Molecular characterization and phylogenetic analysis of small ruminant lentiviruses isolated from Canadian sheep and goats

Background: Small Ruminant Lentiviruses (SRLV) are widespread in Canadian sheep and goats and represent an important health issue in these animals. There is however no data about the genetic diversity of Caprine Arthritis Encephalitis Virus (CAEV) or Maedi Visna Virus (MVV) in this country. Findings: We performed a molecular and phylogenetic analysis of sheep and goat lentiviruses from a small geographic area in Canada using long sequences from the gag region of 30 infected sheep and 36 infected goats originating from 14 different flocks. Pairwise DNA distance and phylogenetic analyses revealed that all SRLV sequences obtained from sheep clustered tightly with prototypical Maedi visna sequences from America. Similarly, all SRLV strains obtained from goats clustered tightly with prototypical US CAEV-Cork strain. Conclusions: The data reported in this study suggests that Canadian and US SRLV strains share common origins. In addition, the molecular data failed to bring to light any evidence of past cross species transmission between sheep and goats, which is consistent with the type of farming practiced in this part of the country where single species flocks predominate and where opportunities of cross species transmissions are proportionately low.

Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.

Genomic heterogeneity of small ruminant lentiviruses detected by PCR

In order to detect a large spectrum of small ruminant lentiviruses, primers for PCR were chosen in conserved parts of the LTR and GAG genes of Icelandic Visna virus 1514 and of the POL gene of caprine arthritis-encephalitis virus. This set of primers was tested in six different caprine arthritis-encephalitis virus (CAEV)- and Maedi-Visna virus isolates of Dutch, American and Swiss origin. The LTR primers allowed the detection of the corresponding fragments of all isolates. The GAG primers allowed amplification of the corresponding fragments of all but the Swiss Maedi-Visna virus strain OLV. Using the POL primers, one Maedi-Visna- and two caprine arthritis-encephalitis virus strains were detected after one round of amplification. Sequencing of the GAG and POL amplification products and comparison to Icelandic Visna virus and CAEV strain CO revealed total heterogeneity of 38% for the GAG- and 28% for the POL fragment. The virus strains studied fall into two groups which are more closely related to one another than to Icelandic Visna virus.

[How the bovine viral diarrhea virus outwits the immune system]

The interaction of bovine viral diarrhea virus (BVD virus) with its host has several unique features, most notably the capacity to infect its host either transiently or persistently. The transient infection stimulates an antiviral immune reaction similar to that seen in other transient viral infections. In contrast, being associated with immunotolerance specific for the infecting BVD viral strain, the persistent infection differs fundamentally from other persistent infections like those caused by lentiviruses. Whereas the latter are characterized by complex viral evasion of the host's adaptive immune response by mechanisms such as antigenic drift and interference with presentation of T cell epitopes, BVD virus avoids the immune response altogether by inducing both humoral and cellular immune tolerance. This is made possible by invasion of the fetus at an early stage of development. In addition to adaptive immunity, BVD virus also manipulates key elements of the host's innate immune response. The non-cytopathic biotype of BVD virus, which is capable of persistently infecting its host, fails to induce type I interferon. In addition, persistently infected cells are resistant to the induction of apoptosis by double-stranded RNA and do not produce interferon when treated with this pathogen-associated molecular pattern (PAMP) that signals viral infection. Moreover, when treated with interferon, cells persistently infected with non-cytopathic BVD virus do not clear the virus. Surprisingly, however, despite this lack of effect on persistent infection, interferon readily induces an antiviral state in these cells, as shown by the protection against infection by unrelated viruses. Overall, BVD virus manipulates the host's interferon defense in a manner that optimises its chances of maintaining the persistent infection as well as decreasing the risks that heterologous viral infections may carry for the host. Thus, since not all potential host cells are infected in animals persistently infected with BVD virus, heterologous viruses replicating in cells uninfected with BVD virus will still trigger production of interferon. Interferon produced by such cells will curtail the replication of heterologous viruses only, be that in cells already infected with BVD virus, or in cells in which the heterologous virus may replicate alone. From an evolutionary viewpoint, this strategy clearly enhances the chances of transmission of BVD virus to new hosts, as it attenuates the negative effects that a global immunosuppression would have on the survival of persistently infected animals.

Viral load, organ distribution, histopathological lesions, and cytokine mRNA expression in goats infected with a molecular clone of the caprine arthritis encephalitis virus

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that causes persistent infection characterized by the appearance of inflammatory lesions in various organs. To define the sites of persistence, 5 goats were infected with a molecular clone of CAEV, and the viral load was monitored by real-time-PCR and RT-PCR in different sites 8 years after infection. The lymph nodes proved to be an important virus reservoir, with moderate virus replication relative to what is reported for lentiviruses of primates. Mammary gland and milk cells were preferred sites of viral replication. The viral load varied significantly between animals, which points to an important role of the genetic background. We found a clear association between occurrence of histopathological lesions and viral load in specific sites. The mRNA expression analysis of several cytokines did not reveal differences between animals that could explain the considerable individual variations in viral load observed.

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

Demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats

Recombination of different strains and subtypes is a hallmark of lentivirus infections, particularly for human immunodeficiency virus, and contributes significantly to viral diversity and evolution both within individual hosts and within populations. Recombinant viruses are generated in individuals coinfected or superinfected with more than one lentiviral strain or subtype. This, however, has never been described in vivo for the prototype lentivirus maedi-visna virus of sheep and its closely related caprine counterpart, the caprine arthritis-encephalitis virus. Cross-species infections occur in animals living under natural conditions, which suggests that dual infections with small-ruminant lentiviruses (SRLVs) are possible. In this paper we describe the first documented case of coinfection and viral recombination in two naturally infected goats. DNA fragments encompassing a variable region of the envelope glycoprotein were obtained from these two animals by end-limiting dilution PCR of peripheral blood mononuclear cells or infected cocultures. Genetic analyses, including nucleotide sequencing and heteroduplex mobility assays, showed that these goats harbored two distinct populations of SRLVs. Phylogenetic analysis permitted us to assign these sequences to the maedi-visna virus group (SRLV group A) or the caprine arthritis-encephalitis virus group (SRLV group B). SimPlot analysis showed clear evidence of A/B recombination within the env gene segment of a virus detected in one of the two goats. This case provides conclusive evidence that coinfection by different strains of SRLVs of groups A and B can indeed occur and that these viruses actually recombine in vivo.

Caprine Arthritis Encephalitis Complex

The caprine arthritis encephalitis virus (CAEV) is a lentivirus that persistently infects goats and sheep. The finding thatCAEV and Maedi-Visna viruses frequently cross the species barrier between goats and sheep, and vice versa, has changedour view of the epidemiology of these viruses that are now referred to assmall ruminant lentiviruses (SRLV).CAEV is transmitted from infected mothers to their offspring, mainly via ingestion of infected colostrum and milk. Thispermits the implementation of control measures based on the segregation ofnewborn kids immediately after birth thatsuccessfully cut the seroprevalence in infected flocks, eliminating CAEV induced clinical disease. CAEV induces overtpathology in about one third of the infected animals. The frequency of affected animals varies in different goat families,pointing to an important genetic component in this disease. The principal manifestations areencephalitis and interstitialpneumonia in young animals,whereas arthritis and mastitispredominate in adult goats. The immunopathologicalmechanisms leading to diseaseare to date unclear and involve the principal components ofthe immune system, i.e., theprofessional antigen presenting cells, which are the principal target of CAEV, and whose activity, e.g., cytokine production,is modulated by the infection, and the B- and T-cell immune responses that are alsomanipulated by the virus.In vivo,infected animals usually have low viral loads, indicating that virus replication istightly restricted by mechanisms thatremain unclear. Finally, the complex biology of SRLV makes them a great challenge for diagnostic laboratories.In this brief review, the literature pertinent toall these aspects is summarized and discussed.

Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools.

Antitumor effect of SIRT1 inhibition in human HCC tumor models in vitro and in vivo

Sirtuins (SIRT1-7) are a highly conserved family of NAD(+)-dependent enzymes that control the activity of histone and nonhistone regulatory proteins. SIRT1 is purposed to promote longevity and to suppress the initiation of some cancers. Nevertheless, SIRT1 is reported to function as a tumor suppressor as well as an oncogenic protein. Our data show that compared with normal liver or surrounding tumor tissue, SIRT1 is strongly overexpressed in human hepatocellular carcinoma (HCC). In addition, human HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for the expression of the sirtuin family members and only SIRT1 was consistently overexpressed compared with normal hepatocytes. To determine its effect on HCC growth, SIRT1 activity was inhibited either with lentiviruses expressing short hairpin RNAs or with the small molecule inhibitor, cambinol. Knockdown or inhibition of SIRT1 activity had a cytostatic effect, characterized by an altered morphology, impaired proliferation, an increased expression of differentiation markers, and cellular senescence. In an orthotopic xenograft model, knockdown of SIRT1 resulted in 50% fewer animals developing tumors and cambinol treatment resulted in an overall lower tumor burden. Taken together, our data show that inhibition of SIRT1 in HCC cells impairs their proliferation in vitro and tumor formation in vivo. These data suggest that SIRT1 expression positively influences the growth of HCC and support further studies aimed to block its activity alone or in combination as a novel treatment strategy.

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep.

In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal's health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.