Rapid and direct detection of hepatitis E virus in raw pork livers by recombinase polymerase amplification assays

Hepatitis E virus (HEV) is a zoonotic pathogen that causes global hepatitis E. Outbreaks of hepatitis E are directly linked to the consumption of pork liver products. Herein reverse transcription recombinase polymerase amplification assays targeting the ORF2 gene were developed for the rapid detection of HEV by integrating the fluorescence detection platform (qRT-RPA) and the visible lateral flow biosensor by naked eyes (LFB RT-RPA). The qRT-RPA assay effectively detected HEV RNA with a limit of detection (LOD) of 154 copies/μl (95%CI: 126–333 copies/µl) in Genie III at 41°C for 20 min. Besides this, the LFB RT-RPA detected the HEV RNA with a LOD of 24 copies/μl (95%CI: 20–57 copies/µl) in an incubator block at 41°C for 20 min. The developed RT-RPA assays also showed good specificity for HEV, with no cross-reactions with any of the other important swine pathogens examined in this work. The performance of the developed RT-RPA assays was validated on 14 HEV RNA-positive and 66 HEV RNA-negative raw pork liver samples identified by a previously described qRT-PCR. Consequently, 11 and 12 samples were HEV RNA-positive as detected by the qRT-RPA and the LFB RT-RPA, respectively. Compared to qRT-PCR, the qRT-RPA and LFB RT- RPA assays revealed a coincidence rate of 96.3 and 97.5% as well as a Kappa value of 0.858 and 0.908, respectively. These results ascertain that the developed RT-RPA assays are effective diagnostic tools for the point-of-care detection of HEV in resource-limited settings.

Introduction
Hepatitis E virus (HEV) is a small, non-enveloped, and single-stranded positive RNA virus that is 7.2 kb in size and belongs to the Hepeviridae family (Wang et al., 2021). HEV is an emerging zoonotic virus transmitted through the fecal-oral route and causes acute or chronic infections (von Wulffen et al., 2018). Its symptoms include mild fever, nausea and vomiting, fatigue and anorexia, abdominal pain, and dark urine. HEV also contributes to extrahepatic manifestations, such as neurological, hematological, and renal conditions (Webb and Dalton, 2019). HEV infection causes self-limiting hepatitis with a mortality rate of approximately 0.2-4%, except for pregnant women and patients with chronic liver disease with a mortality rate of up to 30% (Hennechart-Collette et al., 2021;Raji et al., 2021).
HEV can adapt to a variety of hosts, including pigs, wild boars, deer, rabbits, and camels (Treagus et al., 2021). These animals act as potential reservoirs of HEV, causing the indirect transmission of HEV as well as promoting genetic variation and HEV evolution. Pigs are the primary reservoirs of HEV (Ukuli and Mugimba, 2017). Infected pigs may have no apparent clinical symptoms; however, viruses are shed from their feces or urine route at the early stages of infection. If these excreta are not properly handled, pigs in the same pen infect each other through repeated and direct contact with excreta, thus aggravating HEV infection (Salines et al., 2017). The virus persists in all age groups, from weaners to fatteners (Berto et al., 2012;Jackova et al., 2021). Therefore, HEV transmission is primarily attributed to the consumption of contaminated raw or undercooked pork products, therefore presenting a potential risk to public health. Additionally, excreta harboring HEV can pollute nearby irrigation and coastal waters, resulting in contaminated crops or seafood and thereby increasing the risk of human infection (Salines et al., 2017).
Real-time reverse transcriptase PCR (qRT-PCR) is the most common method for HEV detection (Mykytczuk et al., 2017). This technology involves denaturation, annealing, and the subsequent extension of the target gene. It requires the use of an accurate and stable thermocycler as well as a reliable power suppl and is only limited to well-equipped laboratories (Lobato and O'Sullivan, 2018). Recombinase polymerase amplification (RPA) is a novel enzymatic-based DNA amplification technology where primer annealing and elongation are mediated by different enzymes at constant temperature (Daher et al., 2016). The amplification proceeds at constant temperatures between 37 and 42°C and then typically run to completion within 30 min (Geng et al., 2019). The reaction starts when a recombinase protein uvsX binds to primers, forming a recombinase-primer complex. Subsequently, the complex recombines with homologous sequences in double-stranded DNA, hence forming a D-loop structure and initiating a strand exchange reaction. To prevent primer dissociation, the displaced DNA strand is stabilized by a single-stranded binding protein. Eventually, the amplification is initiated by a stranddisplacing DNA polymerase (Piepenburg et al., 2006;Hill-Cawthorne et al., 2014). The RPA amplification products can be detected through gel electrophoresis, probe-based fluorescence monitoring, and lateral flow dipsticks . Therefore, recombinase polymerase amplification has a significant potential in an underequipped laboratory or in pointof-care diagnostics Davi et al., 2019).
For the rapid detection of HEV, we developed reverse transcription RPA assays targeting the ORF2 gene of HEV by combining fluorescence detection platform (qRT-RPA) and visible lateral flow biosensor by naked eyes (LFB RT-RPA). Thereafter, the assays were validated using raw pork livers.
A total of 626 raw pork liver samples were collected from different regions in Hebei Province between April 2021 and March 2022, i.e., 213 from the different retail markets, 300 from three different pig slaughterhouses, and 113 from the different Bio-Safety Disposal Centers for Dead Livestock and Poultry (BsDC) in Hebei Province. Total RNA extracted from 626 raw pork livers was detected using the previously described qRT-PCR assay (Qiao et al., 2008). Among these samples, 14 were HEV-positive with Ct values ranging from 30.23 to 37.46 and were confirmed as genotype 4d by sequencing the PCR product of a nested PCR assay described (Huang et al., 2002). The clinical performance of the RPA assays was established by testing 14 positive and 66 randomly selected negative samples. Viral and sample DNA/RNA extraction PRV, PCV2, PRRSV, CSFV, PPV, and FMDV viral DNA or RNA were extracted using the TIANamp Virus genomic RNA kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions. For the liver samples, approximately 100 mg of each sample was transferred into a 1.5-ml sterilized centrifuge tube with five 3.0-mm grinding beads and 1 ml of phosphate-buffered saline and then homogenized at 30 frequency/s for 5 min with an MM400 grinding mill (Retsch, Haan, Germany). After 12,000×g centrifugation at 4°C for 10 min, 200 µl supernatant was collected for viral RNA extraction using the Magnetic Viral DNA/RNA Extraction Kit (TIANLONG, Xian, China) with an np986-C Nucleic Acid Extraction System (TIANLONG, Xian, China) following the manufacturer's instructions. The nucleic acids were stored at −80°C for subsequent analysis.

Generation of standard RNA
The pET28a-ORF2 was first linearized with BamHI (TaKaRa, Dalian, China) and then transcribed into RNA using T7RiboMAX ™ Express Large Scale RNA Production System (Promega, USA). Subsequently, the residue plasmid DNA was eliminated using RNase-Free DNaseI (Tiangen, Beijing, China), and the transcribed RNA was purified using the RNA Clean Kit (Tiangen, Beijing, China). The in vitro transcribed standard RNA was quantified as 3.4 × 10 8 copies/ml using a previously established RT-ddPCR assay in our laboratroy. Afterward, 10fold serial dilutions of standard HEV RNA ranging from 3.4 × 10 6 to 3.4 × 10 0 copies/ml were prepared for further studies.
The primers were screened in triplicates based on the TwistAmp ™ amplification guidelines (TwistDx Ltd., Cambridge, UK) to obtain primers with the best performance. In the primary candidate screening, three forward and four reverse candidate primers were designed in the conserved region of the HEV ORF2 gene (Table 1). The different combinations of candidate primers were screened using the ZC BioScience ™ basic kit (ZC BioScience, Hangzhou, China) and then analyzed using 2% gel electrophoresis. Primer pairs with the most product yield and less product/noise ratio were selected for secondary candidate screening, and the probes HEV-RPA-P1 and HEV-RPA-P3 were designed. Thereafter, the primers were screened by keeping the primer length unchanged while moving in one base increment around the selected primers. Therfore, five forward (HEV-RPA-F301-F305) and four reverse primers (HEV-RPA-R301-R304) were obtained. The newly obtained primers were screened using the ZC BioScience ™ exo kit (ZC BioScience, Hangzhou, China). Considering that the position of the forward primer could only be moved within a small range (more mutations and continuous repeating bases around this region), all available primers in this region were considered, and the tertiary candidate screening was not performed. Eventually, the reverse primer was refined by adding and subtracting bases from the 3′ end of the primer HEV-RPA-R301, and four reverse primers (HEV-RPA-R3001-R3004) were obtained. The newly obtained primers were screened similarly to the previous round. All primers and probes in Table 1 were synthesized by Geneary (GenerayBiotech, Shanghai, China).

qRT-RPA assay
The qRT-RPA assay was conducted in a 50-µl volume using the ZC BioScience ™ exo kit (ZC BioScience, Hangzhou, China). The reaction mixture contained 25 ml of rehydration buffer, 2.5 ml of magnesium acetate (280 mM), 2.1 ml of each primer (10 mmol/L), 0.6 ml of exo probe (10 mmol/L), 5 ml of extracted nucleic acid, and 12.7 ml of ddH 2 O. The reaction tubes were immediately mixed and spun down. The qRT-RPA reactions were performed at 41°C for 20 min using a Genie III scanner device (OptiGene Limited, West Sussex, UK). The fluorescence signal was measured at an interval of every 20 s. Samples that yeilded an exponential amplification curve above the negative control threshold were considered positive.

LFB RT-RPA assay
The LFB RT-RPA assay was performed in a 50-µl volume using the GenDxTM RT-LFB kit (GenDx, Suzhou, China). The reaction mixture contained 20 ml of rehydration buffer, 2.0 ml of magnesium acetate (280 mM), 2.1 ml of each primer (10 mmol/ L), 0.6 ml of nfo probe (10 mmol/L), 5 ml of extracted nucleic acid, and 18.2 ml of ddH 2 O. To determine the optimal amplification temperature, the RPA reactions were performed on a metal bath incubator set at 39-45°C for 20 min. Thereafter, the reactions were performed at the optimal temperature for 5, 10, 20, and 30 min to determine the optimal incubation time. After the reaction, 5 ml of RPA products was mixed with 200 ml of ddH 2 O followed by lateral flow biosensor analysis (GenDx, Suzhou, China). The results were considered positive when both the test line and the control line were visible, negative when only the control line was visible, and invalid when the control line was invisible.

Analytical specificity analysis
The specificity of the developed HEV-specific qRT-RPA and LFB RT-RPA assays was examined with 3.4 × 10 4 copies of HEV standard RNA and 6.08 × 10 7 -5.15 × 10 9 copies of DNA or RNA of other important swine viruses including PRV, PCV2, PRRSV, CSFV, PPV, and FMDV. During the analysis, ddH 2 O was chosen to act as the negative control. All the samples were tested in triplicates.

Analytical sensitivity analysis
The sensitivity analysis of the RT-RPA and RT-qPCR assays was performed using 10-fold serial dilutions of standard HEV RNA ranging between 3.4 × 10 6 and 3.4 × 10 0 copies/ml. Each run was repeated eight times. Meanwhile, 3.4 × 10 2 copies/ml of HEV RNA was serially diluted in twofold (one in one to one in four) and tested for the qRT-RPA and RT-qPCR assays in eight replicates, whereas 3.4 × 10 1 copies/ml of HEV RNA was serially diluted in twofold (one in one to one in four) and tested for LFB RT-RPA in eight replicates.
A Probit (predicted proportion positive) analysis (SPSS v22.0, Armonk, USA) with data of the positive samples from each of the eight replications was performed to establish the 95% limit of detection (LOD).

Validation with raw pork livers
A total of 80 raw pork livers from different sources, including 14 HEV-positive livers and 66 randomly selected HEV-negative livers in qRT-PCR, were detected using the qRT-RPA and LFB RT-RPA assays, respectively.

Results
Screening of the optimal primerprobe combinations Three rounds of primer screening were conducted in this study. Three forward and four reverse primers were designed and screened by using ZC BioScience ™ basic kit in the primary screening of primer candidates. Among these, two primer pairs amplified HEV RNA (HEV-RPA-F1/R1-2 and HEV-RPA-F3/ R3). The HEV-RPA-F3/R3 primer pair produced the greatest amount of amplification product and was selected for subsequent experiments (Supplementary Figure S1A). In the secondary screening of primer candidates, five forward (HEV-RPA-F301-F305) and four reverse primer candidates (HEV-RPA-R301-R304) were designed surrounding HEV-RPA-F3 and HEV-RPA-R3, respectively. Reverse primer HEV-RPA-R3 was selected to screen all six forward primers, and the primer with the best fluorescence signal was considered the best forward primer (HEV-RPA-F302) (Supplementary Figure S1B). Subsequently, HEV-RPA-F302 was selected to screen all the five reverse primers; HEV-RPA-R301 generated the best result (Supplementary Figure S1C). In the tertiary candidate screening, four reverse primer candidates (HEV-RPA-R3001-R3004) were designed surrounding HEV-RPA-R301 and were screend using the best forward primer HEV-RPA-F302. HEV-RPA-R301 was again used to generate the best amplification result (Supplementary Figure S1D). Therefore, HEV-RPA-F302/ R301/P3 was subsequently used in the qRT-RPA assay.
In most cases, the similar primer and probe sequences with different residue modifications functioned effectively in both qRPA and LFB RPA assays. However, our data confirmed that HEV-RPA-F302/R301/P3 produced false-positive signals in the LFB RT-RPA assay. Therefore, the primer pair and probe HEV-RPA-F1/R1-2/P1 were selected for the HEV LFB RT-RPA assay.

Performance of the RT-qRPA assay
The specificity of RT-qRPA was evaluated using HEV and six other important swine-associated viruses. Consequently, only HEV RNA revealed a typical fluorescent signal in the RT-qRPA assay, whereas no fluorescent signals were obtained for the other six viruses ( Figure 1A). Similar results were obtained in three repeats.

Optimization of LFB RT-RPA reaction conditions
HEV-specific LFBRT-RPA reaction conditions were optimized using 3.4 × 10 4 copies/ml of HEV RNA as a template. As shown in Figure 2A, the LFB RT-RPA assay worked effectively at a temperature ranging between 39 and 45°C, and the brightest test lane was observed at 41°C. As shown in Figure 2B, the test line was extremely weak in reactions incubated for 5 min, and the test lines were clearer when the incubation duration was over 10 min. The assay performance was improved with a longer reaction time; there was no discernable difference after 20 min. Thus, 41°C and 20 min were set as the optimal conditions for the LFB RT-RPA assay.

Performance of the LFB RT-RPA assay
For the specificity analysis of LFB RT-RPA, only HEV RNA was detected as positive, and no cross-reaction was noted with the other six swine-associated viruses ( Figure 2C). The 10-fold serial dilutions of the HEV RNA that ranged between 3.4 × 10 5 and 3.4 × 10 0 copies/µl were used to evaluate the limit of detection. As shown in Figure 2D, the test line was observed between 3.4 × 10 5 and 3.4 × 10 1 copies/µl of HEV RNA, whereas five of eight, five of eight, and zero of eight were positive at concentrations of 1.7 × 10 1 , 8.5 × 10 0 , and 3.4 × 10 0 copies/µl, respectively. According to Probit regression analysis, the LOD for the LFB RT-RPA assay was 24 copies/ml (95%CI: 20-57 copies/µl) ( Figure 2E).

Performance of RT-RPA assays on raw pork livers
For the 14 HEV RNA-positive livers in the qRT-PCR assay, the qRT-RPA and LFB RT-RPA detected 11 and 12 positive samples, respectively. All 66 randomly selected negative samples were HEV RNA-negative in both qRT-RPA and LFB RT-RPA assays ( Table 2). The coincidence rate between the test results of RT-RPA and RT-qPCR was 96.3%, whereas the coincidence rate between the test results of LFB RT-RPA and RT-qPCR was 97.5%. No significant difference was noted between the qRT-RPA and qRT-PCR assays (p = 0.25). No significant difference was also found between LFB RT-RPA and qRT-PCR assays (p = 0.50). Furthermore, the qRT-RPA and RT-qPCR assays were significantly in agreement (kappa = 0.858 at 95%CI). The LFB RT-RPA and qRT-PCR assays were also significantly in agreement (kappa = 0.908 at 95%CI). The above-mentioned data show that the developed HEV-specific RT-RPA assays had a similar diagnostic performance with qRT-PCR on clinical samples (Table 3).

Discussion
Since its first description in 2006, RPA technology has received significant research attention. At present, RPA is widely used in the detection of pathogens, including bacteria, viruses, and parasites. It fosters innovation in the field of isothermal nucleic acid amplification technology (Euler et al., 2013;Dobnik et al., 2016;Liu et al., 2019).
Suitable primers and probes are critical for the success of an RPA assay. So far, software for designing RPA primers is unavailabe. The RPA primer screening process is similar to PCR, i.e., it involves the selection of the target region, designing the primer candidates, and screening for the best candidate (Lobato and O'Sullivan, 2018). Hairpin structure, primer dimers, and primer-primer interactions should be avoided . Nevertheless, we detected several mutations in the ORF2 gene sequence of HEV. Moreover, the continuous conserved region was short and scattered, limiting the selection of primers and probes for the RPA assay. Thus, the primer conservation was the only factor in the initial design of primers, and the amplification efficiency of primers was experimentally confirmed. After three rounds of screening, HEV-RPA F302/R301/P3 was selected for the qRT-RPA assay, whereas HEV-RPA F1/R1-2/P1 was selected for the LFB RT-RPA assay. Furthermore, the designed primers and probes were compared using the DNASTAR software with representative HEV genotypes 1-4. In the qRT-RPA assay, four to five nucleotide mismatches were detected in HEV-1, 10 nucleotide mismatches were detected in HEV-2, six to seven nucleotide mismatches were detected in HEV-3, and zero to three nucleotide mismatches were detected in HEV-4. In the LFB RT-RPA assay, five to nine nucleotide mismatches were detected in HEV-1, 12 to 13 nucleotide mismatches were detected in HEV-2, six to nine nucleotide mismatches were detected in HEV-3, and four to nine nucleotide mismatches were detected in HEV-4. Previous studies have shown that RPA can tolerate five to nine mismatches in primers and probes without affecting its performance (Boyle et al., 2013;Liu et al., 2019). In this regard, it was difficult for the two RPA assays that we developed to detect HEV-2, but in theory, these would perform well in detecting the other three genotypes (Figure 3). All isolated HEV strains in this study belonged to the genotype 4d, with four to nine nucleotide mismatches from the primers and probes of the RT-RPA assays established in this study    (unpublished data). This indicates that the primers and probes of the RPA method are highly resistant to mutations. Nevertheless, further validation studies are necessary for the other HEV genotypes. After optimizing the reaction conditions, the qRT-RPA and LFB RT-RPA assays display a similar performance to the qRT-PCR assay (Qiao et al., 2008), whereas the LOD obtained from qRT-RPA is distinct with the LFB RT-RPA assay in this study. There are two possible reasons for this phenomenon. One reason is the use of different primer and probe sequences and different nucleases (the qRT-RPA assay uses exonuclease, while the LFB RT-RPA assay uses endonuclease), and the other reason is the different detection methods for the amplification products. The performance of the RT-RPA assays on the raw pork livers of different sources was slightly lower than that of the qRT-PCR assay. However, the RT-RPA demonstrated distinct advantages of rapidness and convenience, suggesting that the developed RT-RPA assays can be used as alternative detection techniques for HEV. Three samples were negative in the RT-RPA assays but weakly positive in qRT-PCR, with Ct values of approximately 34.22-37.04, which contained low amounts of HEV RNA. Our findings are similar to previous reports that detected Mycoplasma hyopneumoniae, bovine ephemeral fever virus, and peste des petits Ruminant's virus through RPA assays (Hou et al., 2018;Yang et al., 2018;Davi et al., 2019). Therefore, target gene concentration should be increased by increasing the sample content in the reaction system to improve the detection performance of the clinical samples.
RPA technology has many incomparable strengths over qPCR technology. Firstly, RPA operates at a relatively low temperature, i.e., body temperature, water bath, or heating blocks (Crannell et al., 2014;Cherkaoui et al., 2021). Secondly, RPA can tolerate up to nine mismatches, hence increasing the selection of primers for viruses with robust nucleic acid sequence mutations (Boyle et al., 2013). Thirdly, RPA reagents are freezedried, and the RPA kit can be stored at room temperature for up to 6 months (Chandu et al., 2016). The above-mentioned benefits enable the RPA assays to be more suitably used at point-of-care (POC) or underequipped laboratory diagnosis and in resource-limited settings or field diagnosis of various pathogens. However, it is necessary to open the reaction tube before lateral flow biosensor analysis. This process may carry over aerosol contamination in fields. In order to reduce potential contamination, the laboratory should have a rigorous partition, and UV irradiation or DNase treatment should be used frequently. In addition, the reaction tubes should be carefully opened and closed, and gloves should be frequently changed during RPA operation.
In conclusion, the RPA assays developed in this work have significant potential for POC detection of HEV and can therefore be used in the field.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the Medical Ethics Committee of Hebei Medical University.


The preparatory phase for ground larviciding implementation for chocerciasis control in the Meme River Basin in South West Cameroon: the COUNTDOWN Consortium alternative strategy implementation trial

Background Onchocerciasis control using ivermectin alone has been achieved in some endemic savannah zones of Africa. In the forest regions, the co-endemicity with Loa loa has led to severe adverse events (SAEs) resulting in poor adherence of community members to ivermectin mass drug administration (MDA). This may jeopardize achieving the interruption of transmission of onchocerciasis. Therefore, to accelerate the elimination of onchocerciasis in L. loa co-endemic zones, alternative treatment strategies (ATS) including ground larviciding may be necessary. This study aimed at identifying Simulium breeding sites, cytospecies, transmission profile, susceptibility of Simulium larvae to insecticide (temephos) and identification of some non-target aquatic fauna prior to the implementation of the COUNTDOWN consortium ground larviciding alternative strategy in the Meme River Basin in South West Cameroon. Methods A topographic map and entomological survey were used to determine breeding sites. Larvae and adults were identified using standard identification keys. Susceptibility tests were carried out on collected larvae by exposing them to decreasing concentrations of temephos and assessing survival rates while the cytospecies were identified using cytotaxonomy. Various entomological indicators were assessed from dissected flies. Fishing was used as proxy to traps to assess some aquatic fauna at different sites. Results Twenty-two breeding sites were prospected in the Meme River Basin with eight productive for larvae. A concentration of 0.5–0.1 mg/l temephos induced 100% larval mortality. As the concentration of temephos decreased from 0.05 to 0.0025 mg/l, mortality of larvae also decreased from 98.7 to 12%. Nine cytospecies were observed in the Meme River Basin; 13,633 flies were collected and 4033 dissected. A total of 1455 flies were parous (36.1%), 224 flies were infected (5.5%), and 64 were infective (1.6%). Aquatic fauna observed included Cyprinus spp., Clarias spp., crabs, tadpoles, beetles and larvae of damsel fly. Conclusions Onchocerciasis is being actively transmitted within the Meme River Basin. Simulium larvae are susceptible to temephos, and nine cytospecies are present. Non-target fauna observed included fishes, frogs, crabs and insects. Besides treatment with ivermectin, vector control through ground larviciding may be a complementary strategy to accelerate onchocerciasis elimination in the study area. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-022-05300-z.

Ekanya et al. Parasites & Vectors (2022) 15:219 Background Onchocerciasis, a parasitic disease caused by the filarial nematode Onchocerca volvulus [1], is a public health problem found in some of the world's most disadvantaged communities in remote, rural sub-Saharan Africa. Adult worms live in subcutaneous nodules and produce millions of microfilariae during their reproductive life span (10-15 years). The microfilariae (mf ) can live between 1 and 2 years in the skin, from where they are taken up by black fly vectors during a blood meal. Mf develops into infective larvae within black fly, which infect another person during a subsequent bite, thus maintaining the transmission cycle. Host inflammatory reactions to dead mf in the skin cause onchocercal dermatological disease and in the eyes ocular disease that may end in blindness. Because of the public health problem and socioeconomic effects of onchocerciasis, large-scale control programmes were created in the past with strategies for its control while in recent years strategies have been directed at its elimination [2]. In 11 West African countries, the Onchocerciasis Control Programme (OCP) aerial larviciding led to the control of onchocerciasis as a public health problem [3].
The advent of ivermectin and its donation by Merck & Co. (NJ, USA) [4] provided the opportunity for its largescale use for the treatment of onchocerciasis by the African Programme for Onchocerciasis Control (APOC) in 20 endemic African countries. Ivermectin mass drug administration (MDA) has led to the interruption of transmission in some foci in Mali, Senegal, Sudan and Uganda [5][6][7][8]. Unfortunately, the same level of achievement has not been made in Cameroon, especially in the South West Region where onchocerciasis is co-endemic with loiasis.
In the South West Region of Cameroon, the persistence of onchocerciasis transmission is due to the existence of abundant breeding sites in fast-flowing rivers, which results in very high numbers of biting black flies transmitting the parasite and poor adherence of the community members to ivermectin mass drug administration [9,10]. The poor adherence to ivermectin MDA is due to the fear of SAEs that follow ivermectin treatment of highly infected Loa loa individuals in co-endemic areas of onchocerciasis and L. loa [10,11]. Faced with these challenges and coupled with the change in paradigm from onchocerciasis control to elimination, it was important for APOC to offer new approaches to improve or complement ivermectin MDA [12]. This led to adoption of alternative treatment strategies (ATS). These ATS include complementary vector control, enhanced community-directed treatment with ivermectin (CDTI), community-directed treatment (CDT) with drug combinations or new drugs and test-and-treat (TNT) strategies [13]. The ATS are essentially directed towards highly endemic onchocerciasis foci where the disease cannot be eliminated by annual CDTI alone.
The Meme River Basin of South West Cameroon has been under CDTI for > 18 years. A situational analysis of onchocerciasis conducted in this region by Wanji et al. [9] demonstrated persistent transmission despite ivermectin MDA with prevalence values as high as 52% [9]. This persistence in onchocerciasis transmission motivated the COUNTDOWN Consortium to evaluate implementation of one of the recommended ATS, ground larviciding, as a potential tool to accelerate the elimination of onchocerciasis in this endemic focus. Ground larviciding is known to significantly reduce the number of biting black flies, thus reducing disease transmission and infection rates. Pre-requisites to ground larviciding are: identification of breeding sites, susceptibility test of Simulium larvae to a larvicide of interest, identification of cytospecies in the river basin and identification of non-target aquatic fauna. In this study, we chose to use temephos (Abate ® 500 EC-BASF, Douala, Cameroon) as the larvicide. Temephos is an organophosphate larvicide which has been shown to be biodegradable and non-toxic to non-target fauna with a good carry during the rainy season. This article presents the breeding sites, identified Simulium cytospecies, current onchocerciasis transmission profile, susceptibility of Simulium damnosum larvae to temephos and a baseline survey of some non-target aquatic fauna present in the Meme River Basin prior to the implementation of the COUNTDOWN Consortium ground larviciding alternative strategy implementation trial.

Study area
Meme River Basin is situated in the rain forest of the South West Region of Cameroon approximately 60 km inland from the Atlantic Ocean. The topography is very diverse; the main feature is a mountain range (Rumpi Hills, characterized by a volcanic ridge culminating at 1764 m with a northeast orientation [14]. The volcanic ridge is broken by several valleys and constitutes a watershed that is the source for several rivers, the Manyu, Meme, Mungo and Ndian. These rivers go down steep slopes generating fast currents and appropriate flow rates that favour the establishment of permanent Simulium breeding sites conducive to high intensity of onchocerciasis transmission. The climate is characterized by 8 months of rainfall and a short dry season from December to March. The annual rainfall varies between 2500 and 4000 mm with annual temperatures ranging from 25 to 32 °C.
River Meme and its tributaries originate from the Rumpi Hills and flow down precipitous slopes some kilometres from the selected communities. The main tributaries of the Meme River are the Mbile River, which flows pass Marumba I and II, and the Uve River in Bakumba and Big Massaka. These tributaries create important Simulium breeding sites as they enter the Meme River.

Identification of breeding sites
Potential Simulium breeding sites in the Meme River were selected using a topographical map of 1:200,000 scale [28] followed by an entomological survey to check for the presence of rapids, trailing vegetation and larvae to confirm that it is a breeding site. The Melange River in Ndian Division was set as the control area, which will be used to monitor the stability of entomological indicators after ground larviciding. The larvae and adults were identified using standard identification keys [15,16]. All breeding sites were geo-referenced using a GPS. The coordinates were used to generate a topographical map of the breeding sites ( Fig. 1a, b).

Study sites
The study was conducted from March to July 2017. River Meme and its tributaries were surveyed, 20 breeding sites from 12 communities (Betenge, Small Massaka Lokando Big Butu, Small Butu, Big Massaka, Kumukumu, Bombele, Bobanda Kwakwa Nake and Marumba 1) were visited, but due to absence of larvae in some sites at the time of the study, only 8 sites from six communities (Betenge, Small Massaka, Big Massaka, Bombele, Kwakwa and Marumba 1) were chosen for fly and larvae collection. Due to accessibility, two sites near one community (Berenge) were surveyed in the Melange River Basin (control area). Flies were collected and dissected from one of the sites that had larvae to monitor the stability of entomological indicators in an untreated area after ground larviciding. Some sites in the Meme River Basin did not have enough larvae, so larvae for cytotaxonomy were collected only from three sites (Big Massaka, Bombele and Kwakwa). In the study arm, the breeding sites chosen were at: • Marumba I (River Mbile, a tributary of Meme). Here, S. damnosum larvae were collected and a fly dissection point was set up at the river bank. • Bombele, where the Meme River runs over several rock features developing rapids, which constitute breeding sites along its way to join the Atlantic Ocean. • Kwakwa (along River Mbile, a tributary of Meme).
Here, many larvae were found and a fly collection point was set up at the bank of the river. • Big Massaka (River Uve, tributary of Meme). A survey of this tributary revealed many breeding sites which contained S. damnosum larvae. A catching point was therefore located in this community. • Betenge, where the Meme runs. A fly collection and dissection point were located in this community • Small Massaka (River Ka, tributary of Meme). A fly collection and dissection point were located in this community

Susceptibility of S. damnosum larvae to temephos (Abate 500EC)
Larvae of S. damnosum were collected from several breeding sites, namely Marumba 1, Big Massaka, Bombele and KwaKwa in the Meme River Basin located in the South West Region of Cameroon. These larvae were exposed using a bowl containing local bottled water (Supermont) to ten concentrations of temephos (0.0005 mg/l to 0.5 mg/l) (Additional file 1: Text S1) for a period of 3 h [17]. After this, mortality was determined by observation of larvae motility response to stimulation [18].

Dosage-mortality regression line
Adjusted mortality for each concentration was calculated utilising the control mortality frequency that ran in parallel with the test. If the control mortality was between 5 and 20%, then Abbot's formula [19] was used to correct the mortality and ensure that other factors were not contributing to the mortality of the larvae. No adjustment was done if control mortality was < 5% and results were discarded for any testing with control mortality > 20%.

Identification of cytospecies of S. damnosum in the Meme River Basin
Cytotaxonomy was used to identify the different cytospecies in the Meme River Basin. Cytotaxonomy requires the extraction and analysis of polythene chromosomes (giant chromosomes caused by cells that have undergone repeated rounds of replication) present in the silk gland of black fly larvae. Larvae for cytotaxonomy were collected from three breeding sites (Big Massaka, Bombele and Kwakwa) because the other breeding site did not have enough larvae for cytotaxonomy. The sixth and seventh stage larvae were collected and put in Carnoy's fixative (3 parts ethanol: 1part acetic acid) that was freshly prepared at the riverside. A minimum of 30 larvae were put in 24 ml of Carnoy. The Carnoy's solution was changed after 3 h and 24 h. The silk glands were dissected followed by hydrolysation to enable all the silk in the glands to be removed. Thereafter, chromosomes were stained for 5 min with Feulgen stain, which stains the DNA pink. Continual addition of orcien to the silk gland occurred to prevent dehydration. The gonads of the larvae were inspected to identify the sex. If male, the gonads are round, but if female, the gonads are elongated in shape. Squashing was done to separate the chromosomes while a light microscope was used to visualize and identify them. Photographs were taken at × 400. Identification of each larval chromosome was done using information from pictorial guide and identification key by Post et al. [20].

Collection and identification of Simulium flies
Simulium flies were collected from seven different sites: Bombele and Betenge (River Meme), Big Massaka (River Uve), Kwakwa and Marumba I (River Mbile), Small Massaka (River Ka) and Berenge (River Melange). Residents of endemic communities for onchocerciasis were trained to carry out collections; Simulium flies landing on exposed legs for a blood meal were captured using suction tubes or mouth aspirators (locally adapted) before they bit [21]. Adult flies were either dissected to determine the parity and infectivity rate or stored in 80% alcohol for future use. Onchocerca volvulus transmission indicators (parous rate, infection rate, infectivity rate and monthly transmission potential) were computed after fly collection and dissection as described by Walsh et al. [22].

Dissection of Simulium flies
Captured flies were killed using chloroform, counted and dissected in physiological saline under a dissecting microscope (Humanscope, HUMAN Diagnostics Worldwide, USA). Flies caught were dissected on an hourly basis to determine parity and infection status. This consisted of holding the fly with a needle in the thorax, piercing the abdomen with a dissecting needle at the posterior end and then pulling out the different internal organs to examine the fat bodies, state of the Malpighian tubules and ovaries to distinguish parous from nulliparous flies (nulliparous ovary is generally clear with follicles that are bigger and have voluminous fatty substance while parous flies generally have yellowish, spotted ovaries because of follicular relicts. The follicles are much smaller with little or no fatty substance). The head, thorax and abdomen of parous flies were further dissected separately and examined for O. volvulus developing larvae (L1, L2 and L3). Any larvae found were counted and recorded on a dissection sheet [23] and the entomological indices computed [22]

Survey of some non-target aquatic fauna
Survey of some non-target aquatic fauna was conducted to obtain a picture mainly of the common fish species and also other common vertebrate as well as invertebrate non-target fauna to serve as indicators to guide measurement of any detrimental effect of temephos during ground larviciding. For this to be done, fishermen and -women were contacted to determine the species of aquatic vertebrates common in the Meme River and its tributaries. Thereafter, they were requested to carry out fishing exercises to determine the vertebrate aquatic fauna found at the different study sites. Locally made fishing nets were placed in water overnight to trap any available vertebrate aquatic fauna as modified from the protocol adopted by OCP in 1991 [24]. During the collection of Simulium larvae, invertebrate aquatic fauna present on the same substrate as the Simulium larvae were observed. Any aquatic fauna observed were grouped as either vertebrate or invertebrate. The vertebrate aquatic fauna were identified with reference to Temegne and Momo [25] while the invertebrate aquatic fauna were identified using a key by Danladi et al. [26].

Data analysis
Fly collection data were entered in a template created in Epi info version 3.5.3 (https:// www. cdc. gov/ epiin fo/ index. html), exported to Microsoft Excel and then later to SPSS version 24 for analysis. The different entomological indicators generated were biting densities, daily biting rate, monthly biting rate, parous rates infection rates, infective rates and monthly transmission potential. The chi-square test for trend was used to establish whether there was any association among parous rate, infection rate and infective rate.
Entomological indices were calculated using the following formulae:

Identification of breeding sites
Twenty-two breeding sites from 13 communities (Berenge, Betenge, Small massaka, Lokando, Big butu, Small butu, Big massaka, Kumukumu, Bombele, Bobanda, Kwakwa, Nake and Marumba 1) in both the control and study arms were prospected based on accessibility (Fig. 1a, b). Nine of these sites from seven communities (Berenge, Betenge, Small Massaka, Big Massaka, Bombele, Kwakwa and Marumba 1) had larvae (productive) at the time of prospection. Breeding sites in Bombele and Betenge (River Meme), Big Massaka (River Uve), Kwakwa and Marumba I (River Mbile) and Small Massaka (River Ka) were identified as the main breeding sites of the study area; as such, fly and larvae collection points were set up at these sites. In the case of larval density, substrates were not calibrated but on average about ten larvae were found per substrate. Simulium damnosum was found to be the only anthropophagic species in the study area.

Susceptibility test results
At a concentration between 0.5 and 0.1 mg/l, a mortality of 100% was recorded. It was observed that as the concentration of temephos decreased (from 0.05 to 0.00025 mg/l), mortality of larvae also decreased uniformly (from 12 to 98.7%) as seen in Table 1. Thus, S. damnosum larvae in these areas demonstrated good susceptibility to temephos (Abate 500EC).
Dosage-mortality regression curve was plotted for susceptibility testing (Fig. 2). The control mortality was set to a concentration of 0.0001 mg/l as taking a logarithm of 0 is undefinable. This shows that the concentration of temephos required to kill 50% of larvae (EC50) is 0.005 mg/l and the concentration required to kill 95% of larvae (EC95) is 0.041 mg/l. The Hill slope (steepness of curve) was not constrained and included the control mortality.

Identification of cytospecies in the Meme River Basin
A total of 328 larvae were collected for cytotaxonomy with 40 from Bombele, 241 from Big Massaka and 47 from Kwakwa. Sixty larvae chosen for chromosome preparation due to their large size and white histoblast. This included 13 from Bombele, 29 from Big Massaka and 18 from Kwakwa. Chromosome preparations of 60 larvae were examined to identify high-quality images where the banding patterns on the chromosome were distinctive and clear. Additionally, some preparations were removed if the chromosomes were tightly coiled or broken. This led to the cytotaxonomic analysis of 15 larvae; 9 S. squamosum subcomplex larvae were found, which were three times the larvae found for any other subcomplex ( Table 2). Four larvae from the S. squamosum subcomplex could not be identified as either male or female. The cytospecies S. soubrense (Beffa form) was observed but has not been previously described in Cameroon (Fig. 3, in blue).

Entomological indicators of O. volvulus transmission
Entomological indices of O. volvulus transmission in the study site are found in Table 3. A total of 13,633 flies were collected and 4033 were dissected. Of the flies dissected, 1455 were parous, giving a parous rate of 36.1%; 224 were infected (infection rate 5.5%) and 64 were infective giving an infective rate of 1.6%. Kwakwa had the highest parous rate (43.7%) while Marumba I recorded the lowest parous rate (17.8%). On the other hand, Bombele had the highest infection rate (10%) while Betenge had the lowest infection rate (1.4%).

Biting density
More flies were caught during the afternoon hours with the peak biting hours for most of the communities between 1 and 3 p.m. This coincides with the peak farming hours. The most flies per hour were caught at Bombele with 1177 flies caught between 2 and 3 p.m. Biting density decrease towards the evening period (Fig. 4).

Daily biting rate (DBR) and monthly biting rate (MBR)
The DBR and MBR for the various collection sites are shown in Table 4. The highest daily and monthly biting rates were recorded at Kwakwa (1311 and 40618 respectively) while the lowest daily and monthly biting rate was recorded at Betenge (142 and 4402 respectively). Figure 5 shows that there was some trend in parasitological indicators. Bombele with the highest parous rate also recorded the highest infection rates and eventually one of the highest infective rates. On the other hand, Betenge had a very high parous rate but recorded the lowest infection rate. As such, a chi-square test of

Fig. 2
Dosage-mortality regression curve of concentrations. The control mortality was set to a concentration of 0.0001 mg/l as taking a logarithm of 0 is undefinable. The Hill slope (steepness of curve) was not constrained and included the control mortality trend indicated that there was no statistically significant association (Chi-square test, x 2 = 2.332, df = 1, P-value = 0.127) among parous rate, infection rate and infective rate.

Survey of some non-target aquatic fauna
The aquatic fauna found in the Meme River Basin were the same in all the study sites at the time of survey. This included two species of fish: Cyprinus (Mbanga  Fig. 3 Observed cytospecies of S. damnosum in the Meme River Basin. The cytospecies S. soubrense (Beffa form) was observed but has not been previously described in Cameroon (in blue) fish) and Clarias sp. (African catfish). Other aquatic fauna observed were crabs and tadpoles. The presence of tadpoles indicated that frog species were also present though no frogs were captured. During the collection of S. damnosum larvae, aquatic Coleoptera (beetle) and the larvae of damsel flies (Odonata: Zygoptera) were also observed on the same substrate.

Discussion
This study was designed to assess entomological indicators of onchocerciasis transmission and the susceptibility of Simulium larvae to temephos prior to the implementation of ground larviciding as an alternative treatment strategy for the acceleration of onchocerciasis elimination in the Meme River Basin. To realise this, we mapped the In this study, exposure of black fly larvae to decreasing concentrations of larvicide (from 0.5 to 0.0005 mg/l) resulted in decreasing mortality of S. damnosum larvae. One hundred percent mortality was achieved at concentration exposures of between 0.5 and 0.1 mg/l and we determined EC 50 of 0.005 mg/l. We therefore conclude that Simulium larvae are susceptible to ABATE 500 EC and this insecticide is effective for use in ground larviciding to accelerate the elimination of onchocerciasis in the study area. This is in line with the study carried out by Kalinga et al. [27] where there was 100% mortality of Simulium larvae to temephos between 0.129 and 0.34 mg/l pre-and 0.144-0.211 mg/l (95% CI, P < 0.05) post-treatment. The concentrations that were lethal to S. damnosum larvae in this study fall within the standard diagnostic dose of ≤ 0.4 mg/l for susceptible S. damnosum (s.l.) populations. This also shows the rapid activity of temephos, which has been shown to be highly efficacious against S. damnosum (s.l.) larvae at concentrations giving 0.05 mg/l at high river discharges (> 25 cubic m/s) and at 0.1 mg/l at low discharges (when insecticide carry is reduced). This is an indication that larvae from the Meme River Basin are highly susceptible to temephos.
Previously within Cameroon, there were eight cytospecies of S. damnosum recorded in the literature [20]. This record gives the most cytospecies that have been recorded in one country in West Africa [28]. This could be because Cameroon is situated in the Gulf of Guinea, which is known for its high biodiversity [29] with many different cytospecies occurring in proximity to different subcomplexes. In this study, the cytospecies S. soubrese (Beffa form) was found in two breeding sites (Kwakwa and Big Massaka). Notably, there have been no published studies that have recorded this cytospecies in Cameroon. However, this cytospecies has been recorded in Nigeria, which is a neighbouring country to Cameroon precisely in the Jos Plateau, which is 500 km from the Meme River Basin, and the Guarara River, which is 600 km away from the Meme River Basin [20]. The addition of S. soubrense (Beffa form) gives a record of nine cytospecies of S. damnosum found in Cameroon (Fig. 3).
There has been a decrease in infected flies from 12.9% in 2015 by Wanji et al. [9] to 5.5%. There has also been a decrease in infectivity from 3.9% in 2015 [9] to 1.6% The availability of fast-flowing rivers in this basin contributes to the large numbers of black flies necessary for high transmission. In this pre-intervention baseline study, biting density was up to 36 bites/person/day, highlighting the need for a focal vector control [30].
In areas of high pre-intervention baseline infection endemicity, it has been suggested that vector control could be used as a complementary intervention strategy [31]. Modelling results using EPIONCHO and ONCHOSIM [32] suggest that in such settings vector control could be used in conjunction with ivermectin MDA to enhance effectiveness or to consolidate the gains made towards the path of onchocerciasis elimination. The aquatic fauna found in the Meme River Basin were similar in all the study sites. Both vertebrate aquatic fauna and invertebrate aquatic fauna were observed. It is expected that this aquatic fauna will not be affected since temephos is relatively non-toxic to fish and other aquatic vertebrates [33].
This study is limited in that it failed to collect quantitative pre-control data for non-target fauna, which would have been compared with post-quantitative data for the sake of environmental impact assessment.

Conclusions
Onchocerciasis is being actively transmitted within the Meme River Basin. Simulium damnosum larvae in this river basin are susceptible to temephos and there are nine cytospecies of S. damnosum complex within the basin. Non-target fauna observed included fishes, frogs, crabs, and insects. Beside treatment with ivermectin, vector control through ground larviciding may complement and accelerate onchocerciasis elimination in the study area.


s2fieldsofstudy: Biology Medicine

text:
Iodine-125 Seeds Inhibit Carcinogenesis of Hepatocellular Carcinoma Cells by Suppressing Epithelial-Mesenchymal Transition via TGF-β1/Smad Signaling

To investigate the radioactive iodine-125 (I-125) seed on migrating and invading of hepatocellular carcinoma (HCC) cells and its mechanism, the irradiation of PLC and Huh7 cells was carried out with I-125 seeds in vitro. Cell counting kit 8 assay was employed to measure cell viability. Cell migration was evaluated by using wound-healing assay. Cell invasion was detected by Transwell assay; RT-PCR and Western blot were used for the detection of the mRNA and proteins of TGF-β1 signaling pathway-related genes. The viability of PLC and Huh7 cells declined in a dose-dependent manner with increasing irradiation from 0 Gy, 2 Gy, 4 Gy, and 6 Gy, to 8 Gy, respectively. The IC50 of PLC and Huh7 cells were 6.20 Gy and 5.39 Gy, respectively, after 24 h of irradiation. Migration and invasion abilities of I-125 group cells were greatly weakened (P < 0.05) comparing with the control group. According to the outcomes of RT-PCR and WB, I-125 seed irradiation significantly inhibited the mRNA and protein expression of N-cadherin, vimentin, TGF-β1, p-Smad2/3, and Snail. But the mRNA and protein expressions of E-cadherin were enhanced. Rescue experiment demonstrates that TGF-β1 activator could reverse the inhibitory effects of I-125 on invasion and migration of cells. The results of in vivo experiments further verified that the I-125 seeds can inhibit the proliferation and TGF-β1 of xenographed PLC cells. In conclusion, I-125 seeds restrain the invasion and migration of HCC cells by suppressing epithelial to mesenchymal transition, which may associate with the inhibition of the TGF-β1 signaling.

Introduction
As one of the most common malignant tumors across the world, hepatocellular carcinoma (HCC) is at the forefront in both incidence rate and mortality rate [1,2]. Surgical resection is still the first option for the stage I or II of HCC. However, typical clinical symptoms can hardly be found in the patient in the early stage of HCC, and most of them were in the middle and late stages when diagnosed. About 70% patients lost the chance to have surgery when they are diagnosed with HCC [1]. Sorafenib, a multienzyme inhibitor, are somehow effective to HCC. But what needs to be mentioned is its drug resistance [3]. At present, there is no effective treatment for advanced HCC [4]. Metastasis of cancer cells mainly explains the failure of treatment, and it is also one of the biggest challenges in the treatment of HCC.
Epithelial-mesenchymal transition (EMT) means the process that epithelial-like cells transform into mesenchymal-like cells under the stimulation of various factors, which has been considered as essential for tumor cell metastasis [5]. The epithelial cells lost polarity after EMT, which leads the links between cells to be interrupted; the movement of cells are enhanced. Finally, the tumor cells invade surrounding tissues and even metastases to other organs [6]. There are various signaling pathways regulate EMT, such as the Wnt/β-catenin pathway, TGF-β1 pathway, and NF-κB Pathway [5]. The TGF-β1 pathway is one of the classical signaling pathways, functioning vital in the occurrence and development of tumor [7]. A large number of studies showed that the TGF-β1 signaling pathway regulates EMT of a variety of tumors and promotes the metastasis of tumor cells [7,8].
With endogenous radiotherapy, I-125 radioactive seed implantation possesses exact curative effects, including high precision, strong conformability, and repeatability, which lead to fewer complications [9,10]. The I-125 seed is one of the most used particle sources, extensively applied in treating different solid tumors. Previous studies have shown that I-125 seed implantation therapy can greatly enhance the survival rate of patients suffering from advanced HCC, especially in those with portal vein tumor thrombus [11,12]. Also, the therapy can be used for other cancer patients who had failed or recurred [13].
Nevertheless, the mechanism of I-125 seeds in treating HCC is not fully elucidated, and especially, the effects of I-125 seeds on the EMT and TGF-β1 signaling pathway in HCC cells have not been investigated. In this study, we explored the function of correct dose of I-125 seed, which may generates certain degree of reactive oxygen species (ROS) inside cells, and in turn to trigger the process of anti-invasion/antimetastasis of HCC by downregulating EMT through the TGF-β1/Smad signaling pathway. We may provide an experimental basis for promoting the I-125 seed implantation therapy and using it as a more effective therapy for HCC.  (Figure 1(a)) were set on the surface of the plate within the appliance, and the seed plate with seeds was set on the support plate above the cell dish. Distance from the bottom of cell dish to the bottom of the seed plate was 5 mm. Those devices were placed in a square container made of 3 mm thick lead, and the wall of the container has four ventilation holes. Subsequently, when in use, 50 I-125 seeds are placed in a square with a side length of 50 mm evenly, and the transverse and longitudinal distance between seeds is 5 mm (Figure 1(b)). The initial activity of iodine 125 seeds was 0.8 mCi. The initial average dose and the dose distribution of each point on the cell dish were calculated by using Monte Carlo model. The treatment planning system (TPS) and the film calibration radiation dose method are used to evaluate the uniformity of the radiation dose distribution at different points on the bottom of the cell dish (Figures 1(c) and 1(d)). The total radiation dose within a specific exposure time or the time (t) required to reach a specific radiation dose can be calculated by the following formula [14]:

Materials and Methods
where D 0 is the initial dose rate. T is a half-life, equal to 1425.6 hours (59.6 days). λ is the decay constant of iodine 125 seeds, and t is the irradiation time (hours).

I-125
Seed Irradiation on PLC and Huh7 Cells. I-125 seed irradiation at a dose of 0, 2, 4, 6, and 8Gy was used to treat PLC and Huh7 cells, respectively. During the irradiation, the module was always placed in the 37°C cell incubator. No treatment for the control group, the I-125 group was treated with 4 Gy irradiation, and I-125+TGF-β1 group was treated with 4 Gy irradiation and TGF-β1 activator (10 μg/ml) (MedChem Express, Cat. SRI-011381, Monmouth, NJ, USA).
3.2. Cell Viability Assay. PLC and Huh7 cell lines of HCC have been shown to be well responded to reactive oxygen species (ROS), and I-125 irradiation universally generates ROS in the cellular mitochondria. Cell viability of PLC and Huh7 cells was conducted using a CCK8 kit (Beyotime, China). Briefly, PLC and Huh7 cells were collected after treating with varied doses (0, 2, 4, 6, and 8 Gy) irradiation. Subsequently, cell density was adjusted to 4 × 10 4 cells/ml; then, 100 μl/well cell suspension was added into a 96 well plate, respectively, and incubated for 24 h at 37°C with 5% CO 2 . Three multiple holes were set in each group. Each well was added with CCK8 (10 μl) solution, and the measurement of absorbance at 450 nm was made.
3.3. Wound Healing Assay. PLC and Huh7 cells were pretreated with 0 Gy and 4 Gy dose irradiation, respectively, at 80% confluence and scraped through the monolayer cells with a 10 μl micropipette tip to make wounds. After being washed with PBS, the cancer cells were cultured in serumfree medium. A microscope was used to visually observe and measure the wound distance (Olympus, Japan), at immediately and 24 h later.

Migration and Invasion
Assay. Different doses (0.4Gy) of irradiation were used to treat PLC and Huh7 cells; migration and invasion assay was conducted following the commercial protocol. Briefly, cell density was adjusted to 1 × 10 6 cells/ml. Later, the upper chamber of Transwell (Corning, USA) was added with 200 μl of cell suspension, and the 24-hour incubation of the lower chamber added with 500 μl of medium with 10% FBS was made at 37°Cwith 5% CO 2 . The culture medium was discarded after being washed with PBS; cells were fixed with 4% of paraformaldehyde and stained with 0.1% of crystal violet. The random selection of five fields for photographing and counting was made. The difference between migration assay and invasion assay was whether the bottom of Transwell is pretreated with diluted Matrigel matrix, or without (Corning, New York, USA).
3.5. Western Blotting Assay. The extraction of total protein was made with RIPA lysate buffer and protease inhibitor, and its concentration was determined by bicinchoninic acid (BCA) assay kit (Beyotime, China). 10% SDS-PAGE (Beyotime, China) separated protein which then transferred to the PVDF membrane. Later, 5% skimmed milk at room temperature was used to block the membrane for 1 h. Following, primary antibodies against E-cadherin, N-cadherin, vimentin (CST, Danvers, MA, USA), TGF-β1, Smad2, and Smad3 (Proteintech Group, USA), p-Smad2, and p-Smad3 (Affinity, Affbiotech Group, USA) were added and incubated overnight at 4°C. Horseradish peroxidase-labeled second antibody (CST, Danvers, MA, USA) was incubated for 1 hour. Protein was detected by enhanced chemiluminescence.
3.6. RT-PCR. After extracted by TRIzol (Takara, Japan), total RNA was transcribed into cDNA reversely. Then, the QRT-PCR was made with SYBR Green fluorescent dye method using cDNA as template. The reaction system was SYBR Green 10 μl; all primers were ordered from Sangon Biotech (Shanghai, China) 0.8 μl, ddH 2 O 7.2 μl, and cDNA 2 μl; and the total system was 20 μl. The qPCR procedure was as follows: 95°C 30 s; 95°C 5 s, 60°C 30 s, 72°C 15 s, 40 cycles; 95°C 15s, 60°C 1 min, and 95°C 15 s. The expression difference of each gene was compared by 2 -ΔΔCT relative quantitative method ðΔCT : the CT value of the target gene − CT value of GAPDH Gene,ΔΔCT value = theΔCT value of the experiment group − theΔCT value of the control groupÞ. The expression of gene in control group is 1, and the relative expression of gene in the experimental group is 2 -ΔΔCT . The primer sequences are shown in Table 1.

Immunofluorescence Assay.
In short, the required cells were seeded on the glass slide. The cells seeded on the glass slide were washed after 24 h of incubation, fixed, and infiltrated for 10 minutes and then blocked with 5% of bovine serum albumin (BSA) for 30 minutes. Subsequently, the antitarget protein antibody E-cadherin, N-cadherin, TGF-β1, and p-Smad3 solution was added and incubated at 4°C overnight. Alexa Fluor 549-conguated secondary antibody (Invitrogen, USA) was used. 4′, 6′-diamino-2-phenylindole (DAPI) (Invitrogen, USA) was adopted to stain cell nuclei. The images were recorded and photographed with a fluorescence microscope (Olympus, Japan).

Nude Mouse Model and Immunohistochemistry Assay.
Ten nude mice (4-6 weeks old) were purchased from local 3 Disease Markers university facility. The subcutaneous inoculation of PLC cells (1 × 10 7 cells/ml) was made to these nude mice. About 10 days later, the subcutaneous transplanted tumor in nude mice was about 250-300 mm 3 . The nude mice were randomly fallen into the control group and I-125 group, with 5 nude mice in each group. In the I-125 group, a 0.8 mCi I-125 seed was implanted into each subcutaneously transplanted tumor center. But the control group was untreated. Then, the length (a) and width (b) of the tumor was measured every four days, and the calculation of tumor volume (V) as the following formula: V = 1/2 a.b 2 was made. After 28 days, mice were euthanized, and the collection of tumors was conducted for further study. The ethics committee of the First Affiliated Hospital of Army Medical University approved all experimental protocols.
After being fixed with 4% paraformaldehyde, xenograft tumor tissues were embedded in paraffin and sectioned. Then, the antibodies against TGF-β1 were stained with DAB horseradish peroxidase chromogenic Kit (Beyotime, China) at room temperature. After being stained with hematoxylin for 30 s, sections were dehydrated and fixed and then sealed with neutral glue. And a microscope was used to observe and photograph all stained images (Olympus, Japan).
3.9. Statistical Analysis. The experimental data and image preprocessing were analyzed by SPSS24.0 (IBM, USA) and GraphPad prism 6.0 software. The significant distinction between the two groups was analyzed by employing the independent t-test, and the distinctions among the groups were analyzed by adopting one-way ANOVA. P < 0:05 or P < 0:01 was of statistical significance.

I-125
Seed Irradiation Suppresses HCC Proliferation, Migration, and Invasion. The dose of I-125 for irradiation was chosen based on growth curve of PLC and Huh7 in normal control and I-125 incubation based on their IC50 in order to see the direct effect of I-125 on HCC proliferation, which shows the cell proliferation decreased as increased radiation dose (Figure 2(a)); the IC50 of PLC and Huh7 cells was 6.20 Gy and 5.39 Gy at 24 hours after irradiation, respectively. In order to observe the effects of I-125 seed on migration and invasion of PLC and Huh7 cells, 4 Gy was used for subsequent experiments. By visual measurement of the confluence cell edges between the strip, and comparing with the control group, the migration ability of PLC and Huh7 cells was weakened after I-125 seed irradiation, and the relative migration ability was reduced to 53% and 65%, respectively (Figures 2(b) and 2(c)). In addition, the result of transwell assay indicated that the I-125 seed irradiation dramatically inhibited the invasion of PLC and Huh7 cells (P < 0:01, Figure 2(d)). All above suggest that I-125 seed irradiation inhibits cell proliferation, migration, and invasion of HCC.

I-125 Seed Irradiation Inhibited EMT of PLC and Huh7
Cells. The morphology of polyhedral fibroblasts was replaced by typical cobblestone like epithelioid cells after I-125 seed irradiation (red arrow, Figure 3(a)), and I-125 seed irradiation also upregulated the protein expression of E-cadherin in PLC and Huh7 cells (Figure 3(b)). Moreover, the mRNA expression of E-cadherin in PLC and Huh7 cells were relatively increased, respectively; however, the mRNA relative expression of N-cadherin and vimentin were decreased (Figure 3(c)). Interestingly, the results of Western blotting assay were similar to the outcomes of RT-PCR (Figures 3  (d) and 3(e)). So, these findings suggest that I-125 seed irradiation can inhibit or reverse the occurrence of EMT in PLC and Huh7 cells.

I-125 Seed Irradiation Inhibits TGF-β1 Signaling
Pathway. The gene expression of TGF-β1, Smad2, and Snail was analyzed according to RT-PCR assays (Figure 4(a)), and the protein expression was detected by WB (Figure 4(b)) and immunofluorescent assay (Figure 4(c)); the levels of TGF-β1 were reduced in PLC and Huh7 cells after I-125 seed irradiation. As I-125 seed irradiation decreased the expression of TGF-β1, p-Smad2, and p-Smad3 (Figures 4(b), 4(d), and 4 (e)) even though did not significantly affect the protein

Disease Markers
Smad3 in the I-125+TGF-β1 group were greatly reduced but that of E-cadherin was increased after subtracts of I-125 group. Simultaneously, the results of the IF assay ( Figures 5(d), 5(e), and 5(f)) were consistent to those of the WB assay. These results suggest that TGF-β1 activator can reverse the I-125 seed irradiation inhibitory effect on the EMT and TGF-β1 signaling pathway in vitro.

Anticancer Role of I-125
Seed Irradiation In Vivo. The size of tumors in I-125 implanted nude mice was increased compared with the control group (Figures 6(a)-6(c)), that is, I-125 seed irradiation greatly restrained the rise of tumor volume and tumor weight, and the difference was statistically significant from 16 days after I-125 seed implantation. Consistently, H & E staining showed that the cell density of the I-125 group was greatly weakened (less emerged), and the nuclear fragmentation and necrosis were significantly increased by comparing with the control group ( Figure 6(d)). In addition, I-125 seed irradiation attenuates the expression of TGF-β1 in xenografts (Figures 6(e) and 6 (f)), which also indicate the I-125 influence the TGF-β1-triggered EMT signaling. Even though TGF-β1 is very decisive in initiation of cell proliferation, migration, and EMT, but it needs to further study because dose, timing, and many other factors in vivo may influence the pathway much more than the study in vitro.

Discussion
Radiotherapy is one of the three commonly used methods for cancer treatment, and about 60% of cancer patients receive radiotherapy. However, due to severe side effects of traditional radiotherapy, Barcelona guidelines do not recommend radiotherapy for HCC. Radioactive seed implantation therapy is to implant I-125 seed into tumor under the guidance of imaging in order to kill tumor cells and to achieve the purpose of tumor treatment. Compared with traditional radiotherapy, it has the characteristics of high target rates and sharp peripheral dose drop, which can significantly decrease the incidence of adverse effects. In addition, several studies have shown that continuous low-dose radiotherapy with I-125 seed has stronger biological effects than conventional high-dose radiotherapy [16][17][18]. The merits of this therapy makes it a comprehensive treatment options for  Figure 5: TGF-β1 activator reverse I-125 seed irradiation inhibitory effects on the EMT and TGF-β1 signaling pathway. TGF-β1 activator reverses the inhibitory role in migration (a) and invasion (b) of PLC and Huh7. TGF-β1 activator reverses the roles in the EMT and TGF-β1 signaling pathway-related markers by WB assay (c) and immunofluorescence assay for PLC (d) and for Huh7 (e) and their relative IOD (integrated optical density) per cell were plotted, respectively (f). The average number of invasive cells was calculated by counting then number of cells in 5 fields per chamber. Data was expressed as mean ± SD (n = 3). The difference was of statistical significance (P < 0:05).
9 Disease Markers patient with advanced HCC. Nevertheless, the mechanisms of radioactive particles in treating HCC keep largely elusive. In this study, we found that the I-125 seed irradiation inhibited the proliferation, invasion, and migration of HCC cell lines. Moreover, the I-125 seed irradiation inhibited EMT of HCC cell lines by partially suppressing TGF-β signal pathway. Also, the I-125 seed irradiation inhibited the growth of HCC in vivo.
Although I-125 radioactive is widely believed to have a gamma ray that triggers mitophagy and a number molecular chain reactions by elevating mitochondria reactive oxygen species (ROS) [19], a group of key signaling molecules plays important roles in the progression of inflammatory disorders as well as anti-inflammation in a dose-dependent manner; thus, antitumor effects of I-125 seed with certain strength of radioactivity can be achieved by inhibiting cell 10 Disease Markers proliferation, inducing apoptosis and autophagy, and arresting the cell cycle [20][21][22]. To better understand the I-125 seed effects on the invasion ability of PLC and Huh7 cells, the viability of PLC and Hu7 cells were detected by using a CCK8 assay; IC50 of 24 h after I-125 irradiation was counted out. The results are consistent with previous studies [23,24]. However, we found that I-125 seed irradiation significantly restrained the proliferation of cancer cells, and this restraint effect was dose-dependent. Moreover, IC50 of PLC and Huh7 cells was 6.20 Gy and 5.39 Gy at 24 hours after irradiation. Furthermore, our results are slightly different from the IC50 reported previously. The possible reason is that different types of tumor cells have different radiosensitivity. According to the previous research, high-dose radiation drives the migration and invasion of tumor cells. In contrast, continuous low-dose radiation of I-125 seed inhibits the migration and invasion of tumor cells [17]. The outcomes indicated I-125 seed irradiation significantly inhibited the migration and invasion of HCC cells below IC50. According to the outcomes, the inhibitory effect of I-125 seed on the migration and invasion of PLC and Huh7 cells is brought not only by radioactive cytotoxicity but also by DNA dam-age or modification on the site of specific regulation of genes transcription [25]. Our results were largely consistent with previous studies, and the change of the gene expression by I-125 seed irradiation inhibited the invasion of nasopharyngeal carcinoma and gastric cancer [26,27].
EMT is considered to be important for tumor cells acquiring the ability of migration and invasion [5]. Through the EMT process, the expression of epithelioid properties related proteins (such as E-cadherin) decreased and the expression of interstitial properties-related proteins (such as N-cadherin and vimentin) increased [6]. According to the past researches, I-125 seed irradiation downregulates the expression of N-cadherin while upregulating the expression of E-cadherin in lung cancer cells and glioma cells, indicating that the I-125 seed irradiation inhibits the occurrence of EMT in those cells [10,28]. In our study, the morphology of EMT has been observed, and shown that I-125 also promotes the expression of E-cadherin and reduces the expression of N-cadherin and vimentin, suggesting that I-125 seed irradiation inhibits the EMT of PLC and Huh7 cells.
TGF-β1 belongs to the TGF-β family, functioning vital in tumor cell proliferation, differentiation, invasion, and apoptosis [29]. TGF-β1 binds to TGF-β II receptor on cell membrane to convert Smad2 and Smad3 into active p-Smad2 and p-Smad3. Subsequently, p-Smad2 and p-Smad3 enter the nucleus to regulate the transcription and expression of corresponding target genes, which induce the EMT and enhance the migration and invasion of tumor cells in the final [7,30]. According to the past researches, radiotherapy inhibits the expression of TGF in tissues, and the inhibitory effect of low-dose radiotherapy is stronger than that of conventional high-dose radiotherapy [31,32]. The outcomes show that I-125 seed irradiation also restrained the expression of TGF-β1. In addition, we found that I-125 seed irradiation inhibited the expression of p-Smad2 and p-Smad3, while the expression of Smad2 and Smad3 was not impacted by I-125 seed irradiation, indicating that I-125 seed irradiation restrained the activity of the TGF-β1/Smad pathway.
However, whether the TGF-β1/Smad signaling pathway function is vital in inhibiting the invasion and metastasis of PLC and Huh7 cells by I-125 seed remains to be further studied. According to the past studies, TGF-β1 activator can activate the TGF-β1 signaling pathway, promote epithelial-mesenchymal transition, and then promote tumor invasion and metastasis [8,33]. Therefore, we studied the role of the TGF-β1 activator in the inhibitory of the I-125 seed-induced cell migration. After adding 10 ng/ml TGF-β1 activator, we found that the number of cell migration and invasion in the I-125+TGF-β1 group was highly larger than that in the I-125 group. At the same time, we found that by comparing with the I-125 group, the expression of TGF-β1 and p-Smad3 in the I-125+TGF-β1 group was greatly enhanced, while the expression of the E-cadherin was decreased. These results indicate that the TGF-β1 activator can at least partially block the I-125 seed irradiation-induced cell migration inhibition by restoring p-Smad3 protein level and further prove that the I-125 seed irradiation inhibits cell migration of HCC cells by suppressing the TGF-β1/Smad signal. We revealed a new mechanism of the I-125 seed irradiation inhibiting the development of HCC.

Conclusion
This study shows that radioactive I-125 seed can restrain the proliferation, migration, and invasion of HCC cells. The inhibitory effect of I-125 seed on EMT was attributed to the suppressing of the TGF-β1 signaling pathway (Figure 7: graphical abstract).

Data Availability
All data generated or analyzed during this study are included in this article.

Ethical Approval
All experimental protocols were approved by the ethics committee of the First Affiliated Hospital of Army Medical University (approval number: AMUWEC20210583).

Conflicts of Interest
The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.