Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus.

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

Construction and characterization of recombinant senecavirus A expressing secreted luciferase for antiviral screening.

Senecavirus A (SVA) is a newly identified picornavirus associated with swine vesicular disease and neonatal mortality. The development of an SVA incorporating an exogenous reporter gene provides a powerful tool for viral research. In this study, we successfully constructed a recombinant SVA expressing Gaussia Luciferase (Gluc), termed rSVA-Gluc. The growth kinetics of rSVA-Gluc in BHK-21 cells were found to be comparable to those of the parental virus, and Gluc activity paralleled the virus growth curve. Genetic analysis revealed stable inheritance of the inserted reporter protein genes for at least six generations. We evaluated the utility of rSVA-Gluc in antiviral drug screening, and the results highlighted its potential as an effective tool for such purposes against SVA. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available on request from the corresponding author.

Long-term co-circulation of multiple influenza A viruses in pigs, Guangxi, China.

Influenza A viruses (IAVs) pose a persistent potential threat to human health because of the spillover from avian and swine infections. Extensive surveillance was performed in 12 cities of Guangxi, China, during 2018 and 2023. A total of 2540 samples (including 2353 nasal swabs and 187 lung tissues) were collected from 18 pig farms with outbreaks of respiratory disease. From these, 192 IAV-positive samples and 19 genomic sequences were obtained. We found that the H1 and H3 swine influenza A viruses (swIAVs) of multiple lineages and genotypes have continued to co-circulate during that time in this region. Genomic analysis revealed the Eurasian avian-like H1N1 swIAVs (G4) still remained predominant in pig populations. Strikingly, the novel multiple H3N2 genotypes were found to have been generated through the repeated introduction of the early H3N2 North American triple reassortant viruses (TR H3N2 lineage) that emerged in USA and Canada in 1998 and 2005, respectively. Notably, when the matrix gene segment derived from the H9N2 avian influenza virus was introduced into endemic swIAVs, this produced a novel quadruple reassortant H1N2 swIAV that could pose a potential risk for zoonotic infection.

The impact of ammonia and microcystin-LR on neurobehavior and glutamate/gamma-aminobutyric acid balance in female zebrafish (Danio rerio): ROS and inflammation as key pathways.

Ammonia and microcystin-LR (MC-LR) are both toxins that can be in eutrophic waters during cyanobacterial blooms. While previous studies have focused on the effects of ammonia exposure on fish neurobehavioral toxicity, little attention has been given to the effects of MC-LR and combined exposures to both. This study exposed adult female zebrafish to ammonia (30 mg/L) and MC-LR (10 µg/L) alone and in combination for 30 days to investigate their neurotoxic effects and underlying mechanisms. Behavioral results showed that exposure to ammonia and MC-LR, both alone and in combination, led to decreased locomotor activity and increased anxiety in fish. Histomorphological analysis revealed the formation of thrombi and vacuolization in the brain across all exposure groups. Exposure to ammonia and MC-LR resulted in significant increases in MDA contents, decreases in Mn-SOD activities, and alterations in GSH contents compared to the control. Single and combined exposure to ammonia and MC-LR also induced the release of inflammatory factors (IL-1ß and TNF-α) by activating the NOD/NF-κB signaling pathway. Furthermore, both ammonia and MC-LR significantly changed the expression of genes related to the glutamatergic and GABAergic systems, elevated Glu and GABA contents, as well as increased the Glu/GABA ratio, indicating that a shift towards increased Glu levels. Overall, these findings suggested that exposure to MC-LR and ammonia, individually and in combination, could decrease locomotor activity and increase anxiety of female zebrafish. This was likely due to brain damage from over-activated ROS and the release of pro-inflammatory cytokines, which led to a disruption in the balance of glutamatergic and GABAergic systems. However, there was no significant interaction between MC-LR and ammonia in fish neurobehavioral toxicity.

Development of a monoclonal antibody specifically recognizing a linear epitope on the capsid protein of the emerging Group III Getah virus.

Getah virus (GETV) is an emerging mosquito-borne alphavirus that can infect horses, pigs and other animals. Given the public health threat posed by GETV, research on its pathogenesis, diagnosis and prevention is urgently needed. In the current study, prokaryotic expression systems were used to express the capsid protein of GETV. This protein was then used to immunize BALB/c mice in order to generate monoclonal antibodies (mAbs). Subsequently, hybridoma cells secreting a mAb (2B11-4) against the capsid protein were obtained using the hybridoma technique. A B cell linear epitope, 18-PAYRPWR-24, located at the capsid protein's N-terminal region was identified using western blotting analysis with the produced mAb, 2B11-4. Sequence alignment indicated that this epitope was highly conserved in group III (GIII) strains of GETV, but varied among the other genotypes. Western blotting showed that mAb 2B11-4 could discriminate Group III GETVs from other genotypes. This study describes the preparation of a mAb against the GETV capsid protein and the identification of the specific localization of B-cell epitopes, and will contribute towards a better understanding of the biological importance of the GETV capsid protein. It will also pave the way for developing immunological detection methods and genotype diagnosis for GETVs.

Construction of and evaluation of the immune response to two recombinant pseudorabies viruses expressing the B119L and EP364R proteins of African swine fever virus.

African swine fever (ASF) is an infectious disease caused by ASF virus (ASFV), which is characterized by high infectivity, rapid onset of disease, and a high mortality rate. Outbreaks of ASFV have caused great economic losses to the global pig industry, and there is a need to develop safe and effective vaccines. In this study, two recombinant pseudorabies virus (PRV) strains, rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L, expressing the EP364R and B119L protein, respectively, of ASFV, were constructed by homologous recombination technology. Western blotting and immunofluorescence analysis showed that these foreign proteins were expressed in cells infected with the recombinant strains. The strains showed good genetic stability and proliferative characteristics for 20 passages in BHK-21 cells. Both of these strains were immunogenic in mice, inducing the production of specific antibodies against the expressed ASFV proteins while providing protection against lethal challenge with PRV. Thus, the recombinant strains rGXGG-2016-ΔgI/ΔgE-EP364R and rGXGG-2016-ΔgI/ΔgE-B119L could be used as candidate vaccines for both ASFV and PRV. In addition, our study identifies two potential target genes for the development of safe and efficient ASFV vaccines, provides a reference for the construction of bivalent ASFV and PRV vaccines, and demonstrates the feasibility of developing a live ASFV vector vaccine.

Construction and characterization of a full-length infectious clone of an emerging senecavirus A strain.

Senecavirus A (SVA) is an emerging virus that causes vesicular disease in pigs. Construction of a full-length SVA cDNA clone is crucial for understanding its replication and pathogenesis. Here, we successfully constructed a CMV-promoter-driven infectious cDNA clone of the SVA isolate SVA/GX/CH/2018, which we named rSVA GX01. Sequence comparison between the pSVA GX01 and the parental isolate (SVA/GX/CH/2018) revealed three single-nucleotide differences. Four-week-old piglets were experimentally infected with either the parental virus or the cloned virus. The results showed that the cloned rSVA GX01 displayed weak pathogenicity in 4-week-old pigs compared to the parental virus SVA CH-GX-01-2018. The infectious clone of SVA will serve as a valuable tool for studying the viral replication cycle and for functional analysis of the viral genome.

Reassortant H9N2 canine influenza viruses containing the pandemic H1N1/2009 ribonucleoprotein complex circulating in pigs acquired enhanced virulence in mice.

The reassortment between avian H9N2 and Eurasian avian-like (EA) H1N1 viruses may have potentially changed from avian-to-mammals adaptation. This study generated 20 reassortant viruses with the introduction of H1N1/2009 internal genes from EA H1N1 virus into H9N2 virus. 12 of these recovered the replication capability both in the lungs and turbinate samples. 10 of 12 obtained PA gene segments from the ribonucleoprotein (RNP) complexes of the EA H1N1 virus, and 3 exhibited extreme virulence. Specially, the combination of PB2, PA and NP genes could overcome the species-specific restriction in human cells. Analysis of the polymerase activities found that introduction of the PA gene resulted in increased polymerase activity. These findings indicated that RNP complexes from EA H1N1 virus could confer an adaptation advantage and high compatibility to avian H9N2 virus. This raises new concerns for public health due to the possible coexistence of H9N2 and EA H1N1 viruses in dogs.

A novel VP1-based enzyme-linked immunosorbent assay revealed widespread Enterovirus G infections in Guangxi, China.

Enterovirus G (EV-G) has recently been shown to affect weight gain and cause neurological symptoms in piglets. However, the serological investigation of EV-G is limited. In this study, we developed a novel serological detection method based on the structural protein, VP1 of EV-G. The intra-assay and inter-assay coefficient variations were 3.2-8.9% and 2.6-8.0%, respectively. There was no cross-reaction of the VP1-based enzyme-linked immunosorbent assay (ELISA) with antisera against the other known porcine viruses. In addition, a comparison was made with other methods including the developed indirect ELISAs based on VP2 and VP3 proteins and western blot (WB) analysis, which demonstrated the reliability of the novel method. Using the VP1-based ELISA, we carried out the first seroepidemiological survey of EV-G in China by testing 1041 serum samples collected from different pig farms in Guangxi from 2019 to 2021. Our results showed that 68.78% of the serum samples and 100% of the pig farms were positive for EV-G, with a relatively high incidence of seropositivity in pigs of different ages. This was specifically evident in fattening pigs and sows, which may suggest that the piglets have experienced an infection with EV-G during their growth process. Our data provide the first serological evidence of EV-G infections in pigs from China and reveal the widespread presence of EV-G infections in Guangxi, China.

Tunable frequency of a microwave mixed receiver based on Rydberg atoms under the Zeeman effect.

Researchers are interested in the sensor based on Rydberg atoms because of its broad testing frequency range and outstanding sensitivity. However, the discrete frequency detection limits its further employment. We expand the frequency range of microwaves using Rydberg atoms under the Zeeman effect. In such a scheme, the magnetic field is employed as a tool to split and modify adjacent Rydberg level intervals to realize tunable frequency measurement over 100 MHz under 0-31.5 Gauss magnetic field. In this frequency range, the microwave has a linear dynamic variation range of 63 dB, and has achieved a sensitivity of 11.72 µV cm-1Hz-1/2 with the minimum detectable field strength of 17.2 µV/cm.. Compared to the no magnetic field scenario, the sensitivity would not decrease. By theoretical analysis, in a strong magnetic field, the tunable frequency range can be much larger than 100 MHz. The proposed method for achieving tunable frequency measurement provides a crucial tool in radars and communication.

Molecular epidemiological and genetic characterization of pseudorabies virus in Guangxi, China.

Pseudorabies virus (PRV) is an important pathogen that can cause harm to the pig population. Since 2011, there have been a number of large-scale outbreaks of pseudorabies on Chinese farms where animals had been vaccinated with the Bartha-K61 vaccine. In order to understand the epidemiological trend and genetic variations of PRV in Guangxi province, China, 819 tissue samples were collected from swine farms where PRV infection was suspected from 2013 to 2019, and these were tested for infectious wild strains of PRV. The results showed a positive rate of PRV in Guangxi province of 28.21% (231/819). Thirty-six wild-type PRV strains were successfully isolated from PRV-positive tissue samples, and a genetic evolutionary analysis was performed based on the gB, gC, gD, gE, and TK genes. Thirty of the PRV strains were found to be closely related to the Chinese variant strains HeN1-China-2012 and HLJ8-China-2013. In addition, five PRV strains were genetically related to Chinese classical strains, and one isolate was a recombinant of the PRV variant and the vaccine strain Bartha-K61. Amino acid sequence analysis showed that all 36 PRV strains had characteristic variant sites in the amino acid sequences of the gB, gC, gD, and gE proteins. Pathogenicity analysis showed that, compared to classical PRV strains, the PRV variant strains were more pathogenic in mice and had a lower LD50. Taken together, our results show that wild-type PRV infections are common on pig farms in Guangxi province of China and that the dominant prevalent strains were those of the PRV variants. The PRV variant strains also had increased pathogenicity in mice. Our data will provide a useful reference for understanding the prevalence and genetic evolution of PRV in China.

Evaluation of packaging capacity at the genomic 2C/3A junction region in Porcine enterovirus G.

Porcine enterovirus G (EV-G) is endogenous to most pig farming countries worldwide. Reports that a papain-like protease (PLP) gene has been naturally inserted into the 2C/3A junction region of the EV-G genome, has increased the potential public health threats from this virus. We constructed a full-length infectious cDNA clone of EV-G, CH/17GXQZ/2017, in order to determine the packaging capacity at the 2C/3A insertion site. Subsequently, recombinants viruses containing the coding tags, GFP, iLOV and His at the 2C/3A junction region, were synthesized. The infectious virus was successfully rescued only with the insertion of the His-tag, which displayed similar virological and molecular properties to its parental strain. This study determined the packaging capacity of the 2C/3A insertion site, and it provides a practical tool for studying the functions and pathogenic mechanisms of EV-G in pigs.

Combined effects of microcystin-LR and rice straw-derived biochar on the hepatic antioxidant capacity of zebrafish: Insights from LC-MS/MS-based metabolomics analysis.

Microcystin-LR (MC-LR) produced by cyanobacteria blooms poses a serious risk to aquatic organisms. Rice straw-derived biochar (BC) is gradually being utilized as an effective adsorbent to remove water pollutants. In the present study, the combined toxicity of MC-LR and BC on hepatic antioxidant capacity and metabolic phenotype of zebrafish (Danio rerio) were conducted due to the increasing concern of eutrophication in aquatic environments. Female zebrafish were exposed to solutions of MC-LR (10 µg/L) and BC (100 µg/L) individually and in combination for 30 days. The results indicated that sub-chronic MC-LR exposure induced oxidative stress and metabolic disorders, with a significant elevation of several amino acids, glucose as well as unsaturated fatty acids. Metabolic pathway analysis showed that the ascorbate and aldarate metabolism and biosynthesis of unsaturated fatty acids were affected under MC-LR stress. Significantly increased MDA levels along with significantly decreased CAT and GPx activities were observed in the MC-LR group. Nevertheless, MDA levels, antioxidant enzyme activities, and the relevant gene expressions (cat1, nrf2a, HO-1, keap1a) returned to baseline in the co-exposure group. These findings revealed that MC-LR resulted in metabolic disorders of protein, sugar, and lipid related to energy production, and BC could relieve MC-LR-induced metabolic disorder and oxidative stress in the liver of zebrafish. However, the potential risk of BC-induced metabolic disorder should not be neglected. Our present results highlight the potential of BC as a tool for mitigating the negative impacts of MC-LR on aquatic organisms in blooms-contaminated water.

Microcystin-LR immersion caused sequential endocrine disruption and growth inhibition in zebrafish (Danio rerio) from fertilization to sexual differentiation completion.

Microcystin-LR (MC-LR) is a highly toxic congener and is also one of the most commonly found. Recent studies have demonstrated that MC-LR can disrupt growth and endocrine in fish, but how it works at the stage of the sex differentiation period had not been determined to date. In this study, zebrafish (Danio rerio) embryos were exposed to MC-LR (0 and 10 µg/L), and sampled at 14, 28, and 42 days post fertilization (dpf), respectively. The results demonstrated that MC-LR caused the growth inhibition of zebrafish at 42 dpf. The expression levels of genes related to the growth hormone/insulin-like growth factor (GH/IGF) and hypothalamic-pituitary-thyroid (HPT) axes, as well as the levels of hormone 3,5,3'- triiodothyronine (T3) and thyroxine (T4), were significantly decreased at all time points. A significant decrease in the ratio of testosterone and estradiol (T/E2) were detected at 28 and 42 dpf in MC-LR group along with changes in genes related to the hypothalamic-pituitary-gonadal (HPG) axis. The result of sex ratio showed that the percentage of females was up to 61.84%, indicating a estrogenic effect induced by MC-LR. The significant changes on hormone levels and gene transcripts occurred mainly in the stage of sex differentiation. The correlation analysis further suggested that key cross-talks among three endocrine axes may be the growth hormone releasing hormone (GHRH), transthyretin (TTR) and gonadotropin releasing hormone (GnRH) signaling molecules. Overall, our findings provide a new insight for understanding the mechanisms by which MC-LR affects fish growth and reproduction during gonadal development.

Near-field antenna measurement based on Rydberg-atom probe.

Current near-field antenna measurement methods are commonly based on metal probes, with the accuracy limited and hard to be optimized due to the drawbacks they suffered, such as large volume, severe metal reflection/interference and complex circuit signal processing in parameter extracting. In this work, a novel method is proposed based on Rydberg atom in the near-field antenna measurement, which can offer a higher accuracy due to its intrinsic character of traceability to electric field. Replacing the metal probe in near-field measurement system by Rydberg atoms contained in a vapor cell (probe), amplitude- and phase- measurements on a 2.389 GHz signal launched out from a standard gain horn antenna are conducted on a near-field plane. They are transformed to far-field pattern and agree well with simulated results and measured results by using a traditional metal probe method. A high precision in longitudinal phase testing with an error below 1.7% can be achieved.

Biological Characteristics and Pathogenicity Analysis of a Low Virulence G2a Porcine Epidemic Diarrhea Virus.

Since the outbreak caused by a porcine epidemic diarrhea virus (PEDV) variant in 2010, the current epidemic of PEDV genotype 2 (G2) has caused huge economic losses to the pig industry in China. In order to better understand the biological characteristics and pathogenicity of the current PEDV field strains, 12 PEDV isolates were collected and plaque purified during 2017 to 2018 in Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate genetic variations, and they were compared with the reported G2a and G2b strains. Phylogenetic analysis of the S protein showed that the 12 isolates were clustered into the G2 subgroup (with 5 and 7 strains in G2a and G2b, respectively) and that they shared 97.4 to 99.9% amino acid identities. Among them, one of the G2a strains, CH/GXNN-1/2018, which had a titer of 106.15 PFU/mL, was selected for pathogenicity analysis. Although piglets infected with the CH/GXNN-1/2018 strain exhibited severe clinical signs and the highest level of virus shedding within 24 h postinfection (hpi), recovery and decreased virus shedding were seen after 48 hpi, and no piglets died during the whole process. Thus, the CH/GXNN-1/2018 strain had low virulence in suckling piglets. Virus neutralizing antibody analysis showed that the CH/GXNN-1/2018 strain induced cross-protection against both homologous G2a and heterologous G2b PEDV strains as early as 72 hpi. These results are of great significance for understanding PEDV in Guangxi, China, and they provide a promising naturally occurring low-virulent vaccine candidate for further study. IMPORTANCE The current epidemic of porcine epidemic diarrhea virus (PEDV) G2 has caused huge economic losses to the pig industry. Evaluation for low virulence of the PEDV strains of subgroup G2a would be useful for the future development of effective vaccines. In this study, 12 field strains of PEDV were obtained successfully, and they were characterized from Guangxi, China. The neutralizing epitopes of the spike proteins and the ORF3 proteins were analyzed to evaluate antigenic variations. One of the G2a strains, CH/GXNN-1/2018, was selected for pathogenicity analysis, and it showed that the CH/GXNN-1/2018 strain had low virulence in suckling piglets. These results provide a promising naturally occurring low-virulent vaccine candidate for further study.

Replication of Porcine Astrovirus Type 1-Infected PK-15 Cells In Vitro Affected by RIG-I and MDA5 Signaling Pathways.

The interferon (IFN) system is an extremely powerful antiviral response in animal cells. The subsequent effects caused by porcine astrovirus type 1 (PAstV1) IFN activation are important for the host's response to viral infections. Here, we show that this virus, which causes mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa in piglets, induces an IFN response upon infection of PK-15 cells. Although IFN-ß mRNA was detected within infected cells, this response usually occurs during the middle stages of infection, after genome replication has taken place. Treatment of PAstV1-infected cells with the interferon regulatory factor 3 (IRF3) inhibitor BX795 decreased IFN-ß expression, whereas the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) inhibitor BAY11-7082 did not. These findings indicate that PAstV induced the production of IFN-ß via IRF3-mediated rather than NF-κB-mediated signaling pathways in PK-15 cells. Moreover, PAstV1 increased the protein expression levels of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in PK-15 cells. The knockdown of RIG-I and MDA5 decreased the expression levels of IFN-ß and the viral loads and increased the infectivity of PAstV1. In conclusion, PAstV1 induced the production of IFN-ß via the RIG-I and MDA5 signaling pathways, and the IFN-ß produced during PAstV1 infection inhibited viral replication. These results will help provide new evidence that PAstV1-induced IFNs may protect against PAstV replication and pathogenesis. IMPORTANCE Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs. However, astrovirus-host interactions are less well studied, particularly with respect to their antagonism of IFN. Here, we report that PAstV1 acts via IRF3 transcription pathway activation of IFN-ß. In addition, the knockdown of RIG-I and MDA5 attenuated the production of IFN-ß induced by PAstV1 in PK-15 cells and increased efficient viral replication in vitro. We believe that these findings will help us to better understand the mechanism of how AstVs affect the host IFN response.

Phylogenetic and Spatiotemporal Analyses of Porcine Epidemic Diarrhea Virus in Guangxi, China during 2017-2022.

Since 2010, porcine epidemic diarrhea virus (PEDV) has swept across China and spread throughout the country, causing huge economic losses. In this study, 673 diarrhea samples from 143 pig farms in Guangxi during 2017-2022 were collected and detected for PEDV. Ninety-eight strains were selected for S1 gene analyses and these strains were classified into four subgroups (G1b, G2a, G2b and G2c), accounting for 1.02 (1/98), 75.51 (74/98), 16.33 (16/98) and 7.14% (7/98) of the total, respectively. Importantly, an increased number of strains in the G2c subgroup was found from 2019 onwards. Bayesian analysis revealed that Guigang may have been the epicenter of PEDVs in Guangxi. In addition, Guigang was identified as the primary hub from which PEDVs spread via two routes, namely Guigang-Wuzhou and Guigang-Laibin. Moreover, several coinfections of novel PEDV variants bearing large deletions in the partial S1 protein and PEDVs possessing an intact partial S1 protein were found in pigs. Further recombination analyses indicated that two of the strains, 18-GXNN-6 and 19-GXBH-2, originated from intra-genogroup recombination. Together, our data revealed a new profile of PEDV in Guangxi, China, which enhances our understanding of the distribution, genetic characteristics and evolutionary profile of the circulating PEDV strains in China.

Development of a recombinant reporter Getah virus for antiviral drug screening assays.

Getah virus (GETV), is an often neglected and re-emerging mosquito-borne RNA virus. GETV can cause illness accompanied with high fever, rash, incapacitating arthralgia and chronic arthritis or encephalitic disease in affected animals. Currently, there is no specific treatment or vaccine against GETV infection. In this study, we developed three recombinant viruses by inserting different reporter protein genes between the Cap and pE2 genes. The reporter viruses exhibited high replication capacity similar to the parental virus. The rGECiLOV and rGECGFP viruses were genetically stable within at least ten rounds of passages in BHK-21 cells. We confirmed that the reporter virus, rGECGFP, facilitated the antiviral assays against GETV by testing it with the known inhibitor, ribavirin. It was also found that the compound, doxycycline, showed an inhibitory effect on GETV replication. In addition, rGECGFP was found to be an authentic mimic of the parental virus infection in 3-day-old mice, but with milder pathogenicity. The reporter viruses will contribute to the assessment of viral replication and proliferation, tracking and elucidating of alphavirus-host interactions. In addition, they will help in the screening of potential antiviral compounds.