Bovine Retroviruses Research Articles

Metagenomics of the midgut microbiome of Rhipicephalus microplus from China

BackgroundTicks, which are ectoparasites of animals, may carry multiple pathogens. The cattle tick Rhipicephalus microplus is an important bovine parasite in China. However, the midgut microbiome of R. microplus from China has not been characterized via metagenomic methods.MethodsRhipicephalus microplus were collected from cattle in the city of Changsha in Hunan province, China. The DNA of the midgut contents was extracted from fully engorged adult female R. microplus. A DNA library was constructed and sequenced using an Illumina HiSeq sequencing platform. SOAPdenovo software was used to assemble and analyze the clean data. The latent class analysis algorithm applied to system classification by MEGAN software was used to annotate the information on the species’ sequences. DIAMOND software was used to compare unigenes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and functional annotation was carried out based on the results of the comparison.ResultsThe dominant phyla in the five samples were Firmicutes, Proteobacteria, and Actinobacteria. Streptococcus, Mycobacterium, Anaplasma, Enterococcus, Shigella, Lactobacillus, Brachyspira, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were the dominant genera in the five samples. The endosymbiotic bacterium Wolbachia was also detected in all of the samples. Mycobacterium malmesburyense, Streptococcus pneumoniae, Anaplasma phagocytophilum, Enterococcus faecium, Shigella sonnei, Enterococcus faecalis, Lactobacillus casei, Brachyspira hampsonii, Pseudomonas syringae, Enterobacter cloacae, and Lactococcus garvieae were the dominant species in the five samples. In addition to these bacterial species, we also detected some eukaryotes, such as Rhizophagus irregularis, Enterospora canceri, Smittium culicis, Zancudomyces culisetae, Trachipleistophora hominis, and viruses such as orf virus, human endogenous retrovirus type W, enzootic nasal tumor virus of goats, bovine retrovirus CH15, and galidia endogenous retrovirus in all of the samples at the species level. The results of the annotated KEGG pathway predictions for the gene functions of the midgut microflora of R. microplus indicated genes involved in lipid and amino acid metabolism, infectious diseases (e.g., Streptococcuspneumonia infection, human granulocytic anaplasmosis, Shigellasonnei infection, Salmonella enterica infection, and pathogenic Escherichia coli infection), and cancer.ConclusionsOur study revealed that the midgut microbiome of R. microplus is not only composed of a large number of bacteria, but that a portion also comprises eukaryotes and viruses. The data presented here enhance our understanding of this tick’s midgut microbiome and provide fundamental information for the control of ticks and tick-borne diseases.Graphical

A novel Betaretrovirus discovered in cattle with neurological disease and encephalitis

BackgroundThe majority of emerging infectious diseases in humans are of animal origin, and many of them are caused by neuropathogenic viruses. Many cases of neurological disease and encephalitis in livestock remain etiologically unresolved, posing a constant threat to animal and human health. Thus, continuous extension of our knowledge of the repertoire of viruses prone to infect the central nervous system (CNS) is vital for pathogen monitoring and the early detection of emerging viruses. Using high-throughput sequencing (HTS) and bioinformatics, we discovered a new retrovirus, bovine retrovirus CH15 (BoRV CH15), in the CNS of a cow with non-suppurative encephalitis. Phylogenetic analysis revealed the affiliation of BoRV CH15 to the genus Betaretrovirus.ResultsBoRV CH15 genomes were identified prospectively and retrospectively by PCR, RT-PCR, and HTS, with targeting of viral RNA and proviral DNA, in six additional diseased cows investigated over a period of > 20 years and of different geographical origins. The virus was not found in brain samples from healthy slaughtered control animals (n = 130). We determined the full-length proviral genomes from six of the seven investigated animals and, using in situ hybridization, identified viral RNA in the cytoplasm of cells morphologically compatible with neurons in diseased brains.ConclusionsFurther screening of brain samples, virus isolation, and infection studies are needed to estimate the significance of these findings and the causative association of BoRV CH15 with neurological disease and encephalitis in cattle. However, with the full-length proviral sequences of BoRV CH15 genomes, we provide the basis for a molecular clone and further in vitro investigation.Graphical

SOCIALIZATION AND GENETIC VARIABILITY AS A DRIVER OF DOMESTICATION (BY THE EXAMPLE OF DOG BREEDS)

Social activity is the basis of interaction between different species in the process of common niche forming, including animal domestication. The increased social activity is universal characteristic of the “domestication syndrome” for different species (M.A. Zeder, 2017). It is assumed that some elements of this increase are due to a certain neotenization of a number of brain metabolic pathways (M. Somel et al., 2009). This is in good agreement with the data on the association of domestication syndrome with the slowing of neural crest cell proliferation (M.A. Zeder, 2015). The syndrome of hypersocialization (Williams-Behren Syndrome — WBS) in humans has been described, associated with hemideletion/hemiduplication of the 7q11.23 region, which includes 25-28 genes whose products are critical for the activity of various aspects of the central nervous system (A. Antonell et al., 2010). It was found that the complex of such genes is located on chromosome 6 in canids, and the domestic dog, considered in recent years as the main model object for studying the genetic mechanisms of domestication (E.A. Ostrander et al., 2019), differs from wolves in the presence of transposon insertions, increased methylation, and reduced gene expression in this region (B.M. von Holdt et al., 2017, 2018; D. Tandon et al., 2019). The aim of this work was to analyze such insertions in the region of the key gene for increased social activity in dogs WBSCR17 (Cfa6.6 and Cfa6.7) in representatives of different breeds and interspecific hybrids with jackals, as well as finding out the presence of mobile genetic elements in these areas. The detected sequences have high homology to the non-autonomous dispersed nuclear element SINEC2A1_CF (94 % homology) and to two regions of endogenous retrovirus 3 the sequences of which are described in humans and cattle (approximately 80 % homology). Data were obtained on the increased variability of the presence and number of insertions into these areas in dogs of different breeds and hybrids, on the presence of homology sites to endogenous human and bovine retroviruses, as well as a short dispersed nuclear element, species-specific for domestic dogs, SINEC2A1_CF, carrying the hexanucleotide AATAAA which contributes to the completion of transcription. These finding suggest the involvement of retroviruses in the formation of an aggregate niche in the domestication process, which leads to increased variability that contributes to the selection of animals with hypersocialization.

О СОЦИАЛИЗАЦИИ И ГЕНЕТИЧЕСКОЙ ИЗМЕНЧИВОСТИ ПРИ ДОМЕСТИКАЦИИ (на примере пород собак)

Social activity is the basis of interaction between different species in the process of common niche forming, including animal domestication. The increased social activity is universal characteristic of the “domestication syndrome” for different species (M.A. Zeder, 2017). It is assumed that some elements of this increase are due to a certain neotenization of a number of brain metabolic pathways (M. Somel et al., 2009). This is in good agreement with the data on the association of domestication syndrome with the slowing of neural crest cell proliferation (M.A. Zeder, 2015). The syndrome of hypersocialization (Williams-Behren Syndrome — WBS) in humans has been described, associated with hemideletion/hemiduplication of the 7q11.23 region, which includes 25-28 genes whose products are critical for the activity of various aspects of the central nervous system (A. Antonell et al., 2010). It was found that the complex of such genes is located on chromosome 6 in canids, and the domestic dog, considered in recent years as the main model object for studying the genetic mechanisms of domestication (E.A. Ostrander et al., 2019), differs from wolves in the presence of transposon insertions, increased methylation, and reduced gene expression in this region (B.M. von Holdt et al., 2017, 2018; D. Tandon et al., 2019). The aim of this work was to analyze such insertions in the region of the key gene for increased social activity in dogs WBSCR17 (Cfa6.6 and Cfa6.7) in representatives of different breeds and interspecific hybrids with jackals, as well as finding out the presence of mobile genetic elements in these areas. The detected sequences have high homology to the non-autonomous dispersed nuclear element SINEC2A1_CF (94 % homology) and to two regions of endogenous retrovirus 3 the sequences of which are described in humans and cattle (approximately 80 % homology). Data were obtained on the increased variability of the presence and number of insertions into these areas in dogs of different breeds and hybrids, on the presence of homology sites to endogenous human and bovine retroviruses, as well as a short dispersed nuclear element, species-specific for domestic dogs, SINEC2A1_CF, carrying the hexanucleotide AATAAA which contributes to the completion of transcription. These finding suggest the involvement of retroviruses in the formation of an aggregate niche in the domestication process, which leads to increased variability that contributes to the selection of animals with hypersocialization.

Linfossarcoma de células B multicêntrico em uma ovelha

Background : : : Lymphosarcoma is a malignant tumor from tumoral cells similar to lymphocytes and its spontaneous development in small ruminants is unusual in Brazil. In cattle, the lymphosarcomas are often associated with infection by bovine leukemia virus (BLV), although sheep are considered as a very susceptible specie to BLV. Spontaneously lymphosarcoma is rare and in most cases it is observed in apparently healthy animals in slaughterhouses. There is no report of this tumor in sheep in southern Brazil. We describe a case of spontaneous multicentric B-cells lymphosarcoma in an ewe. Case : In June 2007, a 4-years-old mixed breed ewe was necropsied after clinical signs as anorexia, weight loss and death in few weeks. This ewe was purchased in September 2006 with another 44 sheep, most females of the same age. Dairy cattle were also raised together with sheep at the same farm. Fragments of liver, lung, spleen, kidneys, brain and parotid lymph node were sent to the Veterinary Pathology Laboratory - Universidade Federal de Santa Maria, Brazil, for histopathological evaluation. Multiply white to yellow nodules, with 0.5 to 2 cm of diameter, were observed in the liver, kidneys, and the parotid lymph node. The nodules were soft and some coalesced and infiltrating the hepatic parenchyma. The kidneys nodules were similar to those of the liver but prominent in the capsule. They were observed infiltrating more intensely the cortical region. The parotid lymph node was 6 x 4 x 3.5 cm size, whitish, multilobulated and the cut surface was totally obliterated by the tumor mass. Histologically, the neoplasm showed intense proliferation of neoplastic lymphoid cells arranged in sheets and a poor fibrovascular stroma, with round small and hyperchromatic nuclei, and scarce eosinophilic cytoplasm compressing the adjacent parenchyma. Immunohistochemistrily, neoplastic cells were strongly positive for anti-CD79a antibody and negative for anti-CD3 antibody, indicating neoplastic cells derived from B lymphocytes. Discussion : The diagnostic of multicentric B-cell lymphosarcoma was made based on gross, histological, and immunohistochemical findings. Spontaneous development of this tumor is uncommon in sheep in Brazil. The multicentric presentation seen in this case is the most common form of lymphosarcoma in sheep, although the thymic and cutaneous forms also occur. Sheep affected by multicentric lymphosarcomas may have symmetrical lymphadenopathy, not observed in this case, and involvement of various organs including liver, kidney, spleen, intestine, skeletal muscle and heart. However, under experimental conditions, these tumors are easily induced after inoculation of bovine retrovirus (BLV) in sheep, by its high sensitivity. Possibly, the spontaneous transmission of BLV from cattle developing the disease in sheep may occur in Brazil as well as in other countries. Epidemiological factors of the disease are not established yet. Despite this, some correlation must exist for the low occurrence of this disease in sheep in properties of southern Brazil, where cattle and sheep are commonly raised together, and the frequency of lymphosarcoma in sheep is low. In this case, the association of BLV with development of the neoplasm could not be confirmed. This is the first report of multicentric B-cell lymphosarcoma in sheep diagnosed in southern Brazil.

Neuropathological survey reveals underestimation of the prevalence of neuroinfectious diseases in cattle in Switzerland

Neuroinfectious diseases in livestock represent a severe threat to animal health, but their prevalence is not well documented and the etiology of disease often remains unidentified. The aims of this study were to generate baseline data on the prevalence of neuroinfectious diseases in cattle in Switzerland by neuropathological survey, and to identify disease-associated pathogens. The survey was performed over a 1-year period using a representative number of brainstem samples (n=1816) from fallen cattle. In total, 4% (n=73) of the animals had significant lesions, the most frequent types of which were indicative of viral (n=27) and bacterial (n=31) etiologies. Follow-up diagnostics by immunohistochemistry, PCR protocols and next-generation sequencing identified infection with Listeria monocytogenes (n=6), ovine herpesvirus 2 (n=7), bovine astrovirus CH13 (n=2), bovine herpesvirus 6 (n=6), bovine retrovirus CH15 (n=2), posavirus 1 (n=2), and porcine astroviruses (n=2). A retrospective questionnaire-based investigation indicated that animals’ owners observed clinical signs of neurological disease in about one-third of cases with lesions, which was estimated to correspond to approximately 85 cases per year in the adult fallen cattle population in Switzerland. This estimate stands in sharp contrast to the number of cases reported to the authorities and reveals a gap in disease surveillance. Systematic neuropathological examination and follow-up molecular testing of neurologically diseased cattle could significantly enhance the efficiency of disease detection for the purposes of estimating the prevalence of endemic diseases, identifying new or re-emerging pathogens, and providing “early warnings” of disease outbreaks.

Bovine Immunodeficiency Virus as a potentiating cofactor for the experimental Bovine Leukemia Virus infection in sheep

Coinfection with bovine retroviruses Bovine Leukemia Virus (BLV) and Bovine Immunodeficiency Virus (BIV) is currently noted in the naturally infected cattle. The effect of BIV as a cofactor for the BLV infectivity is still unknown. In our study we examined the impact of BIV on the BLV in experimentally infected sheep. Four sheep were inoculated with Foetal Bovine Lung cells (FBL) infected with the R29 isolate of BIV and the other four, mock-infected, received uninfected FBL cells. Two weeks later all animals were inoculated intravenously with 5x105 of PBMC of cow persistently infected with BLV. Blood samples were collected every 4 week up to 40th week after inoculation (a.i.). From 4th up to 40th week a.i. antibody titer to BLV in dually infected sheep were almost 1.0 log10 higher than those in BLV group. The BLV infectivity, quantified by BLV-induced syncytia, revealed that the numbers of syncytia in BLV/BIV inoculated animals were higher then those in sheep inoculated with BLV alone. BLV proviral load, determined by a qPCR, increased in the doubly infected animals from 16th to 28th and between 36th and 40th week a.i. Finally semiquantitative RT-PCR disclosed significant increment of BLV tax mRNA at 36th and 40th weeks a.i. in BIV/BLV inoculated sheep. In conclusion these findings confirmed that BIV infection enhance expression of BLV infection in experimentally inoculated sheep.

Polypurine/polypyrimidine sequences with the potential of forming triplexes in the proviral DNA of bovine retroviruses

The perfect interstrand triplexes that could potentially arise in the proviral DNA of two widespread cattle retroviruses such as bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) were determined. The fragments, which formed triplexes at acidic pH, were found in the genomes of both viruses; five fragments were found in BVL and 10 fragments in BIV. One of these fragments (it is localized in the BVL gag gene) might exist like a part of a cruciform structure. Existence of the triplexes was experimentally confirmed by visualization of supercoiled pGEMEX DNA with the use of atomic force microscopy; six fragments with mirror symmetry, which are necessary for formation of intramolecular triplexes, were found. Triplexes represent one of the elements of the signaling mechanisms of the genome function. Maps of triplex location in the cattle retroviral genome were built.

The non-toxic A subunit of Shiga toxin type 1 prevents replication of bovine immunodeficiency virus in infected cells

Shiga toxins are ribosome-inactivating proteins many of which are antiviral. Shiga toxin-producing Escherichia coli (STEC) may be pathogenic to humans, but are carried without ill effects by ruminants. We hypothesize that STEC have antiviral activity in ruminants, and showed previously that the non-toxic subunit A of Shiga toxin 1 (StxA1) acts selectively on cells infected with bovine leukemia virus, without harming normal cells, and that the numbers of intestinal STEC are inversely correlated with viral load in bovine leukemia virus-infected sheep. The purpose of the present study was to characterize StxA1 activity against a second bovine retrovirus, bovine immunodeficiency virus (BIV). Flow cytometry showed that StxA1 treatment induced apoptosis in BIV-infected cells but not in uninfected cells and immunoblot analysis showed that StxA1 curtailed synthesis of Gag p26 protein. A systematic electron microscopy description of BIV infection in fetal bovine lung fibroblasts showed an orderly sequence of changes in cell membrane, endoplasmic reticulum, Golgi, nucleus, and mitochondria, and suggested that the infected cells produce the virus within multivesicular bodies (MVBs). StxA1 interfered with all manifestations of BIV-induced transformation of infected cells into BIV-producing units. BIV-infected cells provided a suitable experimental system for investigation of the mechanism of Stx-antiviral activity.

Differential sensitivity of viruses to the antiviral activity of Shiga toxin 1 A subunit

The non-toxic enzymic A subunit of Shiga toxin 1 (StxA1) reduces expression and replication of the bovine retroviruses, bovine leukemia virus and bovine immunodeficiency virus (BIV). Here, the impact of StxA1 on representative positive and negative stranded RNA viruses was compared. BIV and equine infectious anemia virus were sensitive to picomolar concentrations of StxA1 while poliovirus, rhinovirus, and vesicular stomatitis virus were only marginally sensitive to nanomolar concentrations of toxin. Thus, the length of the reproductive cycle and/or other factors, but not viral encapsulation may play a role in determining sensitivity to StxA1. The effects of StxA1 at concentrations from 0.01 to 10 μg/ml on the most sensitive virus (BIV-infected cultures of fetal bovine lung cells) were analyzed by electron microscopy 48 h post challenge. Cells treated with 0.1 μg StxA1/ml or higher toxin concentrations were similar in appearance and showed progressively fewer viral factories with increasing toxin concentration. However, cells treated with 0.01 μg/ml StxA1 had a radically different appearance, exhibiting smooth cell membranes and high vacuolization. These results showed that complex retroviruses were more sensitive to StxA1 than single-stranded RNA viruses and that StxA1 interfered with retroviral replication in a concentration-dependent manner.

Sensitive and specific detection of bovine immunodeficiency virus and bovine syncytial virus by 5′ Taq nuclease assays with fluorescent 3′ minor groove binder-DNA probes

Sensitive assays are required to detect bovine retroviruses in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5′ Taq nuclease assays using 3′ minor groove binder DNA probes (TaqMan ®MGB) were developed and compared to conventional PCR assays for the sensitive detection of bovine syncytial virus (BSV) and bovine immunodeficiency virus (BIV). Seven beef and dairy herds were screened to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log 10 dilutions of plasmids with inserts containing corresponding provirus sequences. Published PCR assays targeting BIV env sequences did not adequately amplify Australian BIV sequences. Pol sequences from Australian strains of BIV and BSV were used to design TaqMan ®MGB assays, which improved sensitivity 10-fold (BIV) and 100-fold (BSV), respectively, over conventional PCR tests. This is the first report of Australian sequences of BIV and BSV and the first 5′ Taq nuclease assays described to detect these viruses. These methods could be applied to future studies requiring sensitive detection of these two bovine retroviruses.

Viruses and Human Cancer

This book contains papers on the following topics: Immunology and Epidemiology, Biology and Pathogenesis, Models of Pathogenesis and Treatment, Simian and Bovine Retroviruses, Human Papilloma Viruses, EBV and Herpesvirus, and Hepatitis B Virus.

Laboratory Diagnostic Tests for Retroviral Infections in Dairy and Beef Cattle

Detection of bovine retroviruses stretches our diagnostic creativity to its limits. The nucleic acid-based, PCR-amplified assays are finding increased clinical use as the veterinary and livestock industry seek earlier detection of infection for eventual corrective management decisions. We are evolving from a point of disease diagnosis by tumor identification through conventional histopathology, to molecular diagnostics for early identification of retroviral nucleic acid (provirus). The clinical use of antibody-based assays lies in the simplicity of testing large numbers of animals, the relative sensitivity of the assays, and the low cost of testing. Although the pathogenicity of bovine leukemia virus (BLV) for cattle has been well documented, the disease potential for bovine immunodeficiency-like virus (BIV) for cattle is still being determined. Nevertheless, pressure to test for retroviral infections of livestock and, when feasible, removal of these infected animals from the herd will be increased.

Genomic sequence analysis identifies Jembrana disease virus as a new bovine lentivirus.

Jembrana disease virus, the cause of an acute, severe disease in Bali (Bos javanicus) cattle in Indonesia was recently identified as a retrovirus, and possibly a lentivirus. We have produced sequence data representing 598 bp of the pol gene, amplified by PCR from viral cDNA using broadly reactive universal primers for retroviruses and more specific genus-reactive primers for lentiviruses. When the sequence data were compared with that of known lentiviruses and other bovine retroviruses, the closest alignment was with bovine immunodeficiency-like lentivirus (BIV), showing 74% nucleotide sequence identity. This confirmed that JDV is a lentivirus and that it is distinguishable from BIV. The pathogenesis of Jembrana disease is most unusual for a lentivirus infection and differs markedly from that reported for BIV infection.

Bovine immunodeficiency-like virus encodes factors which trans activate the long terminal repeat

Lentiviruses are known to encode factors which trans activate expression from the viral long terminal repeat (LTR); the primary trans activator is the tat gene product. One of the putative accessory genes (tat) of the bovine immunodeficiency-like virus (BIV) bears sequence similarity to other lentivirus tat genes. This finding suggests that BIV may encode a trans-activating protein capable of stimulating LTR-directed gene expression. To test this hypothesis in vitro, BIV LTR-chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed and transfected into three cell lines established from canine, bovine, or lapine tissues that are susceptible to BIV infection. The level of BIV LTR-directed CAT gene expression was significantly elevated in BIV-infected cells compared with uninfected cells. The relatively high basal-level expression of BIV LTR-CAT in uninfected canine and bovine cell lines suggests that cellular factors play a role in regulating BIV LTR-directed gene expression. Additionally, by using a clonal canine cell line in which the BIV LTR-CAT plasmid is stably expressed, BIV LTR-directed CAT expression is elevated 15- to 80-fold by cocultivation with BIV-infected cells, supporting the notion that BIV encodes a trans activator. The relative specificity of this viral activation was assessed by coculturing the clonal BIV LTR-CAT cell line with bovine leukemia virus- or bovine syncytial virus-infected cells; these bovine retroviruses increased expression from the BIV LTR only two- to threefold. Thus, BIV LTR regulatory elements in infected cells, like those of human immunodeficiency virus type 1 and other lentiviruses, are trans activated, presumably through the action of a Tat-like protein and cellular factors.

Some characteristics of a retrovirus isolated from transformed bovine cells

A novel retrovirus has been isolated from bovine cells transformed after cocultivation with leukocytes from an animal suffering from sheep-associated bovine malignant catarrhal fever. Morphologically and in its morphogenesis the virus resembles the type D Mason-Pfizer monkey virus (MPMV) as well as the ovine jaagsiekte retrovirus (JSRV). It is also serologically related to these two viruses, whereas a relationship to other known bovine retroviruses was not detected. However, its density of 1.16 g/ml in sucrose and the preference of its reverse transcriptase for manganese ions are closer to the characteristics of type C retroviruses. Unsuccessful attempts at transmission suggest that the virus may be defective in some function affecting its infectivity.

Role of viruses in the etiology of naturally occurring feline leukemia.

The search to determine if retroviruses cause human leukemia has now been underway for at least 15 years. However, the number of investigators who have conducted direct studies with human materials has been small. By contrast, major emphasis has been given to the study of retrovirus-induced tumors of inbred mice and chickens as models for understanding leukemia of man. At the same time only a limited amount of attention has ben given to the study of the agents that are known to cause leukemia in cats and cattle. Having evolved under natural circumstances in outbred species, the feline and bovine retroviruses would appear to be important agents for study. Presumably information derived about potential mechanisms of leukemogenesis in these species would be applicable to many of the questions one might pose about the etiology of human leukemia. In this context we describe recent findings on the biology and natural history of the feline retroviruses and the diseases they cause.KeywordsFeline Infectious PeritonitisFeline Leukemia VirusPersistent ViremiaCell Membrane AntigenFeLV InfectionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.