Implications and Mechanisms of Antiviral Effects of Lactic Acid Bacteria: A Systematic Review

Background Lactic acid bacteria (LAB) are among the most important strains of probiotics. Some are normal flora of human mucous membranes in the gastrointestinal system, skin, urinary tract, and genitalia. There is evidence suggesting that LAB has an antiviral effect on viral infections. However, these studies are still controversial; a systematic review was conducted to evaluate the antiviral effects of LAB on viral infections. Methods The systematic search was conducted until the end of December 17, 2022, using international databases such as Scopus, Web of Science, and Medline (via PubMed). The keywords of our search were lactic acid bacteria, Lactobacillales, Lactobacillus (as well as its species), probiotics, antiviral, inhibitory effect, and virus. Results Of 15.408 potentially relevant articles obtained, 45 eligible in-vivo human studies were selected for inclusion in the study from databases, registers, and citation searching. We conducted a systematic review of the antiviral effects of the LAB based on the included articles. The most commonly investigated lactobacillus specie were Lactobacillus rhamnosus GG and Lactobacillus casei. Conclusion Our study indicates that 40 of the selected 45 of the included articles support the positive effect of LAB on viral infections, although some studies showed no significant positive effect of LABs on some viral infections.


Introduction
Viral infections have long been of great concerns, and emerging life-threatening viral infections during recent years have highlighted their importance [1].Notable costs of treatment, considerable morbidity and mortality, and possible resistance to chemical drugs have led healthcare providers to seek alternative or adjuvant treatments to improve the cost-efectiveness of treatments and make them more available.Probiotics are among the most popular adjuvant treatments having proven efectiveness in a wide variety of diseases [2,3].
Tey are Gram-positive, non-spore-forming bacteria [7].Some LABs exist as the normal fora of human and animal mucous membranes and colonize the gastrointestinal system (GI), skin, urinary tract, and genitalia [4].LABs have several benefcial roles.In the GI tract, they reduce lactose intolerance, and they have antidiarrheal, antiinfammatory, and antineoplastic activity [8].As well, they have protective roles against peptic ulcers by eradicating H. pylori infection [9].Modulation of immune responses and minimizing the allergic responses are their other benefcial efects [10,11].
Growing evidence supports the antiviral, antibacterial, and antifungal efects of LABs [4,5,12].Several mechanisms of antiviral activity have been proposed, both generally and specifcally, according to certain viruses, but there is no conclusive research to establish the antiviral efects of LABs so far.Te present study aims to systematically review the current literature on the antiviral efects of LABs and provide a comprehensive picture of it.

Materials and Methods
2.1.Search Strategy.Tis study was designed and performed to investigate the antiviral efects of LABs based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyzes (PRISMA) checklist [13].

Inclusion and Exclusion Criteria.
All in-vivo original articles investigating the antiviral efects of LABs were considered eligible.Only studies written in English were included.Animal studies, in vitro studies, case reports, reviews, editorials, commentary, correspondence, and conference articles were excluded.

Study Selection.
All search and screening steps were performed separately by two independent reviewers.Te quality of articles included for data extraction was assessed by the Cochrane tool for experimental research and the Newcastle-Ottawa Scale (NOS) for observational studies [14,15].A third reviewer's opinion was obtained in case of disagreement (shown in Tables 1 and 2).
Te NOS scale assesses the likelihood of bias in prospective studies using the following three domains: participant selection, comparability, and results.A research may receive up to one point for each numbered item in the selection, two points for comparability, and up to two points for outcome categories.For poor, moderate, and excellent study quality, corresponding scores of 0-3, 4-6, and 7-9 were given.
Te Cochrane tool is based on the following: the use of random sequence generation, concealment of condition allocation, blinding of participants and staf, blinding of outcome assessors, completeness of outcome data, and other biases.Each study was given a risk of bias rating: low when there was no worry, uncertain when there was no information, and high when there was concern.

Data Extraction.
Two authors independently extracted the following variables from studies included in this review: the frst author's name, year of publication, type of study, the country of study execution, number of cases and controls, type of bacteria, type of virus, and antiviral efects of lactic acid bacteria on the virus (es).

Results
Te study selection process is shown in Figure 1.According to the fowchart, 15,408 articles were obtained by the primary search.Due to duplication, 12,057 articles were excluded and the 3,337 remaining articles were screened.
In the next step, the title and abstract of the articles were screened in terms of being in vitro or in vivo, as well as the type of research article.Studies that seemed to be in vitro studies, case reports, reviews, editorials, commentary, correspondence, and conference articles were excluded (n � 3081).After screening the title and abstract, 256 articles remained for full-text screening.Ultimately, after exclusion of irrelevant and unavailable studies, 45 eligible articles were selected for full-text data extraction.
Of 45 included articles, 30 were randomized studies, 6 were cohorts, 7 were clinical trials, 1 case-control, and 1 placebo controlled crossover study.
Six of the included articles were conducted in Finland, six in Italy, four in Japan, three in the USA, three in Tanzania, three in China, two in Canada, two in Korea, two in Bolivia, two in UK, two in Belgium, one in each of the Vietnam, Egypt, Iran, Taiwan, Bangladesh, South Africa, India, Indonesia, Mexico, and Agra.Table 3 provides more details about the fnal included articles.

LABs and Antiviral
Efects.L. rhamnosus GG and L. casei were the most LAB probiotics investigated in the literature.While most of the articles confrmed the antiviral efects of LABs, some evidence did not support this idea.Table 4 summarizes the antiviral efects of LABs, including the present application, their efects on viral load/shedding, clinical outcomes, and laboratory modifcations are attributable to LABS (Table 4).Te antiviral mechanism proposed in the studies was summarized into three major categories: (1) direct efect of LAB on viruses (the most common mechanism), (2) production of antiviral compounds, and (3) stimulation of the immune system against viruses.Tese mechanisms are discussed in more detail subsequently.
Another notable aspect of our study is the antiviral effects of LABs in immunocompromised patients.While most of the postulated mechanisms have been about immunocompetent individuals, studies in immunocompromised patients, such as HIV+ patients, dedicate activation of immune cells such as CD4+ cells, amelioration of infammation, and decrease in translocation markers.Te attributable mechanisms would be explained subsequently.

Discussion
As we have stated so far, fve of the selected 45 publications revealed no discernible impact on the viruses.Tese fve 2 International Journal of Microbiology  International Journal of Microbiology International Journal of Microbiology articles were administered L. rhamnosus; two had L. helveticus, and one had B. animalis spp., Lactis in their administered probiotics in addition.Te major explanation of this discrepancy could be the limited research population, the pathogen change, the comparison to vaccination, the varied study techniques from earlier studies, and the various months of patient enrollment in the study.According to the current evidence, central mechanisms of LAB antiviral effects can be categorized into three major groups: (1) the direct interactions between LABs and viruses, (2) the production of bioactive antiviral agents, and (3) the induction of interferon-associated mechanisms.We have summarized these mechanisms in Figure 2 and discuss each mechanism in more detail.
(1) Te direct interactions between LABs and viruses Direct interactions between the LABs and viruses are the primary mechanism of virus inactivation.Te direct antiviral efect of LAB is applied via both adsorption and trapping, which are strain-dependent mechanisms and can inhibit viruses in a nonspecifc and perhaps specifc manner [5,61].Botić et al.
showed that L. paracasei, L. rhamnosus, L. plantarum, and L. reuteri could trap vesicular stomatitis virus (VSV) [61].Also, L. gasseri has direct antiviral ability against simplex type 2 (HSV-2) [62].Some Lactobacillus strains can trap HIV virions by binding their glycoprotein gp120 to the mannose sugar-rich "dome" at the end of the HIV attachment proteins.Tese results suggest the existence of identical mechanisms in inhibiting viruses by LAB strains [63].Notably, attaching LABs to the cell surface leads to blocking the binding of viruses to their receptors on the cell surface, preventing viruses from entering the cell in the early stages of infection [64,65].
Moreover, M cells ingest LABs, producing interferon type I (type I IFN), which activates dendritic cells [78].Activated dendritic cells trigger multiple critical intracellular signaling pathways, resulting in T-cell activation, viral replication inhibition, and production of immunomodulatory cytokines [79].
Production and secretion of IFNs provide a high level of antiviral protection [80].Te Janus kinase-Sign Transducer and Activator of Transcription (JAK-STAT) signaling cascade is activated by type I and type III IFNs, which phosphorylate STAT1 and STAT2 [81].Phosphorylated STATs and the IFN Regulatory Factor-9 (IRF9) form the IFN-Stimulated Gene Factor 3 (ISGF3) complex [80].Te ISGF3 complex enters the cell nucleus and enhances the transcription of genes that have IFN-Stimulated Response Element (ISRE) in their promoters [80].Tese genes might have antiviral or modulatory efects on the infammation and cytokine production pathways [80].Type II IFNs also cause STAT1 homodimerization, which favors ISG promoters with gamma-activated sequence (GAS) containing promoters [80].All result in the induction and activation of several antiviral agents, including the protein kinase RNA- International Journal of Microbiology activated (PKR), ribonuclease 2-5A pathway, and numerous apoptotic pathways which inhibit the virus binding to cells, viral particle penetration into cells, and the release of the nucleocapsid from an envelope [82].Disruption of transcription and translation processes of the structural viral proteins prevents virion formation or budding of viruses [83].
Another key mediator of IFN antiviral efects is interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein that plays a signifcant role in counteracting viruses by conjugating to the viral and cellular proteins and marking them for destruction [84].Tree enzymes mediate the ISG15 conjugation with the target protein; E1 activating enzyme (Ube1L), E2 conjugating enzyme (UbcH8), and E3 ligase enzyme (either HERC5, or EFP, or TRIM25).Tese enzymes establish a covalent bond between the C-terminal glycine of the mature form of ISG15 and the target protein lysine [84][85][86].Conjugation of ISG15 with viral proteins leads to impaired protein function, promotes protein degradation, and prevents oligomerization of viral proteins [87].IFNs increase the poly-SUMOylation and ISGylation of both viral and cellular proteins [88].As well, the ISG15 can modify and inhibit the viral mRNA translation by strengthening the attachment of translation suppressor proteins such as eIF4E homologous protein (4EHP) to the viral mRNA cap [89].ISG15 is also secreted from various cells [90].Te extracellular ISG15 can also act as an antiviral cytokine by producing and secreting antiviral factors such as type III IFNs, nitric oxide (NO), and reactive oxygen species (ROS), which induce apoptosis in virus-infected cells [87,91,92].Binding ISG15 to the lymphocyte functionassociated antigen 1 (LFA-1), located on immune cells, results in cell proliferation, maturation, and production of IFN-c and IL-10 [93][94][95][96][97]. Bioactive agents and LAB particles are essential ligands for toll-like receptors (TLRs) [98].TLRs' activation results in T-cell activation and cytokine production via mitogen-activated protein (MAP) kinases and NF-kB signaling pathways in dendritic cells [99].MAP kinases directly phosphorylate the transcription factor AP-1, a key player in T-cell activation [100][101][102].Te phosphorylated AP-1 heterodimerizes in the nucleus and binds to the IL-2 promoter and enhance its expression which promotes the T-cell activity [102].

Conclusion
According to the current literature, LABs have considerable antiviral activities which afect viruses both directly and indirectly.Although our study provides an overview of the antiviral activities of the LABs as one of the most important human gut microbiota, it has some limitations: the applicability of using LABs as antiviral adjuvants in clinical practice has not been fully investigated, and so it is not justifable by our study.As well, the potential adverse efects of the therapeutic use of LABs and their delivery system as a therapeutic agent are not investigated.We propose further studies to investigate these concerns and promote our knowledge about more efcient antiviral agents.

Figure 2 :
Figure 2: A summary of the mechanisms of the antiviral efects of LABs.Te frst process involves interactions between LABs and viruses that trap or adhere viruses to the surface of the host cell, preventing them from adhering to cells.Te generation of bioactive antiviral substances such as H2O2, lactic acid, bacteriocins, exopolysaccharides, etc., is the second mechanism.Te third mechanism involves interferon-associated mechanisms, which include (a) activation of dendritic cells, which results in T-cell activation, production of cytokines, and inhibition of viral replication; (b) activation of the JAK-STAT signaling cascade, which results in transcription of genes with antiviral and immunomodulatory efects, as well as binding of ISG15 to viral proteins, which causes them to be destroyed; and (c) binding of bioactive substances to the toll-like receptor.

Table 1 :
Quality assessment of the observational studies included in the meta-analysis (the NOS tool).

Table 2 :
Quality assessment of the experimental studies included in the meta-analysis (the Cochrane tool).

Table 3 :
A summary of the included articles' characteristics.

Table 4 :
Summary of the applied clinical conditions, efects of LAB supplementation on viral load/shedding, clinical signs, and symptoms in comparison with the standard treatment strategies.