Compartmentalization of Subtype A17 of Small Ruminant Lentiviruses between Blood and Colostrum in Infected Goats Is Not Exclusively Associated to the env Gene.

The compartmentalization of small ruminant lentiviruses (SRLVs) subtype A17 was analyzed in colostrum and peripheral blood leukocyte cells of three naturally infected goats. This study aimed to analyze heterogeneity of the SRLV env (V4V5) gene, which encodes neutralizing epitopes of SU glycoprotein, the gag gene encoding capsid protein (CA), and LTR, a noncoding region, responsible for determination of cell tropism. Compartmentalization was assessed using six established tree or distance-based methods, including permutation test to determine statistical significance. We found statistical evidence of compartmentalization between blood and colostrum in all infected goats although phylogenetic evidence of such compartmentalization was not obvious. Our study demonstrated that compartmentalization is not exclusively specific to the env gene, as we revealed that gag and LTR sequences are also compartmentalized between blood and colostrum. The work also confirms the combined use of different methods as essential for reliable determination of intrahost viral compartmentalization. Identifying and characterizing distinct viral subpopulations and the genetic evolution of SRLV in specific anatomical sites enhances our overall understanding of SRLV pathogenesis, immune control, and particularly virus transmission.

Phenotypic Lentivirus Screens to Identify Antiviral Single Domain Antibodies.

Our understanding of infection biology is based on experiments in which pathogen or host proteins are perturbed by small compound inhibitors, mutation, or depletion. This approach has been remarkably successful, as, for example, demonstrated by the independent identification of the endosomal membrane protein Niemann-Pick C1 as an essential factor for Ebola virus infection in both small compound and insertional mutagenesis screens (Côté, Nature 477:344-348, 2011; Carette et al., Nature 477:340-343, 2011). However, many aspects of host-pathogen interactions are poorly understood because we cannot target all of the involved molecules with small molecules, or because we cannot deplete essential proteins. Single domain antibody fragments expressed in the cytosol or other organelles constitute a versatile alternative to perturb the function of any given protein by masking protein-protein interaction interfaces, by stabilizing distinct conformations, or by directly interfering with enzymatic activities. The variable domains of heavy chain-only antibodies (VHHs) from camelid species can be cloned from blood samples of animals immunized with the desired target molecules. We can thus exploit the ability of the camelid immune system to generate affinity-matured single domain antibody fragments to obtain highly specific tools. Interesting VHH candidates are typically identified based on their affinity toward immobilized antigens using techniques such as phage display.The phenotypical screening approach described here allows the direct identification of VHHs that prevent infection of cells with influenza A virus (IAV) or other pathogens. The VHH repertoire is cloned into a lentiviral vector, which is used to generate pseudo-typed lentivirus particles. Target cells are transduced with the lentivirus, so that every cell inducibly expresses a different VHH. This cell collection is then challenged with a lethal dose of virus. Only the cells which express a VHH that prevents infection by targeting virus proteins or host cell components essential for infection will survive. We can thus identify critical target molecules including vulnerable epitopes and conformations, render target molecules accessible to informative perturbation studies, and stabilize intermediates of virus entry for detailed analysis.

The concentration of copper, zinc, manganese and selenium in the serum and liver of goats with caprine arthritis-encephalitis.

Concentrations of four trace elements, copper (Cu), zinc (Zn), manganese (Mn) and seleni- um (Se), have thus far proven to be affected by lentiviral infections in people and rhesus monkeys. As small ruminant lentivirus (SRLV) infection is responsible for one of the most important goat diseases, caprine arthritis-encephalitis (CAE), we evaluated serum and liver concentrations of Cu, Zn, Mn, Se in goats severely affected by symptomatic CAE and compared them with litera- ture reference intervals. Serum and liver samples of dairy goats euthanized due to severe clinical form of CAE were collected and screened for the concentration of Cu, Zn, Mn (54 serum sam- ples, 22 liver samples), and Se (36 serum samples, 22 liver samples) using flame atomic absorption spectrometry for Cu, Zn, Mn and graphite furnace atomic absorption spectroscopy for Se. In both serum and liver samples concentration of Zn was the highest, followed by Cu concentration, and then by Mn and Se. There was no relationship between serum and liver concentrations of trace elements. Liver concentrations of all four trace elements and serum Cu concentration fell within literature reference intervals, although liver Se concentration was mainly in the lower marginal range (between 0.4 and 1.0 mg/L). Serum Zn concentration was elevated (>1.2 mg/L) in all goats, serum Mn concentration was elevated (>0.04 mg/L) in 42 (78%) goats and serum Se concentra- tion was elevated (>1.6 mg/L) in 13 (36%) goats. Concluding, severe symptomatic CAE does not appear to be associated with the level of any of the four trace elements.

Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses.

The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins (S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected Vero- CCL-81 (monkey kidney), Huh-7 (human liver), and PK-15 (pig kidney) cells efficiently. CCL94 (cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.

Interspecific transmission of small ruminant lentiviruses from goats to sheep.

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

Interspecific transmission of small ruminant lentiviruses from goats to sheep

This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

An optimized lentivirus-mediated RNAi screen reveals kinase modulators of kinesin-5 inhibitor sensitivity.

Abstract: Induction of RNA interference (RNAi) in human cells has enabled comprehensive functional annotation of the human genome via reverse genetic screens. Here we describe an optimized semiautomated method to produce, titrate, and screen large collections of short hairpin RNA (shRNA)-containing lentiviral vectors. We also present results from a pilot lentiviral RNAi screen for kinases whose silencing modulates sensitivity to a mitotic spindle protein kinesin-5 inhibitor (kinesin-5i). Our screen identified three distinct serine/threonine kinase 6 shRNA vectors within our library as enhancers of kinesin-5i-mediated HT29 cell growth inhibition. In contrast, three distinct shRNAs targeting cell division cycle 2/cyclin-dependent kinase 1 resulted in kinesin-5i resistance. These results demonstrate the feasibility of screening with large collections of lentiviral vectors to identify drug enhancers and suppressors.

Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats.

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.

Evidence of proviral clearance following postpartum transmission of an ovine lentivirus.

Lentiviral transmission by transfer of infected colostrum and/or milk is considered to be highly efficient. In this study, postpartum transmission of ovine progressive pneumonia virus (OPPV) from 10 naturally infected ewes to their 23 lambs was followed from the perinatal period throughout a four-year period. The lambs were allowed to suckle from their dam from birth through 32 weeks of age. Virus was tracked by virus isolation, quantitative PCR (qPCR), and anti-OPPV antibody responses as measured by cELISA. Cell-associated OPPV was isolated from colostrum/milk cells in 7 out of 10 ewes and provirus envelope (env) loads ranged 8 to 10(5) copies/mug DNA in colostrum/milk cells from the 10 ewes using qPCR. Provirus env loads were also detected in the peripheral circulation of 21 lambs at 8 weeks and two lambs at 22 weeks. The qPCR product at 8 weeks was confirmed as the transmembrane (tm) gene of OPPV by cloning and sequencing. Both cELISA titers ranging from 325 to 3125 and cross-neutralizing antibody titers ranging from 6 to 162 to seven different OPPV strains were found in the colostrum of the 10 ewes. Furthermore, cELISA titers in serum from lambs remained detectable through 32 weeks following the clearance of provirus at 24 weeks. After 32 weeks, both provirus and anti-OPPV antibody responses have subsequently remained undetectable through 4 years of age. These data suggest the clearance of cell-associated lentiviruses from lamb circulation after passive transfer of antibody via colostrum.

Seven new ovine progressive pneumonia virus (OPPV) field isolates from Dubois Idaho sheep comprise part of OPPV clade II based on surface envelope glycoprotein (SU) sequences.

Seven new ovine progressive pneumonia virus (OPPV) field isolates were derived from colostrum and milk of 10 naturally OPPV-infected sheep from the US Sheep Experiment Station in Dubois, Idaho, USA. Sixteen sequences of the surface envelope glycoprotein (SU) from these seven Dubois OPPV field isolates and SU sequence from OPPV WLC1 were obtained, aligned with published SRLV SU sequences, and analyzed using phylogenetic analysis using parsimony (PAUP). Percent nucleotide identity in SU was greater than 95.8% among clones from individual Dubois OPPVs and ranged from 85.5 to 93.8% between different Dubois OPPV clones. SU sequences from Dubois OPPVs and WLC1 OPPV had significantly higher percent nucleotide identity to SU sequences from the North American OPPVs (85/34 and S93) than caprine-arthritis encephalitis virus (CAEVs) or MVVs. PAUP analysis also showed that SU sequences from the Dubois OPPVs and OPPV WLC1 grouped with other North American OPPVs (85/34 and S93) with a bootstrap value of 100 and formed one OPPV clade II group. In addition, Dubois and WLC1 SU amino acid sequences had significantly higher identity to SU sequences from North American OPPVs than CAEV or MVV. These data indicate that the seven new Dubois OPPV field isolates along with WLC1 OPPV are part of the OPPV clade II and are distinct from CAEVs and MVVs.