exogenous retroviruses. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections. The genome of all vertebrates is heavily colonized by “endogenous” retroviruses (ERVs). ERVs derive from retrovirus infections of the germ cells of the host during evolution, leading to permanent integration of the viral genome into the host DNA. Because ERVs are integrated in the host genome, they are transmitted to subsequent generations like any other host gene. The function of endogenous retroviruses is not completely clear, but some ERVs can block the replication cycle of horizontally transmitted “exogenous” pathogenic retroviruses. These observations lead to the hypothesis that ERVs have protected the host during evolution against incoming pathogenic retroviruses. Here, by characterizing the evolutionary history and molecular virology of a particular group of endogenous betaretroviruses of sheep (enJSRVs) we show a fascinating series of events unveiling the endless struggle between host and retroviruses. In particular, we discovered that: (i) two enJSRV loci that entered the host genome before speciation within the genus Ovis (∼ 3 million y ago) acquired, after their integration, a mutated defective viral protein capable of blocking exogenous related retroviruses; (ii) both these transdominant enJSRV loci became fixed in the host genome before or around sheep domestication (∼ 10,000 y ago); (iii) the invasion of the sheep genome by ERVs of the JSRV/enJSRVs group is still in progress; and (iv) new viruses have recently emerged (less than 200 y ago) that can escape the transdominant enJSRV loci. This study strongly suggests that endogenization and selection of ERVs acting as restriction factors is a mechanism used by the host to fight retroviral infections.

The presence of Anaplasma phagocytophilum , a tick-transmitted zoonotic pathogen, was investigated in Sardinia using a molecular approach. Phylogenetic analysis revealed that Sardinian strains are genetically distinct from the two lineages previously described in Europe and are closely related to strains isolated in different areas of the United States.

Few data are available on the prevalence and molecular typing of species belonging to the genus Anaplasma in Mediterranean ruminants. In this study, PCR analysis and sequencing of both 16S rRNA and groEL genes were combined to investigate the presence, prevalence, and molecular traits of Anaplasma spp. in ruminants sampled on the Island of Sardinia, chosen as a subtropical representative area. The results demonstrate a high prevalence of Anaplasma spp. in ruminants, with animals infected by at least four of six Anaplasma species ( Anaplasma marginale , A. bovis , A. ovis , and A. phagocytophilum ). Moreover, ruminants host a number of neutrophil-tropic strains genetically closely related to the canine pathogen A. platys . The high Anaplasma spp. prevalence and the identification of as-yet-unclassified neutrophil-tropic strains raise concerns about the specificity of serological tests routinely used in ruminants and provide additional background for reconstructing the evolutionary history of species genetically related to A. phagocytophilum .

A survey of Anaplasma species in small ruminants is still lacking in North African countries. In this study, the presence of A. phagocytophilum , A. phagocytophilum –related species, and A. ovis was investigated in a total of 563 healthy small ruminants (303 goats and 260 sheep), from 25 randomly selected flocks sampled in Tunisia. Anaplasma spp. and A. ovis overall infection rates were 95.0% and 93.8% in sheep and 69.6% and 65.3% in goats, respectively. A. phagocytophilum was not detected in any of tested animals. A total of 20 sheep (7.7%) and 144 goats (47.5%) were infected by Anaplasma strains genetically related to A. phagocytophilum . Both in sheep and goats A. ovis prevalence was higher in adults (≥2 years) than in young (<2 years) subjects ( p = 0.001 and 0.002 for goats and sheep, respectively). In sheep, A. ovis prevalence was higher in ewes with respect to rams ( p = 0.010). The A. ovis infection rate was significantly lower in goats of the local breed ( p = 0.049) and it was higher in goats infested by ticks than in not infested animals ( p = 0.005). Genetic analysis of the msp4 gene of A. ovis indicated the presence of strains shared by Tunisian sheep and goats. Sequence analysis and phylogenetic studies on the basis of the 16S rRNA gene provided evidence for the circulation of at least two different potentially novel species genetically related to A. phagocytophilum in Tunisian small ruminants. These findings cause concern about specificity of serological tests used for detection of A. phagocytophilum in ruminants and provide additional information for elucidating pathogenesis and molecular epidemiology of A. phagocytophilum and related species.

Ovine betaretroviruses include Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). JSRV and ENTV represent a unique class of oncogenic retroviruses that induce tumors of the respiratory tract. JSRV and ENTV are highly related but induce different diseases. Expression of the JSRV envelope (Env) induces transformation of rodent fibroblasts in vitro and phosphorylation of Akt, a central player in the phosphatidylinositol 3-kinase (PI-3K)/Akt signal transduction pathway. However, little information is available on the molecular biology of ENTV. In this study, we initially assessed whether the ENTV Env has the same properties as the homologous JSRV protein. We performed entry and interference assays using retroviral vectors pseudotyped with either the JSRV or the ENTV Env and sheep choroid plexus cells, choroid plexus cells stably expressing the JSRV Env protein, human 293T cells, mouse NIH 3T3 cells, or NIH 3T3 cells expressing human hyaluronidase 2 (HYAL2), the cellular receptor for JSRV. The results obtained indicated that ENTV and JSRV share the same receptor in sheep cells and that they can use human HYAL2 as a cellular receptor in mouse cells. The ENTV Env induces transformation of rodent fibroblasts in vitro. As with the JSRV Env, the tyrosine at position 590 is critical for ENTV Env-induced cell transformation, and Akt is phosphorylated in ENTV Env-transformed cells but not in the parental cell lines. Thus, ovine betaretroviruses share a common mechanism of cell transformation. We further investigated the relevance of Akt activation in cells transformed by ovine betaretroviruses. A PI-3K inhibitor blocked Akt phosphorylation in JSRV Env-transformed cells, suggesting a possible involvement of PI-3K in JSRV and ENTV Env-induced cell transformation. In addition, phosphorylated Akt was detected in a cell line derived from a lung tumor of a sheep with naturally occurring ovine pulmonary adenocarcinoma.