Rapid, Specific Detection of Alphaviruses from Tissue Cultures Using a Replicon-Defective Reporter Gene Assay

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.


Introduction
Alphaviruses are a group of 30 mosquito-borne arboviruses belonging to the genus Alphavirus in the family Togaviridae [1][2][3]. Some alphaviruses, such as Sindbis virus (SINV) [4] and Chikungunya virus (CHIKV) [5,6], can cause fever, arthritis, rash, and other symptoms. Alphaviruses such as Western equine encephalitis virus (WEEV) [7], Eastern equine encephalitis virus (EEEV) [8], and Venezuelan equine encephalitis virus (VEEV) [9] can cause fever and viral encephalitis in humans. Another alphavirus, Getah virus (GETAV), causes pyrexia, skin eruptions, edema of the hind limbs, and enlargement of the submandibular lymph nodes in horses or swine [10]. Alphaviruses are distributed globally, and have caused several epidemics, including the largescale CHIKV infection in the Indian Ocean region in 2005 and the ensuing global infection [11][12][13]. The high pathogenicity of alphaviruses makes them a substantial international public health threat; consequently, a variety of alphaviruses (EEEV, WEEV, and VEEV) have been classified as potential biological warfare agents that could threaten public security [14]. Therefore, a rapid, specific detection technique for alphavirus pathogens plays an important role in monitoring infectious diseases and public security.
Recently, several virus detection techniques have been developed using virus-specific reporter gene assays that efficiently and sensitively detect the corresponding viruses, such as rubella and herpes simplex [15][16][17][18][19][20]. In this study, we established a rapid, specific method for detecting alphaviruses using a reporter gene assay that uses a defective replicon derived from the XJ-160 virus. In this assay, the structural genes of SINV were replaced by reporter genes, and a deletion was made in the non-structural protein 4 (nsP4) gene to construct defective replicons of SINV. On infection with an alphavirus, the alphavirus components induce the expression of the reporter gene in the cells transfected with the replicon-defective plasmid. This simple, rapid, specific method using defective replicons can quickly discriminate whether an unknown sample contains an alphavirus from the first passage in tissue cultures.
Reporter gene expression assays showed that specific green fluorescence was observed after BHK-21 cells were transfected with pVaXJ-EGFP (Figure 2A), while no EGFP expression was observed following transfection with the defective replicon pVaXJ-EGFPDnsp4 ( Figure 2B). Similarly, high luciferase activity was detected after BHK-21 cells were transfected with the pVaXJ-GLuc replicon, while only background levels of luciferase were detected after transfection with the defective replicon pVaXJ-GLucDnsp4 ( Figure 2C). These results indicate that the defective replicons did not express reporter genes normally because of the 1139 bp deletion in the nsP4 region.

Detection capability of defective replicon cassettes
As mentioned previously, because of the deletion in the nsP4 gene, the defective replicons could not replicate and express reporter genes normally after being introduced into cells. However, when transfected cells were infected with an alphavirus, the non-structural protein expressed by the infecting alphavirus could act on the defective replicons in trans and result in the expression of the reporter genes. Using this reporter assay, an alphavirus in an undefined sample should be detected by observing the expression of the reporter genes. A schematic diagram of alphavirus detection using defective replicons is shown in Figure 3.
To confirm the detection capability of the defective replicons, BHK-21 cells were transfected with pVaXJ-EGFPDnsp4 and then infected with 1 MOI of XJ-160 virus. EGFP expression could be observed 44 h post-infection under a fluorescence microscope ( Figure 4A), while no expression of EGFP was observed in the control group with no virus infection ( Figure 4B). Luciferase assays indicated that the luciferase activity could be detected 6 h postinfection, reaching a peak value at 44 h post-infection, and gradually decreasing after 44 h. The luciferase activity 44 h postinfection in the experimental group was 10 times higher than that in the pVaXJ-GLucDnsp4 control group ( Figure 4C). Both the qualitative and quantitative results suggest that the defective replicon-defective reporter gene assay could detect SINV XJ-160 in infected cells.

Specificity of the replicon-defective alphavirus detection method
To determine the specificity of the replicon-defective alphavirus detection method, we attempted to detect a variety of alphaviruses and non-alphaviruses. EGFP expression assays indicated that BHK-21 cells produced green fluorescence when the reportertransfected cells were infected with three strains of SINV (XJ-160, YN87448, and MX10), CHIKV (SD08Pan), and GETAV (HB0234) ( Figure 5B, 5C, 5D, 5E, 5F), and that no green fluorescence was observed after infection with the Japanese encephalitis virus (JEV, P3) or Tahyna virus (TAHV, XJ0625) ( Figure 5G, 5H). The luciferase activity assay showed that GLuc expression activity was significantly increased in the alphavirus infection group, compared to pVaXJ-GLucDnsp4 in the control group without any virus infection. The reporter plasmidtransfected BHK-21 cells infected with XJ-160, YN87448, MX10, SD08Pan, or HB0234 virus expressed GLuc with activities that were 10.2, 9.1, 7.4, 5.6, and 5.3 times higher, respectively, than that of the control group. Following infection with JEV (P3) or TAHV (XJ0625) viruses, the GLuc activities were similar to that of pVaXJ-GLucDnsp4 in the control group ( Figure 5I). These results show that the defective replicon-based method can detect alphaviruses from tissue culture, while other RNA viruses, such as the P3 flavivirus and XJ0625 bunyavirus gave negative results with this system.

Sensitivity of the replicon-defective alphavirus detection method
To determine the sensitivity of the alphavirus detection method, several alphaviruses with a titer of 1610 5 PFU/ml were serially diluted (from 10 21 to 10 27 ), resulting in the addition of 10 4 , 10 3 , 10 2 , 10, 1, 0.1, and 0.01 PFU to the plates, respectively. BHK-21 cells, transfected with each of the defective replicons, were infected with the alphaviruses and assayed for expression of the reporter protein. Luciferase activity assays indicated that the GLuc activity    reached a peak 44 h post-infection, and decreased gradually with the number of PFUs used for the infection. When the virus load was reduced to 1 PFU (XJ-160 or YN87448) or 10 PFU (MX10), the activity measured at 44 h was obviously above that of pVaXJ-GLucDnsp4 in the control group (Figure 6), while the GLuc activity was enhanced until other alphaviruses increased to 10 2 or 10 3 PFU, indicating that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses and between 10 2 and 10 3 PFU for the two other alphaviruses.

Discussion
All viruses have specific pathways for replication and expression of the viral RNA genome. Using this knowledge, it is possible to induce the specific expression of a reporter gene inserted into the viral genome. Therefore, under the control of virus-specific events, we can detect these viruses by measuring the reporter enzyme. This strategy provides a simple, effective, and virus-specific detection technique [22][23][24].
The genome structures and replication strategy specific to alphaviruses were used to construct an alphavirus-inducible reporter gene expression system. The alphavirus genome is a single strand of positive-sense RNA, which has two open reading frames: the 59-end open reading frame that encodes non-structural proteins (nsP1-4) and the 39-end open reading frame that encodes structural proteins (capsid proteins and envelope proteins). Nonstructural proteins are the viral replicases/transcriptases, which are responsible for the replication and transcription of the viral genomic RNA, as well as the synthesis of subgenomic RNA [25]. nsP4 has RNA-dependent RNA polymerase activity, which is highly conserved in the genus Alphavirus, and plays a major role in viral RNA replication [26][27][28]. After an alphavirus enters a host cell, ribosomes bind to the positive-strand genomic RNA and produce non-structural proteins. Using the viral replicases, the negative-strand RNA is synthesized using the positive-strand RNA as a template. Next, subgenomic RNA is synthesized using the negative-strand RNA as a template. Finally, the subgenomic RNA is used as a template to translate and synthesize structural proteins to assemble virus particles [29]. The well-described genome structure and replication strategy of alphaviruses enabled us to construct a replication reporter system by replacing the structural genes with reporter genes to form the plasmids pVaXJ-EGFP and pVaXJ-GLuc. To form the replicon-defective reporter system, we deleted 1139 bp of the nsP4 coding frame using the AclI site, forming the replicon-defective reporter plasmids pVaXJ-EGFPDnsp4 and pVaXJ-GLucDnsp4. As nsP4 plays an indispensable role in viral genome replication, the defective replicon could not replicate normally and express reporter genes after they were introduced into cells. By contrast, if the transfected cells were infected with an alphavirus, the nsP4 proteins produced by the alphavirus could act on the defective replicons in trans, leading to the expression of the reporter genes. In addition, the nsP4 gene is highly conserved in the genus Alphavirus [30]. Recently, the nsP4 coding region of Alphavirus RNA was chosen as the target for genus-specific RT-PCR, and all alphaviruses, including Barmah Forest, Chikungunya, Mayaro, O'nyong-nyong, Ross River, Semliki Forest, Sindbis, Eastern equine encephalitis, Western equine encephalitis, and Venezuelan equine encephalitis viruses, were positive using the generic Alphavirus PCR [31]. Therefore, the defective replicons constructed using SINV XJ-160 can be used to detect a variety of alphaviruses.
We used the method to detect a variety of alphaviruses and nonalphaviruses from tissue culture. The results showed that the system could detect alphaviruses, including the Sindbis, Chikungunya, and Getah viruses. By contrast, the system gave a negative response to other RNA viruses, such as the Japanese encephalitis and Tahyna viruses. The system could detect alphaviruses with a viral load as low as 1-10 3 PFU, indicating that this detection technique has a high specificity and sensitivity for alphaviruses. Sindbis viruses can be divided into three genotypes, the Palearctic/Ethiopian (P/E), Oriental/Australian (O/A), and Western-Australian (W/A), and the three strains of Sindbis viruses used in this study, YN87448, XJ-160, and MX10, belong to the P/ E, W/A, and O/A genotypes, respectively [32]. Therefore, these strains represent the different known genotypes of SINV. Olivo et al. constructed a similar SINV defective replicon system, in which all four non-structural proteins gene were deleted [33]. Unlike the nsP4 gene, the other three nsP genes share low homology among alphaviruses, so the detection system constructed by Olivo et al. could only be used to detect Sindbis virus, rather than other alphaviruses. In this study, we constructed a replicondefective alphavirus detection system that is suitable for detecting a variety of alphaviruses, since we deleted only the majority of the nsP4 region, which is highly conserved in alphaviruses.
Presently, two methods are applied for the detection and identification of unknown viruses, including the detection of viral antigens and viral genes. To identify viral antigens, virus isolates must be incubated in sensitive cells, and then different virus antibodies are used to determine the virus type. This detection process is time consuming and it is difficult to determine the standard of the detection method. The PCR-based viral gene detection method is sensitive, but the chemical treatment of samples in PCR detection leads to viral lysis, making it impossible to isolate and culture the virus, which is undesirable when the virus sample is scarce. Compared with these methods, only a very small amount of sample needs to be incubated with cultured cells in our replicondefective alphavirus detection method. In addition, the cytopathic effect (CPE) caused by alphavirus is usually observed more than 40 h post-infection [10,21,34], while our detection method can detect alphaviruses 6 h post-infection with the GLuc assay. Therefore, the replicon-defective alphavirus detection method can quickly, sensitively and specifically detect alphaviruses without affecting the viral multiplication capacity, allowing the isolation and cultivation of scarce viruses for use in subsequent studies.
We constructed a simple, rapid, color-visible replicon-defective detection assay for the detection of alphaviruses. These characteristics make it possible to use this detection technique in clinical examinations, epidemiological surveillance, and the rapid screening of viral biological warfare agents. If we select a packaging cell line that can express the defective replicon stably, the detection process will be even simpler and more convenient. The establishment of this detection method also provides a reference for the development of new detection methods for other RNA viruses.  [21,34] were maintained at 37uC under 5% CO 2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml each of penicillin and streptomycin.

Construction of defective XJ-160 replicons
The vector pVaXJ, a DNA-based expression vector, was constructed by placing the recombinant genome of XJ-160 virus under the control of the human cytomegalovirus (CMV) promoter of plasmid pVAX1, in which the viral structural genes were replaced by a polylinker cassette to allow the insertion of heterologous genes. The cassettes pVaXJ-EGFP and pVaXJ-GLuc, expressing enhanced green fluorescence protein (EGFP) or Gaussia luciferase (GLuc), respectively, were constructed by cloning the EGFP or GLuc gene into pVaXJ. Then, the defective repliconbased plasmids pVaXJ-EGFPDnsp4 and pVaXJ-GLucDnsp4 were constructed by digesting pVaXJ-EGFP or pVaXJ-GLuc, respectively, with AclI to delete 1139 bp in the nsP4 gene, which is highly conserved in Alphavirus and plays a major role in viral RNA replication through its RNA-dependent RNA polymerase (RDRP) activity [26][27][28].

Plaque assay
The observations of plaque morphology and determination of virus titer were performed using a plaque assay, as described previously [34]. Briefly, BHK-21 cells were grown to confluence in a 6-well plate to which 0.5 ml of diluted virus sample was added. After a 1-h incubation at 37uC, the supernatant was removed, and 2 ml of culture cover medium (26DMEM containing 2% FBS equivalently mixed with 2% UltraPure agarose) were added. Then, the plate was turned over and incubated at 37uC in a humidified atmosphere with 5% CO 2 . When the cytopathic effect was seen under a microscope, another 2 ml of culture cover medium containing 10% neutral red (Sigma, USA) was added and the plates were incubated at 37uC under 5% CO 2 for 6 h. The size and number of plaques were then recorded and the number of plaques was used to calculate the virus titer.

Transfection and reporter gene expression
In 24-well plates, 1610 5 BHK-21 cells were transfected with 0.8 mg pVaXJ-EGFPDnsp4 or pVaXJ-GLucDnsp4 using Lipofectamine 2000 reagent (Invitrogen, USA), and the BHK-21 cells were infected with viruses at 6 h post-transfection. To observe EGFP expression, the medium was removed from the transfected cells and replaced with enough phosphate-buffered saline to keep the cells wet. The nucleuses were stained with 49, 6-diamidino-2-phenylindole (DAPI). Then, the cells were examined using an Olympus IX51 fluorescence microscope. Micrographs were taken using an Olympus DP70 camera. To perform the GLuc activity assay, 20 mL of culture supernatant were collected at different time points and reacted with BioLux GLuc substrate in BioLux GLuc assay buffer from the BioLux TM Gaussia Luciferase Assay Kit (New England Biolabs). GLuc activity was detected by a Modulus luminometer (Promega), and the raw data, expressed as relative light units (RLU), were recorded.