In vitro Susceptibility of Human Cell Lines Infection by Bovine Leukemia Virus.

Evidence of the presence of bovine leukemia virus (BLV) in human beings and its association with breast cancer has been published in the literature, proposing it as a zoonotic infection. However, not enough evidence exists about transmission pathways nor biological mechanisms in human beings. This study was aimed at gathering experimental evidence about susceptibility of human cell lines to BLV infection. Malignant and non-malignant human cell lines were co-cultured with BLV-infected FLK cells using a cell-to-cell model of infection. Infected human cell lines were harvested and cultured for 3 to 6 months to determine stability of infection. BLV detection was performed through liquid-phase PCR and visualized through in situ PCR. Seven out of nine cell lines were susceptible to BLV infection as determined by at least one positive liquid-phase PCR result in the 3-month culture period. iSLK and MCF7 cell lines were able to produce a stable infection throughout the 3-month period, with both cytoplasmic and/or nuclear BLV-DNA visualized by IS-PCR. Our results support experimental evidence of BLV infection in humans by demonstrating the susceptibility of human cells to BLV infection, supporting the hypothesis of a natural transmission from cattle to humans.

Risk factor for breast cancer development under exposure to bovine leukemia virus in Colombian women: A case-control study.

Viruses have been implicated in cancer development in both humans and animals. The role of viruses in cancer is typically to initiate cellular transformation through cellular DNA damage, although specific mechanisms remain unknown. Silent and long-term viral infections need to be present, in order to initiate cancer disease. In efforts to establish a causative role of viruses, first is needed to demonstrate the strength and consistency of associations in different populations. The aim of this study was to determine the association of bovine leukemia virus (BLV), a causative agent of leukemia in cattle, with breast cancer and its biomarkers used as prognosis of the severity of the disease (Ki67, HER2, hormonal receptors) in Colombian women. An unmatched, observational case-control study was conducted among women undergoing breast surgery between 2016-2018. Malignant samples (n = 75) were considered as cases and benign samples (n = 83) as controls. Nested-liquid PCR, in-situ PCR and immunohistochemistry were used for viral detection in blood and breast tissues. For the risk assessment, only BLV positive samples from breast tissues were included in the analysis. BLV was higher in cases group (61.3%) compared with controls (48.2%), with a statistically significant association between the virus and breast cancer in the unconditional logistic regression (adjusted-OR = 2.450,95%CI:1.088-5.517, p = 0.031). In this study, BLV was found in both blood and breast tissues of participants and an association between breast cancer and the virus was confirmed in Colombia, as an intermediate risk factor.

Bovine leukemia virus discovered in human blood.

BACKGROUND: Bovine leukemia virus (BLV) infection is widespread in cattle globally and is present in marketed beef and dairy products. Human infection with BLV has been reported in breast and lung cancer tissues and was significantly associated with breast cancer in 3 case-control studies. The purpose of this current research was to determine if BLV is present in human blood cells and if antibodies to BLV are related to blood cell infection. METHODS: Standard liquid PCR and Sanger DNA sequencing were used to test for BLV in buffy coat cells (leukocytes and platelets) of blood specimens from 95 self-selected female subjects. Enzyme-linked immunosorbent assay (ELISA) for IgG, IgM, and IgA was used to detect antibodies to BLV in the plasma of the corresponding blood samples. RESULTS: BLV DNA was detected in the buffy coat cells of blood in 33/95 (38%) of the subjects by PCR and DNA sequencing. IgG antibodies were detected in 30/95(32%), IgM in 55/95(58%), and IgA in 30/95(32%) of the subjects. There was no significant correlation between presence of the antibodies and presence of BLV DNA. CONCLUSIONS: This first report of BLV in human blood raises the question of whether infection of leukocytes could conceivably lead to leukemia as it does in infected cattle. Also, system wide circulation of infected blood cells could facilitate BLV transit to various internal tissues/organs with potential for their infection and subsequent development of cancer. The most likely route of BLV transmission to humans would be zoonotic, as a foodborne infection. Although eradicated from cattle in some countries, BLV still has a high rate of infection in the Americas, the Middle East, and parts of Europe and Asia. This report of BLV in the blood layer containing human leukocytes/platelets adds important information which could be useful to elucidate possible routes of transmission of BLV to humans and to prevent further human infection.

Breast Cancer Gone Viral? Review of Possible Role of Bovine Leukemia Virus in Breast Cancer, and Related Opportunities for Cancer Prevention.

This article is a literature review of research that explored the association of bovine leukemia virus (BLV) infection in humans with breast cancer. It summarizes and evaluates these publications. This review does not provide absolute proof that BLV is a cause of breast cancer, but, based on well-respected epidemiologic criteria for causation, it does suggest that BLV infection could be a breast cancer risk factor. Any expansion of the current understanding of breast cancer risk factors may increase possibilities to implement primary prevention strategies. The environmental role that BLV-infected cattle may play as a reservoir for infectious BLV offers possibilities for reducing or eliminating potential transmission of BLV from cattle to humans, and/or eliminating the reservoir.

Bovine leukemia virus linked to breast cancer but not coinfection with human papillomavirus: Case-control study of women in Texas.

BACKGROUND: Bovine leukemia virus (BLV) and human papillomavirus (HPV) were previously identified in human breast tissue and have been associated with breast cancer in independent studies. The objective of the current study was to test for the presence of BLV and HPV in the same breast tissue specimens to determine whether the viruses were associated with breast cancer either singly or together. METHODS: Archival formalin-fixed paraffin-embedded breast tissue sections from 216 women were received from The University of Texas MD Anderson Cancer Center along with patient diagnosis. In situ polymerase chain reaction and/or DNA hybridization methods were used to detect targeted DNA segments of BLV and HPV. Standard statistical methods were used to calculate age-adjusted odds ratios, attributable risk, and P values for the trend related to the association between presence of a virus and a diagnosis of breast disease. RESULTS: Women diagnosed with breast cancer were significantly more likely to have BLV DNA in their breast tissue compared with women with benign diagnoses and no history of breast cancer. Women with breast pathology classified as premalignant and no history of breast cancer also were found to have an elevated risk of harboring BLV DNA in their breast tissue. HPV status was not associated with malignancy, premalignant breast disease, or the presence of BLV in the breast tissues. CONCLUSIONS: The data from the current study supported previous findings of a significant association between BLV DNA in breast tissue and a diagnosis of breast cancer, but did not demonstrate oncogenic strains of HPV associated with breast cancer or the presence of BLV DNA in breast tissue. The authors believe the findings of the current study contribute to overall knowledge regarding a possible causal role for viruses in human breast cancer. Cancer 2018;124:1342-9. © 2017 American Cancer Society.

Best practices in cell culture: an overview.

This overview describes a series of articles to provide an unmet need for information on best practices in animal cell culture. The target audience primarily consists of entry-level scientists with minimal experience in cell culture. It also include scientists, journalists, and educators with some experience in cell culture, but in need of a refresher in best practices. The articles will be published in this journal over a six-month period and will emphasize best practices in: (1) media selection; (2) use and evaluation of animal serum as a component of cell culture medium; (3) receipt of new cells into the laboratory; (4) naming cell lines; (5) authenticating cell line identity; (6) detecting and mitigating risk of cell culture contamination; (7) cryopreservation and thawing of cells; and (8) storing and shipping viable cells.

Bovine leukemia virus linked to breast cancer in Australian women and identified before breast cancer development.

Bovine leukemia virus (BLV), a common virus of cattle globally, was believed for decades not to infect humans. More recent techniques (in situ PCR and DNA sequencing) enabled detection of BLV in human breast tissue, and determination of its significant association with breast cancer in a US population. Using similar techniques to study 96 Australian women, we report here detection of retrotranscribed BLV DNA in breast tissue of 40/50(80%) of women with breast cancer versus 19/46(41%) of women with no history of breast cancer, indicating an age-adjusted odds ratio and confidence interval of 4.72(1.71-13.05). These results corroborate the findings of the previous study of US women with an even higher odds ratio for the Australian population. For 48 of the subjects, paired breast tissue samples, removed 3-10 years apart in two unrelated procedures, were available. For 23/31 (74%) of these, in which the first specimen was diagnosed as nonmalignant (benign or premalignant) and the second as malignant, BLV was already present in benign breast tissue years 3-10 years before the malignancy was diagnosed. This is consistent with the supposition of a causative temporal relationship between BLV infection and subsequent development of cancer.

Exposure to Bovine Leukemia Virus Is Associated with Breast Cancer: A Case-Control Study.

BACKGROUND: Age, reproductive history, hormones, genetics, and lifestyle are known risk factors for breast cancer, but the agents that initiate cellular changes from normal to malignant are not understood. We previously detected bovine leukemia virus (BLV), a common oncogenic virus of cattle, in the breast epithelium of humans. The objective of this study was to determine whether the presence of BLV DNA in human mammary epithelium is associated with breast cancer. METHODS: This was a case-control study of archival formalin fixed paraffin embedded breast tissues from 239 donors, received 2002-2008 from the Cooperative Human Tissue Network. Case definition as breast cancer versus normal (women with no history of breast cancer) was established through medical records and examination of tissues by an anatomical pathologist. Breast exposure to BLV was determined by in situ-PCR detection of a biomarker, BLV DNA, localized within mammary epithelium. RESULTS: The frequency of BLV DNA in mammary epithelium from women with breast cancer (59%) was significantly higher than in normal controls (29%) (multiply- adjusted odds ratio = 3.07, confidence interval = 1.66-5.69, p = .0004, attributable risk = 37%). In women with premalignant breast changes the frequency of BLV DNA was intermediate (38%) between that of women with breast cancer and normal controls (p for trend < .001). CONCLUSIONS: Among the specimens in this study, the presence of amplified BLV DNA was significantly associated with breast cancer. The odds ratio magnitude was comparable to those of well-established breast cancer risk factors related to reproductive history, hormones, and lifestyle and was exceeded only by risk factors related to genetics (familial breast cancer), high dose ionizing radiation, and age. These findings have the potential for primary and secondary prevention of breast cancer.

Bovine leukemia virus DNA in human breast tissue.

Bovine leukemia virus (BLV), a deltaretrovirus, causes B-cell leukemia/lymphoma in cattle and is prevalent in herds globally. A previous finding of antibodies against BLV in humans led us to examine the possibility of human infection with BLV. We focused on breast tissue because, in cattle, BLV DNA and protein have been found to be more abundant in mammary epithelium than in lymphocytes. In human breast tissue specimens, we identified BLV DNA by using nested liquid-phase PCR and DNA sequencing. Variations from the bovine reference sequence were infrequent and limited to base substitutions. In situ PCR and immunohistochemical testing localized BLV to the secretory epithelium of the breast. Our finding of BLV in human tissues indicates a risk for the acquisition and proliferation of this virus in humans. Further research is needed to determine whether BLV may play a direct role in human disease.

Using interorganizational partnerships to strengthen public health laboratory systems.

Due to the current economic environment, many local and state health departments are faced with budget reductions. Health department administrators and public health laboratory (PHL) directors need to assess strategies to ensure that their PHLs can provide the same level of service with decreased funds. Exploratory case studies of interorganizational partnerships among local PHLs in California were conducted to determine the impact on local PHL testing services and capacity. Our findings suggest that interorganizational forms of cooperation among local PHLs can help bolster laboratory capacity by capturing economies of scale, leveraging scarce resources, and ensuring access to affordable, timely, and quality laboratory testing services. Interorganizational partnerships will help local and state public health departments continue to maintain a strong and robust laboratory system that supports their role in communicable disease surveillance.

Bovine leukemia virus infection in a juvenile alpaca with multicentric lymphoma.

A 13-month-old alpaca (Vicugna pacos) was presented for mandibular masses and weight loss. Histopathology of biopsy tissue was consistent with lymphoma. The alpaca was euthanized and necropsy revealed lymphoma masses in multiple organs. Immunohistochemistry for T- and B-cell typing was inconclusive. Serology and in-situ polymerase chain reaction hybridization were positive for bovine leukemia virus.

Are viruses associated with human breast cancer? Scrutinizing the molecular evidence.

The three viruses most studied as possible causes of human breast cancer are mouse mammary tumor virus-like sequences (MMTV-LS), Epstein-Barr virus (EBV), and oncogenic (high risk) types of human papilloma virus (HPV). The first step in fulfilling traditional criteria for inferring that a cancer is caused by a virus is to demonstrate the virus in the affected tissue. Molecular techniques, compared to host antibody assessment and immunohistochemistry, are the most definitive in establishing viral presence. Results of 85 original molecular research investigations to detect one or more of the three viruses have been extremely divergent with no consensus reached. We evaluated the methodology of these studies for the following: type of molecular assay, DNA/RNA quality control, positive and negative assay controls, type of fixation, genome targets, methods for preventing and detecting molecular contamination, pathology of specimens processed, sample size, and proportion of specimens positive for the viral genome region targeted. Only seven of the studies convincingly demonstrated the presence of an oncogenic virus biomarker (EBV: 4/30 studies (13%); HPV 3/29 studies (10%), whereas 25 convincingly demonstrated absence of the virus studied (MMTV-LS: 4/25 (16%); EBV: 15/30 (50%); 6/29 (21%). The remainder of the studies suffered shortcomings, which, in our opinion, prevented a definitive conclusion. Only one of the studies compared frequency of the virus in breast tissue of breast cancer patients versus appropriate normal control subjects with no history of breast cancer. None of the studies were designed as epidemiologic studies to determine if the presence of the virus was significantly associated with breast cancer. Based on our evaluation, the data in the publications reviewed here remain preliminary, and do not justify a conclusion that MMTV-LS, HPV, or EBV are causally associated with breast cancer. However, they form a valuable basis for redirecting future studies.

Limited evidence of human papillomavirus in [corrected] breast tissue using molecular in situ methods.

BACKGROUND: Human papillomavirus (HPV) has been proposed as an etiologic agent of breast cancer based on numerous reports of high-risk (oncogenic) HPV types in malignant breast tissues. However, most of those studies used standard and nested solution polymerase chain reaction (PCR) techniques, both of which are disadvantaged by vulnerability to laboratory contamination from positive control DNA and the inability to localize the signal to a specific cell type. To overcome these drawbacks, the authors of this report explored the use of in situ molecular methods of viral detection to reassess the frequency of HPV in malignant breast tissue. METHODS: In situ hybridization (ISH) was used with probes that were specific for the capsid region of 12 oncogenic HPV types, and in situ PCR (IS-PCR) was used with primers that were specific for the capsid region of HPV-16, which is the most common oncogenic HPV type. These methods were resistant to molecular contamination and allowed identification of the positive cell type. The specimens examined were malignant tissues from patients with 70 breast cancer patients at The University of Texas M. D. Anderson Cancer Center in Houston, Texas. RESULTS: HPV was observed in 4 of 70 specimens (5.7%) using ISH and in 2 of 70 specimens (2.9%) of specimens using IS-PCR. Concordance between the 2 methods was high for negative specimens; both methods yielded negative results in 66 of 70 specimens (94.3%). However, there was no concordance for the few positive specimens, probably because of differences in sensitivity and the targeted HPV types. CONCLUSIONS: Oncogenic (high-risk) HPV types were present in malignant breast epithelium very infrequently and, thus, may be causative agents of only a relatively small proportion of all breast cancers.

Epstein-Barr virus is seldom found in mammary epithelium of breast cancer tissue using in situ molecular methods.

Epstein-Barr virus (EBV) has been proposed as a possible etiological agent of breast cancer based on 21 reports of EBV in malignant breast tissues. Most of these studies used standard and nested solution polymerase chain reaction (PCR) techniques, both disadvantaged by susceptibility to contamination from laboratory EBV, and the inability to localize the signal to a specific cell type. To avoid these issues, we used in situ molecular methods of viral detection to reassess the frequency of EBV in malignant breast tissue. We used a commercial in situ hybridization (ISH) system with an EBER genome target, and a non-commercial in situ PCR (IS-PCR) method using primers specific for the BamH1 region. The assays were performed on malignant breast tissue sections from 70 breast cancer patients at the M. D. Anderson Cancer Center, Houston, TX. EBV was found in mammary epithelial cells, the cell type from which most breast cancers arise, in 2/70 (2.9%) of specimens using IS-PCR and in none of the specimens using ISH. Based on these findings that EBV was present in human mammary epithelial cells so infrequently, it is unlikely to play a causative role in most types of breast cancer.

Effect of flash-heat treatment on antimicrobial activity of breastmilk.

BACKGROUND AND OBJECTIVES: The World Health Organization recommends human immunodeficiency virus (HIV)-positive mothers in resource-poor regions heat-treat expressed breastmilk during periods of increased maternal-to-child transmission risk. Flash-heat, a "low tech" pasteurization method, inactivates HIV, but effects on milk protein bioactivity are unknown. The objectives were to measure flash-heat's effect on antimicrobial properties of lactoferrin, lysozyme, and whole milk and on the digestive resistance of lactoferrin and lysozyme. METHODS: Flash-heated and unheated breastmilk aliquots from HIV-positive mothers in South Africa were "spiked" with Staphylococcus aureus and Escherichia coli and then cultured for 0, 3, and 6 hours. Lysozyme and lactoferrin activities were determined by lysis of Micrococcus luteus cells and inhibition of enteropathogenic E. coli, respectively, measured spectrophotometrically. Percentages of proteins surviving in vitro digestion, lactoferrin and lysozyme activity, and bacteriostatic activity of whole milk in heated versus unheated samples were compared. RESULTS: There was no difference in rate of growth of E. coli or S. aureus in flash-heated versus unheated whole milk (p = 0.61 and p = 0.96, respectively). Mean (95% confidence interval) antibacterial activity of lactoferrin was diminished 11.1% (7.8%, 14.3%) and that of lysozyme by up to 56.6% (47.1%, 64.5%) by flash-heat. Digestion of lysozyme was unaffected (p = 0.12), but 25.4% less lactoferrin survived digestion (p < 0.0001). CONCLUSIONS: In summary, flash-heat resulted in minimally decreased lactoferrin and moderately decreased lysozyme bioactivity, but bacteriostatic activity of whole milk against representative bacteria was unaffected. This suggests flash-heated breastmilk likely has a similar profile of resistance to bacterial contamination as that of unheated milk. Clinical significance of the decreased bioactivity should be tested in clinical trials.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy.

INTRODUCTION: We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells. METHODS: HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR). RESULTS: vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues. CONCLUSIONS: We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Mother to child transmission of HIV-1 in a Thai population: role of virus characteristics and maternal humoral immune response.

The objective of this study was to investigate factors influencing mother to child transmission of HIV-1 in Thailand, where HIV-1 CRF01_AE, the major subtype in Southeast Asia, predominates. Samples from 84 HIV-1 infected, anti-retroviral treatment-naïve, non-breast feeding mothers, 28 who transmitted HIV-1 to their babies (transmitters) and 56 who did not (non-transmitters), were studied for maternal humoral immune response and virus characteristics. Maternal humoral immune response was measured by lymphocyte phenotyping; neutralizing antibodies to laboratory HIV-1 MN strain and two clinical isolates; peptide binding antibody to gp41 and V3 from strains CRF01_AE, B, and MN; autologous antibodies; and quasispecies diversity. Virus characteristics studied were viral load, co-receptor usage, and viral replication capacity. No significant difference between transmitters and non-transmitters was found for any parameter of maternal humoral immune response. However, viral load and viral replication capacity were significantly higher in transmitters versus non-transmitters and were not correlated with each other. This suggests that viral replication capacity may be a transmission factor independent of viral load, which is already well established as a risk factor for transmission of HIV-1. All except four viral isolates used the CCR5 co-receptor. This is one of few studies of vertical transmission in a population where HIV-1 CRF01_AE predominates. The data suggest that in this population the maternal humoral immune response was not important in preventing transmission at parturition, but that virus characteristics were key factors, and that viral replication capacity may contribute to birth-associated mother to child transmission of HIV-1.

Natural genetic variations in bovine leukemia virus envelope gene: possible effects of selection and escape.

Bovine leukemia virus (BLV) is an oncogenic virus widespread in cattle. It belongs to the genus Deltaretrovirus of the family Retroviridae along with human and simian T-lymphotropic viruses. Here we report the addition of 28 new sequences to the current literature of 16 full-length BLV envelope gene sequences. The phylogenetic clustering, genotyping, and geographic distribution of BLV env variations corresponded in most cases. Most natural variations are mapped to the surface of the proposed conformational models of BLV gp51 N-terminus and gp30 external domain, overlapping with or adjacent to immunogenic epitopes. Analyses for evidence of possible selection pressures suggest the BLV env is under stringent negative selection overall, while strong positive selection is indicated for immunogenic epitope G. Natural env deletions bounded by similar flanking sequences were observed in multiple isolates and would result in truncated signal peptides, missing gp51, and aberrant coding frames for other proteins.

Potential evolutionary influences on overlapping reading frames in the bovine leukemia virus pXBL region.

Bovine leukemia virus contains a pXBL region encoding the 3' parts of four regulatory proteins (Tax, Rex, G4, R3) in overlapping reading frames. Here we report the pXBL polymorphisms of 30 isolates from four countries. Rates of overall and synonymous substitutions were consistently lower, and nucleotide/amino acid composition bias and codon bias higher, in more-overlapped than in less-overlapped regions. Ratios of nonsynonymous/synonymous substitutions were lowest in the tax gene and its subregions. The 5' parts of the four genes showed selection patterns corresponding to their genomic context outside of the pXBL region. Longer G4 variants due to a natural stop codon mutation had additional triple overlap with reduced sequence variability. These data support the concept that a higher level of overlapping in coding regions correlates with greater evolutionary constraint. Tax, the most conserved among the four regulatory proteins, showed purifying selection consistent with its importance in the viral life cycle.