Phytomedicines to Target Hepatitis B Virus DNA Replication: Current Limitations and Future Approaches

Hepatitis B virus infection (HBV) is one of the most common causes of hepatitis, and may lead to cirrhosis or hepatocellular carcinoma. According to the World Health Organization (WHO), approximately 296 million people worldwide are carriers of the hepatitis B virus. Various nucleos(t)ide analogs, which specifically suppress viral replication, are the main treatment agents for HBV infection. However, the development of drug-resistant HBV strains due to viral genomic mutations in genes encoding the polymerase protein is a major obstacle to HBV treatment. In addition, adverse effects can occur in patients treated with nucleos(t)ide analogs. Thus, alternative anti-HBV drugs of plant origin are being investigated as they exhibit excellent safety profiles and have few or no side effects. In this study, phytomedicines/phytochemicals exerting significant inhibitory effects on HBV by interfering with its replication were reviewed based on different compound groups. In addition, the chemical structures of these compounds were developed. This will facilitate their commercial synthesis and further investigation of the molecular mechanisms underlying their effects. The limitations of compounds previously screened for their anti-HBV effect, as well as future approaches to anti-HBV research, have also been discussed.


Introduction
Hepatitis B infection, which is caused by the hepatitis B virus (HBV), is a common life-threatening, infectious disease that affects the liver. When the disease evolves from an acute infection to chronic hepatitis, it can result in cirrhosis, hepatocellular carcinoma (HCC), and eventually death if not properly treated. Although vaccination is an effective means of origin and their effects against HBV DNA replication, and we reviewed them thoroughly. ChemDraw Professional 16.0 was used to draw all chemical structures.

Terpenoid Compounds
Terpenes are linear or cyclic compounds consisting of five-carbon isoprene basic structural units (saturated or unsaturated). Isoprene units can assemble in several different ways, thereby resulting in a wide variety of secondary metabolites. Terpenoids are modified terpenes with different functional groups, and oxidized methyl groups are moved or removed at various positions in these compounds. With varying structures that result in diverse functionality, terpenoids are potential biologically active compounds, and many of them exhibit inhibitory activities against various human cell lines; for example, Taxol and its derivatives are used as anticancer drugs due to their inhibitory effects on cancer cell proliferation [23]. The structures of various terpenoid compounds that interfere with HBV DNA replication are presented in Figure 1. A triterpenoid saponin (1) was extracted from the Tibetan herb, Potentilla anserina, using ethanol, and its in vivo anti-HBV effects were evaluated in Peking ducklings. Reportedly, this compound inhibited duck hepatitis B (DHBV) DNA replication [24]. Furthermore, saponins were extracted from the traditional Chinese herbal medicinal plant, Abrus cantoniensis Hance (AC), and their anti-HBV effects were evaluated both in vitro and in vivo [25]. The saponin extract decreased HBV DNA production in both HepG2.2.15 cells and C57BL/6 mice infected with recombinant HBV [25]. In mice treated with the saponins, the percentage of CD4+ and T cells in their spleens and serum IFN-γ levels were also upregulated [25]. Ganoderic acid (2), isolated from Ganoderma lucidum, inhibited HBV replication in HepG2.2.15 cells when they were treated at 8 µg/mL for up to eight days [26]. Two terpenoid compounds, astataricusone B (3) and epishionol (4), were isolated from the roots and rhizomes of Aster tataricus, and found to exhibit inhibitory activity against HBV DNA replication, with IC 50 (half-maximal inhibitory concentration) values of 2.7 and 30.7 µM/L, respectively [27]. Hemslecin A (5), extracted from flowering plants of the genus Hemsleya, inhibited HBV DNA replication, with an IC 50 value of 11.2 µM (SI = 5.8) in HepG2.2.15 cells [28]. The climbing vine swallowwort, Cynanchum auriculatum, reportedly contains the terpenoid compound caudatin (6), which inhibits HBV DNA replication, with an IC 50 value of 40.62 mM/L (SI = 6.0) [29]. Glycyrrhizin (7) and its metabolite, glycyrrhetinic acid (GA) (8), are the main constituents of Licorice roots (Glycyrrhizae glabra), and exhibit inhibitory activity against HBV DNA replication (IC 50 = 39.28 µM/L) [30]. Helioxanthin (HE-145) (9) was extracted from the heartwood, Taiwania cryptomerioides hayata, and inhibited HBV replication in HCC cells. HE-145 reportedly decreased HBV DNA-binding activity in HepA2 cells, with a unique anti-HBV mechanism, and, thus, is a potential anti-HBV agent [31]. Asiaticoside (10), isolated from Hydrocotyle sibthorpioides, significantly reduced HBV transcription, replication, as well as cccDNA levels, by suppressing the promoter activities of the core S1, S2, and X genes in HepG2.2.15 cells [32]. In addition, the phytocompound methyl helicterate (MH) (11), isolated from the Chinese herb Helicteres angustifolia, significantly reduced cccDNA and viral RNA levels in HepG2.2.15 cells and DHBV-infected ducklings [33]. Lupane-type triterpenoids, including betulinic acid (12), were isolated from T. conophorum seeds, characterized, and their anti-HBV effects were investigated in rat models and HepG2 cells. These compounds showed hepatoprotective and cytotoxic activities, which may be due to their interaction with HBV, as the compounds exhibited high binding affinities for the virus [34].

Flavonoid Compounds
Flavonoids constitute a diverse range of polyphenolic structures found in many natural substances, such as fruits, vegetables, grains, bark, roots, stems, flowers, tea, and wine. Currently, flavonoids are considered indispensable tools in the field of human physiology due to their anti-oxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties. The flavonoids of medicinal interest can be classified into several groups, such as flavonols, flavones, flavanones, isoflavones, and anthocyanidins [35]. The chemical structures of some flavonoid compounds affecting HBV DNA replication are shown in Figure 2. Camellia sinensis (green tea), which contains epicatechin (ECG), epigallocatechin (EGC), epicatechin (EC), catechin (C), and epigallocatechin gallate (EGCG) (13)(14)(15)(16)(17), exhibits significant antiviral activity through various mechanisms. The antiviral effects of

Phenolic and Polyphenolic Compounds
Phenolic and polyphenolic compounds constitute one of the largest and most widespread plant secondary metabolite groups, and they possess significant antiviral and antioxidant properties. Although polyphenols include a wide range of molecules that exhibit different biological activities, their core structure is composed of a benzene ring and a hydroxyl functional group [43]. The chemical structures of various phenolic and polyphenolic compounds of plant origin, which affect HBV DNA replication, are presented in Figure 3. The in vitro anti-HBV effects of the polyphenolic extract of Geranium carolinianum L. (PPGC) were evaluated in HepG2.2.15 cells, and the extract was found to decrease HBsAg and HBeAg secretion, with IC 50 values of 46.85 µg/mL and 65.60 µg/mL, respectively. Southern blot analysis further confirmed that PPGC decreased plasma and liver DHBV DNA levels in infected ducklings in a dose-dependent manner [44]. The plant Oenanthe javanica (OJ) is widely used for the treatment of hepatitis; the HepG2.2.15 cell line and a DHBV infection model were used as in vitro and in vivo models, respectively, to investigate the anti-HBV effects of its total phenolic extract. It was found that OJ significantly inhibited HBV replication in HepG2.2.15 cells and inhibited DHBV replication in ducks [45]. Several quinic acid derivatives, including 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoyl-muco-quinic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, and 5-O-(E)-p-coumaroylquinic acid (24)(25)(26)(27)(28)(29), extracted from the aerial parts of Lactuca indica L. (Compositae), effectively decreased HBV DNA levels in HepG2.2.15 cells [46]. Chlorogenic acid (30) and its related compounds, which are found in the leaves and fruits of dicotyledonous plants such as the coffee plant, exhibit antiviral activity. Chlorogenic acid, quinic acid (31), and caffeic acid (32) reportedly inhibit HBV DNA replication in the HepG2.2.15 cell line [47]. Protocatechuic aldehyde (PA) (33), isolated from Salvia miltiorrhiza, reduced HBV DNA release in HepG2.2.15 cells in a dose-and time-dependent manner. At the doses of 25, 50, or 100 mg/kg (administered intraperitoneally, twice daily), PA reduced viremia in ducks infected with DHBV [48]. Mulberrofuran G (34), isolated from the root bark of Morus alba L., exhibited moderate inhibitory activity against HBV in HepG2.

Enyne Compounds
Enynes, which are organic compounds consisting of a carbon-carbon double bond (C=C) and a carbon-carbon triple bond, can inhibit HBsAg and HBeAg secretion. The chemical structures of some enyne compounds affecting HBV DNA replication are shown in Figure 4. Artemisia capillaris (Yin-Chen) contains enynes (36)(37), and its extracts were

Enyne Compounds
Enynes, which are organic compounds consisting of a carbon-carbon double bond (C=C) and a carbon-carbon triple bond, can inhibit HBsAg and HBeAg secretion. The chemical structures of some enyne compounds affecting HBV DNA replication are shown in Figure 4. Artemisia capillaris (Yin-Chen) contains enynes (36)(37), and its extracts were evaluated in HepG2.2.15 cells to determine the active components responsible for its anti-HBV effects. The results showed that the compound atractylodin (38), isolated from this herb, significantly inhibited HBV DNA replication, with an IC 50 value of 9.8 µM (SI > 102), and the hydroxyl and glycosyl groups were found to be responsible for maintaining this activity [51]. evaluated in HepG2.2.15 cells to determine the active components responsible for its anti-HBV effects. The results showed that the compound atractylodin (38), isolated from this herb, significantly inhibited HBV DNA replication, with an IC50 value of 9.8 μM (SI > 102), and the hydroxyl and glycosyl groups were found to be responsible for maintaining this activity [51].

Lignan Compounds
The non-flavonoid polyphenols known as lignans are widely distributed in the plant kingdom. Lignans exhibit antiviral, antioxidant, antibacterial, and antifungal activities in animal models. Lignans have diverse and complex chemical structures, although they are essentially dimers of phenylpropanoid units (C6-C3) linked by the central carbons of their side chains [60]. The chemical structures of some lignan compounds that affect HBV DNA replication are shown in Figure 6. The methanol extract of Streblus asper roots contains honokiol (47) and (7′R, 8′S, 7′R, 8′S)-erythron-strebluslignanol G (48), which reportedly show significant anti-HBV activity. These compounds significantly inhibit HBV DNA

Lignan Compounds
The non-flavonoid polyphenols known as lignans are widely distributed in the plant kingdom. Lignans exhibit antiviral, antioxidant, antibacterial, and antifungal activities in animal models. Lignans have diverse and complex chemical structures, although they are essentially dimers of phenylpropanoid units (C 6 -C 3 ) linked by the central carbons of their side chains [60]. The chemical structures of some lignan compounds that affect HBV DNA replication are shown in Figure 6. The methanol extract of Streblus asper roots contains honokiol (47)

Xanthone Compounds
Xanthones are cyclic oxygenated compounds that display numerous bioactive properties, including antimicrobial, antitubercular, antitumor, antiviral, and antioxidant properties. Their health-promoting effects are mainly attributed to their tricyclic scaffold [64]. The chemical structures of some xanthone compounds affecting HBV DNA replication are shown in Figure 7. Xanthone compounds (50-52) extracted from Curcuma xanthorrhiza exhibit inhibitory effects at the post-entry stage of HBV infection. The effects of this plant extract were evaluated in Hep38.7 Tet cells; HBV DNA was quantified using an IC-RT-qPCR assay and the percentage inhibition was determined compared to that in the untreated control group. At 50 μg/mL, the C. xanthorrhiza extract reduced HBV DNA levels by 30% [65]. The xanthone compound mangiferin (53), isolated from Swertia mussotii, also induced a significant reduction in HBV DNA replication, with IC50 values ranging from 0.01 to 0.13 mM [66,67]. Cao et al. reported that dihydroxy-3,5-dimethoxyxanthone (54) and norswertianolin (55) isolated from Swertia yunnanensis exhibit inhibitory effects against HBV DNA replication in HepG2.2.15 cells, and methylation or glycosylation of the hydroxyl group of the compounds might be responsible for this inhibitory effect [38,66]. Another xanthone compound, 1,5,8-Trihydroxy-3-methoxyxanthone (56), isolated

Xanthone Compounds
Xanthones are cyclic oxygenated compounds that display numerous bioactive properties, including antimicrobial, antitubercular, antitumor, antiviral, and antioxidant properties. Their health-promoting effects are mainly attributed to their tricyclic scaffold [64]. The chemical structures of some xanthone compounds affecting HBV DNA replication are shown in Figure 7. Xanthone compounds (50-52) extracted from Curcuma xanthorrhiza exhibit inhibitory effects at the post-entry stage of HBV infection. The effects of this plant extract were evaluated in Hep38.7 Tet cells; HBV DNA was quantified using an IC-RT-qPCR assay and the percentage inhibition was determined compared to that in the untreated control group. At 50 µg/mL, the C. xanthorrhiza extract reduced HBV DNA levels by 30% [65]. The xanthone compound mangiferin (53), isolated from Swertia mussotii, also induced a significant reduction in HBV DNA replication, with IC 50 values ranging from 0.01 to 0.13 mM [66,67]. Cao et al. reported that dihydroxy-3,5-dimethoxyxanthone (54) and norswertianolin (55) isolated from Swertia yunnanensis exhibit inhibitory effects against HBV DNA replication in HepG2.2.15 cells, and methylation or glycosylation of the hydroxyl group of the compounds might be responsible for this inhibitory effect [38,66].

Tropolone Compounds
Tropolone is a seven-membered aromatic-ringed compound that includes a cyclic ketone functional group (cyclohepta-2,4,6-trien-1-one substituted by a hydroxy group at position 2) (57) (Figure 8). It is a toxin produced by the agricultural pathogen Burkholderia plantarii. It is an antibacterial and antifungal compound [69]. β-Thujaplicinol (58), extracted from Western Red Cedar heartwood (Thuja plicata, Thuja occidentalis, and Chamaecyparis obtusa), reportedly suppressed the replication of HBV strains of genotypes A and D in Huh7 cells transfected with HBV replication-competent plasmids by inhibiting RNAseH activity, with an estimated EC 50

Polysaccharide Compounds
Polysaccharides are natural polymeric compounds that exist as starch or cellulose in plants. Recent studies on polysaccharides have shown that they exhibit a wide range of biological activities, including immune enhancing, antiviral, and anti-inflammatory effects, as well as anti-HBV effects. Des(rhamnosyl) verbascoside (3,4-dihydroxybenzene ethanol-4-O-caffeoyl-β-D-glucoside) (59) is the main phenylethanol glycoside found in Lindernia ruellioides (Colsm.) Pennell (Figure 9), and its anti-HBV effects were evaluated in HepG2.2.15 cells. It significantly downregulated the expression of the HBV X protein (HBx) and inhibited DNA replication in a dose-dependent manner. In addition, it improved cell survival following H2O2-induced hepatocyte injury [71]. Another polysaccharide, heteropolysaccharide (FP-1), isolated from flaxseed hull using the hot water extraction method, reportedly exerted immunomodulatory effects by upregulating the mRNA expression levels of TNF-α, nitric oxide (NO), IL-6, and IL-12 in murine macrophages, and inhibited HBV DNA replication in HepG2.2.15 cells [72].

Others
Several compounds that could not be categorized under the classes described above have also been reported to decrease HBV DNA replication ( Figure 10). Coumarin, which is distributed in various plant varieties, was isolated by Huang et al. from Microsorum fortunei (Moore) (60-62), and its anti-HBV effects were evaluated in HepG2.2.15 cells and DHBV-infected ducklings [73]. HBV quantification in the coumarin-treated cells and ducklings revealed a significant decrease in HBV DNA compared to the control groups [73]. Cananga odorata (63), an Indonesian plant, was shown to reduce HBV DNA and HBsAg secretion in Hep38.7-Tet cells, with an IC50 of 56.5 μg/mL [74].

Polysaccharide Compounds
Polysaccharides are natural polymeric compounds that exist as starch or cellulose in plants. Recent studies on polysaccharides have shown that they exhibit a wide range of biological activities, including immune enhancing, antiviral, and anti-inflammatory effects, as well as anti-HBV effects. Des(rhamnosyl) verbascoside (3,4-dihydroxybenzene ethanol-4-O-caffeoyl-β-D-glucoside) (59) is the main phenylethanol glycoside found in Lindernia ruellioides (Colsm.) Pennell (Figure 9), and its anti-HBV effects were evaluated in HepG2.2.15 cells. It significantly downregulated the expression of the HBV X protein (HBx) and inhibited DNA replication in a dose-dependent manner. In addition, it improved cell survival following H 2 O 2 -induced hepatocyte injury [71]. Another polysaccharide, heteropolysaccharide (FP-1), isolated from flaxseed hull using the hot water extraction method, reportedly exerted immunomodulatory effects by upregulating the mRNA expression levels of TNF-α, nitric oxide (NO), IL-6, and IL-12 in murine macrophages, and inhibited HBV DNA replication in HepG2.2.15 cells [72].

Polysaccharide Compounds
Polysaccharides are natural polymeric compounds that exist as starch or cellulose in plants. Recent studies on polysaccharides have shown that they exhibit a wide range of biological activities, including immune enhancing, antiviral, and anti-inflammatory effects, as well as anti-HBV effects. Des(rhamnosyl) verbascoside (3,4-dihydroxybenzene ethanol-4-O-caffeoyl-β-D-glucoside) (59) is the main phenylethanol glycoside found in Lindernia ruellioides (Colsm.) Pennell (Figure 9), and its anti-HBV effects were evaluated in HepG2.2.15 cells. It significantly downregulated the expression of the HBV X protein (HBx) and inhibited DNA replication in a dose-dependent manner. In addition, it improved cell survival following H2O2-induced hepatocyte injury [71]. Another polysaccharide, heteropolysaccharide (FP-1), isolated from flaxseed hull using the hot water extraction method, reportedly exerted immunomodulatory effects by upregulating the mRNA expression levels of TNF-α, nitric oxide (NO), IL-6, and IL-12 in murine macrophages, and inhibited HBV DNA replication in HepG2.2.15 cells [72].

Others
Several compounds that could not be categorized under the classes described above have also been reported to decrease HBV DNA replication ( Figure 10). Coumarin, which is distributed in various plant varieties, was isolated by Huang et al. from Microsorum fortunei (Moore) (60)(61)(62), and its anti-HBV effects were evaluated in HepG2.2.15 cells and DHBV-infected ducklings [73]. HBV quantification in the coumarin-treated cells and ducklings revealed a significant decrease in HBV DNA compared to the control groups [73]. Cananga odorata (63), an Indonesian plant, was shown to reduce HBV DNA and HBsAg secretion in Hep38.7-Tet cells, with an IC50 of 56.5 μg/mL [74].

Others
Several compounds that could not be categorized under the classes described above have also been reported to decrease HBV DNA replication ( Figure 10). Coumarin, which is distributed in various plant varieties, was isolated by Huang et al. from Microsorum fortunei (Moore) (60)(61)(62), and its anti-HBV effects were evaluated in HepG2.2.15 cells and DHBVinfected ducklings [73]. HBV quantification in the coumarin-treated cells and ducklings revealed a significant decrease in HBV DNA compared to the control groups [73]. Cananga odorata (63), an Indonesian plant, was shown to reduce HBV DNA and HBsAg secretion in Hep38.7-Tet cells, with an IC 50 of 56.5 µg/mL [74].

Limitations of Previous Studies
The majority of studies that focused on screening phytomedicines and phytochemicals for downregulating HBV replication are mostly preliminary (Tables 1 and 2). Several studies have demonstrated the anti-HBV effects of total plant extracts. However, the specific compounds responsible for these effects, as well as their structures, have not been determined. In addition, most of the studies were conducted using in vitro systems such as hepatoma cells transiently transfected with HBV replication-competent plasmids or stably HBV-producing HepG2.2.15 cells. Only a few of the studies were conducted using in vivo models such as Peking ducklings infected with DHBV or in transgenic mice. Another significant limitation of almost all these studies is that none of them elucidated the precise mechanism underlying the HBV DNA replication-inhibitory effects of the screened phytomedicines and phytochemicals. The HBV replication process is highly complex and has several steps ( Figure 11). Some of the most important steps in HBV DNA replication and virion production include the production of cccDNA from partial dsDNA, the transcription of the cccDNA template leading to the four RNA strands, pgRNA encapsidation, reverse transcription within the capsid, synthesis of (+) strand DNA within the capsid, assembly of surface proteins, and virion release. Phytomedicines and phytochemicals that interfere with any of these steps of HBV DNA replication and virion production may decrease DNA levels and the virion copy number. However, almost none of the studies reviewed here investigated or described the specific step at which the compounds exerted their effects. The mechanisms by which these phytomedicines and phytochemicals affect HBV-derived factors and signaling pathways, as well as the host immune system, are yet to be investigated.

Limitations of Previous Studies
The majority of studies that focused on screening phytomedicines and phytochemicals for downregulating HBV replication are mostly preliminary (Tables 1 and 2). Several studies have demonstrated the anti-HBV effects of total plant extracts. However, the specific compounds responsible for these effects, as well as their structures, have not been determined. In addition, most of the studies were conducted using in vitro systems such as hepatoma cells transiently transfected with HBV replication-competent plasmids or stably HBV-producing HepG2.2.15 cells. Only a few of the studies were conducted using in vivo models such as Peking ducklings infected with DHBV or in transgenic mice. Another significant limitation of almost all these studies is that none of them elucidated the precise mechanism underlying the HBV DNA replication-inhibitory effects of the screened phytomedicines and phytochemicals. The HBV replication process is highly complex and has several steps ( Figure 11). Some of the most important steps in HBV DNA replication and virion production include the production of cccDNA from partial dsDNA, the transcription of the cccDNA template leading to the four RNA strands, pgRNA encapsidation, reverse transcription within the capsid, synthesis of (+) strand DNA within the capsid, assembly of surface proteins, and virion release. Phytomedicines and phytochemicals that interfere with any of these steps of HBV DNA replication and virion production may decrease DNA levels and the virion copy number. However, almost none of the studies reviewed here investigated or described the specific step at which the compounds exerted their effects. The mechanisms by which these phytomedicines and phytochemicals affect HBVderived factors and signaling pathways, as well as the host immune system, are yet to be investigated.     Figure 11. HBV replication cycle showing the possible steps in the DNA replication process that can be affected by phytomedicines/phytochemicals. The "?" mark indicates the possible steps where the phytomedicines may show inhibitory activity towards HBV DNA replication.

Future Approaches
Several studies have focused on evaluating the viral DNA replication-associated anti-HBV effects of phytomedicines and phytochemicals. However, accurate structures of the identified compounds, experimental animals with in vivo and de novo infection conditions, or clinical trials in humans are lacking. Moreover, the exact mechanisms of action of these compounds have not been elucidated. Thus, further studies should be performed to determine the specific structures of compounds that have been reported to exert significant inhibitory effects against HBV DNA replication. The specific steps of the HBV DNA replication process affected by the different compounds should be investigated by Southern blotting, Northern blotting, or Western blotting in combination with quantitative PCR. The stimulation of host factors or signaling pathways and immune system in the phytomedicines/phytochemicals treated and replicating HBV cell and host need to be properly investigated. In vivo investigations in experimentally HBV-infected animal models such as mice, monkeys, or chimpanzees, treated with different drugs of plant origin, would be the ideal approach for the clinical examination of the effects of these phytomedicines and phytochemicals. Finally, a comparative study using the most effective phytomedicines and phytochemicals in in vitro and in vivo systems is needed before validating their effects in HBV-infected patients.

Future Approaches
Several studies have focused on evaluating the viral DNA replication-associated anti-HBV effects of phytomedicines and phytochemicals. However, accurate structures of the identified compounds, experimental animals with in vivo and de novo infection conditions, or clinical trials in humans are lacking. Moreover, the exact mechanisms of action of these compounds have not been elucidated. Thus, further studies should be performed to determine the specific structures of compounds that have been reported to exert significant inhibitory effects against HBV DNA replication. The specific steps of the HBV DNA replication process affected by the different compounds should be investigated by Southern blotting, Northern blotting, or Western blotting in combination with quantitative PCR. The stimulation of host factors or signaling pathways and immune system in the phytomedicines/phytochemicals treated and replicating HBV cell and host need to be properly investigated. In vivo investigations in experimentally HBV-infected animal models such as mice, monkeys, or chimpanzees, treated with different drugs of plant origin, would be the ideal approach for the clinical examination of the effects of these phytomedicines and phytochemicals. Finally, a comparative study using the most effective phytomedicines and phytochemicals in in vitro and in vivo systems is needed before validating their effects in HBV-infected patients.