Human betaretrovirus (HBRV), homologous to mouse mammary tumor virus (MMTV), and human breast cancer: a significant epidemiological association.

Introduction
Breast cancer is a major global health challenge, affecting millions of individuals and imposing significant emotional and economic burdens on families and healthcare systems. It has now surpassed lung cancer as the most commonly diagnosed cancer worldwide, accounting for 1 in 8 cancer diagnoses and 2.3 million new cases annually across both sexes. In 2020 alone, nearly 685,000 women died from breast cancer, making it responsible for 16% of all cancer-related deaths among women [1]. The increasing incidence, particularly in transitioning countries, highlights the urgent need for effective public health interventions and innovative research strategies [2].
Epidemiological studies have identified a range of determinants contributing to breast cancer development. From an etiological standpoint, hereditary germline mutations—particularly in the BRCA1, BRCA2, and TP53 genes—represent causal factors that directly predispose to malignant transformation [3]. A family history of breast cancer similarly reflects an underlying genetic susceptibility, even in the absence of identified mutations [3]. In contrast, several pathogenetic or promoting factors influence the biological progression of the disease. Estrogens play a central role in breast cancer pathogenesis by stimulating mammary epithelial proliferation and enhancing the effects of other cofactors [4]. Obesity, particularly in postmenopausal women, increases endogenous estrogen production through aromatization in adipose tissue [5]. Likewise, exogenous hormone use and physical inactivity may amplify estrogenic effects [6]. The association between alcohol consumption and breast cancer remains debated; any potential contribution is likely mediated through increased estrogen synthesis [7]. Race and ethnicity are not considered intrinsic biological risk factors but may reflect socioeconomic disparities influencing access to early diagnosis, prevention, and treatment [8].
Despite these known risk factors, the causes of breast cancer remain unclear. Emerging evidence suggests that certain viruses may play an etiological role in the disease. Notably, Human Papillomavirus (HPV), Epstein–Barr Virus (EBV), Bovine Leukemia Virus (BLV), and Mouse Mammary Tumor Virus (MMTV) are all recognized oncogenic viruses [9–12]. Among these, MMTV has a well-established role in mammary tumorigenesis in mice, primarily through insertional mutagenesis, which activates oncogenes and disrupts tumor suppressor genes. In humans, viral infections may contribute to cancer progression not only by interacting with host factors such as immunosuppression or genetic predisposition but also by directly inducing somatic mutations that drive tumor development [13]. The detection of MMTV-like sequences in benign breast tissue before malignancy further supports its potential role in breast oncogenesis [14].
MMTV, a betaretrovirus historically identified as the Bittner virus, was first associated with mammary tumors in mice in the early 20th century [15–18]. In 1933, a non-chromosomal factor influencing tumor incidence was recognized, and by 1939, Bittner demonstrated transmission through milk, establishing a viral etiology [15, 19–22]. MMTV was formally characterized as a transmissible retrovirus in 1958 [23–28]. Subsequent studies revealed particles morphologically identical to MMTV in human milk [29] , and identified viral sequences in human breast carcinomas sharing over 90% homology with murine MMTV [30–34]. In 1972, MMTV sequences were detected in 66% of human invasive breast cancers, but were absent in normal tissue [35], and later studies confirmed elevated anti-MMTV antibodies and proviral integration in tumor samples [36–38]. These findings prompted recognition of a related human virus, initially termed Human Mammary Tumor Virus (HMTV) and subsequently designated Human Betaretrovirus (HBRV) [39]. Detection of HBRV sequences in human saliva and ancient remains suggests long-term adaptation to the human host, possibly originating from cross-species transmission approximately 10,000 years ago [40, 41].
Mechanistic studies have demonstrated that MMTV induces mammary tumors in mice through proviral integration near proto-oncogenes such as Wnt1, Fgf/Fgfr, and Notch4, leading to their dysregulation and uncontrolled proliferation [42, 43]. The viral envelope (env) gene encodes an immunoreceptor tyrosine-based activation motif (ITAM) with intrinsic transforming potential in mammary epithelial cells [44]. At the same time, hormone-responsive elements in the long terminal repeat (LTR) region link viral replication to hormonal regulation and tumorigenesis [45]. In humans, HBRV sequences exhibit over 90% homology with murine MMTV, particularly within the env and LTR regions, and have been shown to infect and replicate in human breast epithelial cell lines [38, 46–48]. These findings provide biological plausibility for a viral contribution to breast cancer pathogenesis.
To maintain terminological consistency with recent literature, the virus formerly referred to as MMTV-like virus (MMTV-LV) or Human Mammary Tumor Virus (HMTV) is referred to here as Human Betaretrovirus (HBRV). These terms denote the same viral entity, closely related to MMTV but adapted to humans [16].
Although methodological variability has contributed to differences in reported detection rates, an expanding body of molecular evidence supports an etiological role for HBRV in human breast cancer. HBRV sequences have been identified in benign breast tissue before malignancy and shown to integrate into human breast epithelial cells [49]. Notably, HBRV positivity is significantly higher in sporadic than hereditary breast carcinoma—30.3% versus 4.2% in one study [50]—and has been detected in ductal carcinoma in situ (DCIS), suggesting a potential role in early tumorigenesis [14, 51]. To clarify these associations and quantify the strength of evidence, the present systematic review and meta-analysis was conducted to assess the relationship between HBRV infection and the risk of human breast cancer.

Methods

Study question
This study aims to investigate the potential relationship between HBRV and breast cancer. Additionally, it examines how various factors, including gene detection methods, sample types, and geographical distribution, influence the detection and association of HBRV with breast cancer.

Search strategy
This study was conducted following PRISMA guidelines [52]. A comprehensive literature search was performed across multiple online databases, including MEDLINE (PubMed), Web of Science, Scopus, and EMBASE, with studies retrieved up to October 2025. The search strategy combined Medical Subject Headings (MeSH) terms and free-text keywords using Boolean operators (AND, OR). The following search terms were applied in the title and abstract fields: (“Mouse Mammary Tumor Virus” OR “MMTV*” OR “Human Mammary Tumor Virus” OR “HMTV” OR “Human Betaretrovirus” OR “HBRV”) AND (“Human Breast Cancer” OR “Human Breast Carcinoma”). Boolean operators were used to enhance precision and comprehensiveness, and the wildcard symbol (*) was employed to capture variations in terminology (e.g., MMTV, MMTV-like, MMTV-LV).
Two independent reviewers screened and evaluated the identified studies. To assess the level of agreement between these reviewers, the kappa coefficient was calculated.

Inclusion and exclusion criteria
Studies were included if they investigated the prevalence of HBRV in breast cancer patients or if they were case-control studies examining the association between HBRV and breast cancer.
The exclusion criteria comprised review articles, studies that relied exclusively on serological methods for HBRV detection, and letters to the editor. Additionally, full-text articles published in languages other than English were excluded, although studies with an English abstract were considered eligible for inclusion.

Data extraction
Data extraction was independently conducted by two reviewers using a standardized data extraction sheet based on the Cochrane Consumers and Communication Group template (accessible at http://cccrg.cochrane.org/author-resources). Duplicate studies from different databases were identified and counted only once.
For each included study, relevant details were extracted, including the authors, publication year, country, and continent, sample type, detection method, total number of cases, positive cases, total controls, and positive controls The screening and data extraction process was conducted in three stages: title, abstract, and full-text review to ensure accuracy and completeness.

Evaluation of study quality and risk of bias
The quality and risk of bias in case-control studies were assessed using the Newcastle-Ottawa Quality Assessment Scale (NOS) [53]. The Newcastle-Ottawa Scale (NOS) comprises eight items distributed across three key domains: selection, comparability, and outcome (or exposure). Based on their NOS scores, studies were categorized into three quality levels: high quality (low risk of bias) for scores of 6 or higher, moderate quality (moderate risk of bias) for scores between 4 and 6, and low quality (high risk of bias) for scores below 4.
To ensure consistency in study evaluation, all researchers were trained in the application and interpretation of the NOS criteria. Each study was independently assessed by two reviewers, with any discrepancies resolved by a third reviewer. Publication bias was evaluated using funnel plots.

Assessment of heterogeneity and statistical analysis
A meta-analysis was performed using RevMan 5.1, with forest plots generated to visualize the findings. The I² test was employed to evaluate heterogeneity among studies. An I² value of approximately 25% indicated low heterogeneity, while a value of around 50% suggested moderate heterogeneity. A higher value, close to 75%, was considered indicative of substantial heterogeneity across the included studies.
The Mantel-Haenszel method was employed to calculate the pooled odds ratio (OR) and 95% confidence intervals (CIs). Additionally, studies were stratified into subgroups to enable methodological comparisons.

Results

Literature search and study characteristics
The detailed process of study selection is illustrated in Fig. 1. The initial database search identified 764 records, including 185 from MEDLINE, 202 from Scopus, 180 from Web of Science, and 197 from EMBASE. After removing duplicate entries, 215 studies remained for further evaluation.

Additionally, the titles and abstracts of the studies were screened to determine their relevance in exploring the association between HBRV and breast cancer. At this stage, irrelevant studies, letters to the editor, review articles and a meta-analysis were excluded, leaving 47 studies for full-text review. After a comprehensive evaluation, 2 studies with potential duplicate data were identified and therefore excluded. This resulted in the inclusion of 45 studies in the qualitative synthesis, with 26 studies deemed suitable for meta-analysis. The characteristics of the included studies are summarized in Table 1.

The kappa coefficient of 0.95 indicated a high level of agreement between the two independent investigators during the screening and selection process.

Quality assessment
The quality assessment of included studies, conducted using the Newcastle-Ottawa Scale (NOS), is summarized in Table 1.

Publication bias and heterogeneity
The initial forest plot indicated a high level of heterogeneity (I² = 82%) (Additional File 1). The corresponding funnel plot suggested the presence of an outlier (Fig. 2). To address potential publication bias, a sensitivity analysis was conducted, wherein each study was systematically excluded to assess its impact on the overall meta-analytic results and heterogeneity (I² statistic).
Following the exclusion of the identified outlier [64], a revised forest plot was generated, demonstrating a substantial reduction in heterogeneity (I² = 22%) (Fig. 3).

Association between HBRV detection and the presence of breast cancer
The initial meta-analysis, which included 26 case-control studies comprising 28 datasets, revealed a significant association between HBRV and breast cancer: The fixed-effects model yielded an OR of 4.92 (95% CI: 4.00–6.04, p < 0.00001) and the random-effects model produced an OR of 9.68 (95% CI: 4.91–19.08, p < 0.00001) (Additional File 1). However, heterogeneity was high (I² = 82%).
After excluding the outlier, the updated forest plot was drawn: The fixed-effects model OR increased to 11.95 (95% CI: 8.78–16.25, p < 0.00001), and the random-effects model OR was 10.21 (95% CI: 6.96–14.98, p < 0.00001) (Fig. 3).
With the removal of the outlier, heterogeneity was significantly reduced (I² = 22%), strengthening the robustness of the findings. These results indicate that HBRV is significantly associated with an increased risk of breast cancer.

Impact of detection methods on HBRV and the presence of breast cancer
To assess whether different detection techniques influenced the association between HBRV and breast cancer, a subgroup analysis was conducted (Fig. 4).

The low I² and Chi² values indicated no significant heterogeneity among the subgroups. The pooled ORs for different HBRV gene detection methods varied across studies. PCR yielded an OR of 11.18 (95% CI: 7.57–16.49), while Nested PCR showed a higher OR of 19.15 (95% CI: 6.61–55.46). Semi-nested PCR produced an OR of 9.99 (95% CI: 4.94–20.17), and Fluorescent nested-PCR (FN-PCR) demonstrated an OR of 16.10 (95% CI: 5.99–43.29). These findings highlight variations in detection efficiency among different PCR techniques.

Impact of sample type on HBRV detection and the presence of breast cancer
Further subgroup analysis was conducted to evaluate the effect of sample type on HBRV detection and its association with breast cancer risk (Fig. 5). The study by Zenit-Zhuravleva was not included as it utilized both Frozen tissue and FFPE together [62].

The high I² and Chi² values indicated substantial heterogeneity across the subgroups. The pooled ORs varied based on the type of tissue sample analyzed. Frozen tissue demonstrated a higher OR of 18.00 (95% CI: 10.03–32.31), whereas Formalin-Fixed Paraffin-Embedded (FFPE) tissue exhibited a lower OR of 9.78 (95% CI: 6.79–14.10). Due to the high heterogeneity observed, direct comparisons between subgroups were not feasible.

Impact of geographic location on HBRV detection and the presence of breast cancer
A final subgroup analysis categorized studies based on the geographic origin of the samples (Fig. 6).

The low I² and Chi² values indicated no significant heterogeneity within subgroups. The pooled ORs varied across continents, indicating potential geographical differences in HBRV prevalence and its association with breast cancer. North America exhibited the highest OR at 24.75 (95% CI: 11.19–54.73), followed by Europe with an OR of 15.02 (95% CI: 5.91–38.21). Africa, Asia and Oceania had similar ORs of 9.53 (95% CI: 3.90–23.27), 8.61 (95% CI: 4.45–16.66) and 9.45 (95% CI: 5.77–15.45), respectively. In contrast, South America presented the lowest OR at 6.31 (95% CI: 0.78–50.93). These findings suggest potential regional differences in HBRV prevalence, transmission, or detection methodologies, warranting further investigation.

Discussion
The association between Human Betaretrovirus (HBRV, formerly MMTV-LV/HMTV) and human breast cancer has been a subject of scientific inquiry for several decades. This interest is driven by the well-established role of MMTV as an etiological agent of mammary tumors in both natural and experimental mouse models. Given their close genetic and structural similarity, it has been hypothesized that comparable mechanisms may contribute to breast cancer pathogenesis in humans.
The findings of this study demonstrate a significant association between HBRV detection and the presence of breast cancer, consistent with the conclusions of a recent systematic review and meta-analysis [12]. Subgroup analyses indicate that detection rates vary according to methodology, sample type, and geography—reflecting both biological and technical variability. Despite consistent reports of HBRV sequences in human breast cancer tissues, some studies have failed to detect the virus [76–81]. These discrepancies may reflect genuine geographic variation in viral prevalence, differences in population exposure (e.g., urban versus rural cohorts), or inclusion of hereditary breast cancer cases, which are typically virus-negative and may dilute detection frequencies [72, 82–84]. Notably, several investigations have reported a decline in viral positivity from ductal carcinoma in situ (DCIS) to invasive breast carcinoma, suggesting that HBRV may play a role in early tumorigenesis but become less detectable as tumors progress [84]. Methodological limitations, such as variable PCR sensitivity and template quality, may further contribute to inconsistent detection [85, 86].
A key technical challenge is the generally low viral load observed in human samples. Quantitative PCR analyses have shown a progressive reduction in viral copy number from DCIS to invasive carcinoma, consistent with the possibility that the virus initiates oncogenic events early but is subsequently lost or transcriptionally silenced [51]. Earlier studies raised concerns about potential laboratory contamination as an explanation for viral detection in human breast cancer tissues [87]. However, these concerns are now considered outdated, as numerous recent molecular investigations have demonstrated that the detected HBRV sequences are genuine and not the result of murine contamination. Importantly, modern analyses employ rigorous contamination controls, including murine DNA–free protocols and assays confirming the absence of murine mitochondrial DNA and intracisternal A-particle sequences in HBRV-positive samples [14, 40]. The detection of HBRV across multiple continents further supports its genuine presence and human adaptation [47].
The biological behavior of MMTV and HBRV displays notable parallels but also critical distinctions. In mice, exogenous MMTV is transmitted through milk and induces mammary tumors via insertional mutagenesis, whereas endogenous MMTV (Mtv) represents integrated proviral elements that rarely produce infectious particles [88–90]. In humans, the exogenous form—HBRV—is distinct from the endogenous retrovirus family HERV-K [91]. HBRV sequences have been identified in the milk of approximately 5% of lactating women and in saliva from both healthy individuals and breast cancer patients, suggesting potential routes of vertical or horizontal transmission [40, 92] Nevertheless, epidemiological data do not support breastfeeding as a major transmission route, and the instability of MMTV in human milk further argues against this mechanism [93, 94]. Reports of MMTV-like sequences in companion animals remain limited and should be interpreted cautiously as evidence of homology rather than zoonotic transmission [95, 96]. These findings should be interpreted with caution and understood in the context of HBRV being a human virus homologous to murine MMTV, rather than evidence of direct zoonotic transmission. Comparative studies in companion animals may nevertheless offer useful insights into viral evolution and host interactions, but additional research is required to clarify their relevance to human infection.
Experimental studies have demonstrated that HBRV can infect, integrate, and replicate in human breast epithelial cells [97]. Early reports detected MMTV-like RNA in up to 80% of invasive breast cancers [35] and similar frequencies in DCIS [51], suggesting infection occurs early in tumorigenesis, with lower prevalence (~ 40%) in invasive carcinomas [85]. Mechanistically, HBRV likely contributes to oncogenesis through several complementary pathways. The primary mechanism is insertional mutagenesis, wherein proviral integration near proto-oncogenes or tumor suppressor genes dysregulates transcription and promotes malignant transformation. In murine tumors, MMTV integration commonly activates Wnt and Fgf family genes, driving proliferative signaling cascades [98, 99]. Analogous long terminal repeat (LTR)-mediated enhancer effects may occur in human cells, altering local chromatin structure and transcriptional regulation [100, 101].
The viral envelope (Env) protein also exhibits intrinsic oncogenic activity through an immunoreceptor tyrosine-based activation motif (ITAM) that activates Src-family kinases, promoting proliferation, epithelial–mesenchymal transition, and inhibition of apoptosis [44, 102, 103]. The Env gene thus functions as an oncogene-like viral component capable of initiating transformation even without complete viral replication. Additionally, the viral superantigen (Sag) alters T-cell repertoires and modulates the immune microenvironment, facilitating chronic inflammation and immune evasion [104]. Interactions with host restriction factors, such as APOBEC3 enzymes, further illustrate the complex interplay between the virus and host defenses: while APOBEC3-mediated editing can inhibit replication, it may also induce genomic mutations and instability in infected cells, contributing indirectly to oncogenesis [105, 106]. Collectively, these mechanisms—including insertional mutagenesis, LTR-driven transcriptional dysregulation, Env-induced signaling and transformation, superantigen-mediated immune modulation, and APOBEC-associated genomic instability—provide multiple, biologically plausible routes through which HBRV may contribute to human breast cancer pathogenesis.
Hormonal and genetic interactions appear central to HBRV’s oncogenic potential. Estrogen and progesterone enhance HBRV LTR transcriptional activity, promoting viral replication and integration under hormonal stimulation [45]. This mechanism may explain the predominance of HBRV positivity in estrogen receptor–positive breast cancers and its rarity in hereditary BRCA1/2-mutated tumors, where carcinogenesis arises from intrinsic genomic instability rather than viral activity. The higher prevalence of HBRV in DCIS than in invasive lesions further supports a model in which infection contributes to early tumor initiation but diminishes as malignancy progresses [51].
Breast cancer incidence exhibits notable geographic variability, with higher rates observed in Western countries compared to lower rates in parts of Asia and Eastern Europe [107]. While lifestyle and environmental factors contribute to these patterns, molecular evidence suggests that viral and genetic components may also be involved. Historically, such variation was attributed to the distribution of Mus domesticus, a mouse species carrying infectious MMTV strains overlapping areas of higher HBRV prevalence [72, 108]. However, modern genomic studies indicate that HBRV sequences have been present in humans for millennia, consistent with ancient cross-species transmission followed by long-term adaptation Consequently, current geographic variation likely reflects regional differences in viral persistence, host genetics, and detection methodology rather than ongoing zoonosis.
Beyond its established association with breast cancer, HBRV sequences have also been detected in liver tissue from patients with primary biliary cholangitis (PBC), where viral gene expression and replication markers suggest a possible role in autoimmune pathogenesis [39, 109, 110]. This association was pivotal in recognizing the virus as Human Betaretrovirus (HBRV), distinct from murine MMTV. While reports have occasionally described viral detection in other malignancies, such as ovarian or prostate cancers, these findings remain preliminary and outside the scope of the present review.
Previous studies have demonstrated that MMTV can infect human mammary epithelial cells in vitro, establishing a productive infection capable of generating infectious progeny virions [111, 112]. Moreover, subsequent research showed that a second MMTV strain (C3H) is also capable of infecting human mammary epithelial cells, though less efficiently than the GR strain [48]. These findings collectively support the potential for MMTV to infect and replicate within human cells, reinforcing its proposed oncogenic relevance.
The ability of MMTV to integrate into the human genome and induce the transformation of normal breast epithelial cells into malignant phenotypes further supports its oncogenic potential [48]. Additionally, MMTV proteins have been found to target T and B lymphocytes in Peyer’s patches, while the inactivation of the APOBEC3B enzyme—ordinarily involved in retrovirus restriction—has been implicated in breast cancer progression [44, 106, 113].
Several studies have reported the co-detection of HBRV sequences with other oncogenic viruses—including human papillomavirus (HPV), Epstein–Barr virus (EBV), and bovine leukemia virus (BLV)—in breast cancer tissues [61, 75]. These findings indicate that multiple viral infections can coexist within the same tumor, although a direct cooperative or causal relationship remains unproven. It is plausible that viral co-infections could modulate the tumor microenvironment through processes such as chronic inflammation, cytokine signaling, and epithelial–mesenchymal transition (EMT) — mechanisms that are well documented in other virus-driven cancers [114]. Viral oncoproteins and non-coding RNAs, including virus-regulated microRNAs, can further perturb host gene networks involved in inflammation and phenotypic plasticity [114, 115]. In this context, HBRV might act as one element of a complex viral milieu that influences tumor behavior and host immune responses, rather than as an isolated etiologic agent [83].
Recent advances in transcriptomic analyses provide new tools to elucidate viral activity in cancer. Large-scale RNA-seq studies have now revealed locus-specific and subtype-dependent expression of human endogenous retroviruses (HERVs) in breast cancer, including HERV-K (HML-2) loci that correlate with tumor subtype and gene regulation [116]. For example, the regulatory influence of HERV-K LTR regions on neighboring host genes has been shown in breast cancer datasets via RNA-seq, suggesting potential enhancer or promoter activity [117]. Applying similar transcriptomic methods to HBRV-positive tumors could clarify whether viral sequences are transcriptionally active, define integration sites, and distinguish between latent and biologically active infection, thereby advancing our understanding of viral contribution to carcinogenesis.
A remaining question is how to reconcile the low detectability of viral sequences in human tumors with the robust insertional mutagenesis model seen in mice. In contrast to murine infection, which involves high viral titers and widespread integration, human infection appears to occur at low copy numbers and may rely on alternative mechanisms such as transient (“hit-and-run”) transformation, Env-driven signaling, or chronic inflammatory modulation [83, 102]. These models could explain how viral influence initiates transformation without persistent, high-copy integration detectable in all tumor cells.
While the pooled odds ratios observed in this meta-analysis indicate a strong statistical association between HBRV and breast cancer, the magnitude of effect (OR > 10 in some subgroups) may reflect methodological heterogeneity, including differences in assay sensitivity, contamination control, and study design. These results demonstrate association rather than causation. Rigorous, prospective studies employing standardized molecular protocols and blinded analyses are required to determine whether HBRV plays a direct etiologic role or acts as a secondary participant in tumor biology.
Although several reviews have examined this topic, most were narrative in nature and lacked standardized bias assessment. The present study addresses this limitation by combining quantitative meta-analysis with formal quality evaluation, providing an updated, objective measure of the strength of association across populations.
Overall, the accumulating evidence implicating HBRV in human breast cancer underscores the need for harmonized research approaches. Discrepancies in viral detection highlight the importance of standardized contamination-controlled assays, confirmatory in situ hybridization, and next-generation sequencing. Future research should focus on defining the temporal relationship between infection and tumor development, characterizing host–virus interactions—including immune and hormonal regulation—and elucidating potential molecular targets. A clearer understanding of these mechanisms may inform novel preventive or therapeutic strategies for virus-associated breast cancer subtypes.

Supplementary Information