Pteropine Orthoreovirus (PRV7S) does not Establish Persistent lnfection in NP460 Cells but with lnflammatory Response Triggered＊

LOH Mun Khin Alson ,SIEW Zhen Yun ,LEONG Pooi Pooi ,KOH Rhun Yian ,CHYE Soi Moi ,WONG Siew Tung ,and VOON Kenny

Pteropine orthoreovirus (PRV) is a nonenveloped double-stranded RNA (dsRNA) virus of the genus Orthoreovirus under the family Reoviridae.The PRV genome is composed of 10 dsRNA segments,including three large (L) segments,three medium (M) segments,and four small (S) segments surrounded by an icosahedral capsid[1].Furthermore,PRV is a fusogenic virus characterized by the capability of the induction of cell-cell fusion and syncytia formation[1,2].The fusogenic property of orthoreovirus is often correlated with the cytopathicity,virulence potential and pathogenesis of reovirus[1,3].Continuous systemic surveillance and genomic characterization of PRV are important to monitor PRV’s virulence and genetic reassortment[2].However,the mechanism of PRV pathogenesis in humans is still poorly understood.

PRV was isolated from fruit bats and patients with respiratory symptoms varying from mild to severe.In Malaysia,studies have shown that some human populations have developed antibodies to the virus,indicating that it has been transmitted from bats to humans in the past[1,2].These findings suggest that PRV has evolved to cross the species barrier (zoonosis) between bats and humans through genetic reassortment.Apart from genetic analysis,the study of PRV pathogenesis and host immune response is important and may contribute to strategies against this highly transmissible reovirus with concerns.

Studies demonstrated that different cell lines have different susceptibilities and innate immune responses against PRV infection[3,4].These studies suggested that inflammation plays a role in the immune response towards PRV infection.Besides,persistent infection of reovirus may contribute to its pathogenesis and replication.Persistent reovirus infection has been studied in different settings,mainly mammalian orthoreovirus (MRV),but also avian orthoreovirus (ARV) and piscine orthoreovirus.However,there is a lack of evidence indicating whether PRV can establish persistent infectionin vitroandin vivo.Interestingly,a recent study by Tee et al.(2023) demonstrated that PRV may replicate and remain in human oral keratinocytes (OKF-6) and non-carcinoma nasopharyngeal (NCNP) epithelial cells (NP69) for a relatively long period[2].Hence,in our study,we examined the capability of PRV7S to establish persistent infection in NCNP cell lines(NP460 and NP69) at a multiplicity of infection (MOI)0.1.The inflammatory response of NP460 cells towards PRV7S infection was also determined by using a membrane-based inflammatory cytokine antibodies array.

Microscopic evaluation (Figure 1A) of PRV7Sinfected NP460 cells from 1 day post-infection (dpi)to 35 dpi indicated that the infected NP460 cells were able to proliferate without any apparent morphological changes.However,starting from 14 dpi,floating cells were observed from the infected NP460 cell culture and the extent of cell depletion gradually increased to 35 dpi.Interestingly,CPE(cytopathic effect,e.g.syncytia formation) was not observed in infected NP460 cells throughout the evaluation.To determine if NP460 were persistently infected by PRV7S,surviving cells on 35 dpi were passaged into a new culture dish with fresh medium for another 7 days.At 42 dpi,no viable cell was observed from the culture dish.The infected NP460 was compared to two nasopharyngeal carcinoma(NPC) cell lines (CNE1,TW01) and NP69 cells(Figure 1B).Results showed that CNE1 and TW01 cells have a higher susceptibility towards PRV7S infection compared to NP460 and NP69 cells.Infected CNE1 and TW01 cells presented complete CPE at 5 dpi and 7 dpi,respectively,while NP460 and NP69 cells remained viable.In contrast to NP460 cells,NP69 cells presented morphological changes such as the elongation and expansion of cell structure,and reduced cell cytoplasmic body volume.At 14 dpi,no viable NP69 cells were observed.This study confirms that the NCNP cell lines,NP460 and NP69 cells are less susceptible to PRV infection compared to CNE1 and TW01 cells.The result obtained regarding NP69 cells agrees with the previous findings[2].Reovirus preferentially multiplies in RAS-activated cells,because these cells express more junctional adhesion molecule (JAM),a crucial receptor for reovirus attachment and entry.Moreover,RAS activation may increase autophagy,which is desirable for reovirus replication[1].CNE1 and TW01 cells are known to have RAS-activating mutations which lead to the activation of the RAS signalling pathway,which can promote cell growth,survival,and proliferation.Hence,CNE1 and TW01 cells are more susceptible to PRV7S infection as demonstrated previously[5].In contrast,NP460 cells are less susceptible to PRV7S infection in comparison to the RAS-activated NPC cells,thus limits the PRV7S replication.

Figure 1.Microscopy evaluation of NP460,NP69,TW01 and CNE1 cells infected with MOI 0.1 of PRV7S.(A) Microscopy evaluation of PRV7S-infected NP460 from 1 dpi to 35 dpi.PRV7S-infected NP460 cells were able to proliferate without any apparent morphological changes.From 14 dpi onwards,floating cells were observed from the PRV7Sinfected NP460 cell culture and the extent of cell depletion gradually increased to 35 dpi.(B)Microscopy evaluation of PRV7S-infected NP69,TW01 and CNE1 cells from 1 dpi to 14 dpi.Infected CNE1 and TW01 showed full CPE at 5 dpi and 7 dpi,respectively,while NP69 cells remained viable.Full CPE was observed from NP69 cell culture at 14 dpi.Red outlines indicate a subsequent passage of cell cultures.All micrographs are at 40× magnification and all scale bars represent 50 μm;dpi: day postinfection;MOI: multiplicity of infection;CPE:cytopathic effect.

Since PRV7S persist in NP460 cells relatively longer than the other three cells.A study demonstrated that reovirus infection can have several long-term effects on normal cells[6].Moreover,reovirus may establish persistent infection in certain cell types,and both acute and persistent reovirus infection may trigger the upregulation of several pro-inflammatory cytokines.However,the regulations of cytokines and chemokines may not solely depend on viral replication.Pro-inflammatory cytokines play a crucial role in activating the immune system and triggering inflammation in response to various stressors[6].In the case of persistent reovirus infection,these cytokines may lead to chronic inflammation and damage to the host tissue,which could contribute to the development of other diseases or conditions.Furthermore,reovirus infection in normal cells activates PKR,a protein kinase that is usually involved in anti-viral response and interferon's antiproliferative response[1].PRV7S is a potential oncolytic virus,and it is important to investigate its capability to establish persistent infection in normal cells because this may affect one's overall health.It is also interesting to study does an aberrant viral replication occurs in PRV7S-infected NP460 cells because NP460 cell is relatively less susceptible to PRV7S infections compared to CNE1,TW01 and NP69 cells.Aberrant viral replication refers to viral replication that deviates from the normal course of a viral infection such as defective viral particles,change in the host environment and mutation in viral genes.The replication of PRV7S in NP460 cells was investigated by detecting the PRV sigma 1/A gene and infectious viral particles released by the infected NP460 cells.Real-time PCR (qPCR) results(Figure 2A) showed PRV sigma 1/A gene detected from PRV7S-infected NP460 cells starting from 1 dpi to 42 dpi.The sigma 1/A gene of reovirus encodes the viral attachment protein σ1,which is responsible for binding to the host cell receptor and initiating the process of viral entry into the cell[1].Based on our observation,PRV7S entry into the NP460 cells resulted in an increased trend in PRV sigma 1/A viral RNA from 1 dpi to 7 dpi,while the decreasing trend in the viral RNA detected from 7 dpi to 42 dpi was suggested to be a switch from virus entry phase to virus replication cycle.According to TCID50assay results (Figure 2B),the presence of infectious PRV7S viral particles in the supernatant was verified and it was able to infect the Vero cells.At 3 dpi,the supernatant has the lowest PRV7S titer of 2.20 ±0.47 TCID50/mL.The peak amount of infectious virus was detected on 7 dpi and 10 dpi at 7.2 ± 0.33 TCID50/mL.Based on the results,the replication and infection capacity of PRV7S produced by infected NP460 cells is not compromised as the PRV gene is detectable and the progeny PRV7S produced is still infectious.We suggest that when more NP460 cells are infected by PRV,more progeny virus will be released which leads to a higher virus titre in the supernatant.Concurrently,PRV replication will exhaust infected NP460 cells and reduce the NP460 cell viability.Hence,the NP460 cell viability and cell viral RNA load were decreased from 7 dpi to 42 dpi,and high viral titer was detected in the culture supernatant from 7 dpi to 35 dpi as well.These results suggest that PRV7S replicates by lytic cycle in NP460 cells and releases infectious viral particles into the cell culture supernatant.Therefore,PRV7S viral replication and infectivity were not compromised in the NP460 cells.Remarkably,PRV7S-infected NP460 cells did not cause any syncytia formation,which is a hallmark of fusogenic virus infection[1],and no other significant morphological changes were observed.

Figure 2.Viral RNA and extracellular detection of PRV7S from infected NP460 cell culture.(A)qPCR detection of PRV sigma 1/A gene expression in PRV7S-infected NP460 cells.(B)TCID50 quantification of PRV7S from infected NP460 cell culture supernatant.At 1 dpi and 42 dpi,the virus load in the supernatant did not reach the minimal detectable threshold.Hence,determined as undetectable (n.d.).The data are expressed as mean ± SD of three independent experiments.

Besides the study of persistent infection,investigating the immune response of NCNP cells against oncolytic virus infection is also important.Utilizing oncolytic viruses poses safety concerns of off-target effects,whereby the oncolytic virus could infect and damage normal (healthy) cells[1].Understanding how normal cells respond to oncolytic virus infection is critical to ensure that these viruses are safe and do not cause undesired harm.The innate immune response plays a critical role in controlling reovirus infections through the activation of pro-inflammatory cytokines.Upon reovirus infection,the viral dsRNA is recognized by several pattern recognition receptors (PRR),which activate downstream signalling cascades and transcription factors,ultimately,leading to the production of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor alpha(TNF-α)[7].These cytokines are responsible for recruiting and activating immune cells and controlling viral replication and spread.To examine the inflammatory response of NP460 cells towards PRV7S infection,the cell culture supernatant from non-infected NP460 and PRV7S-infected NP460 cells at 35 dpi was tested with a membrane-based cytokine antibody array.Infected NP460 cells at 35 dpi were the last passage with viable PRVinfected NP460 observed,thus selected for inflammatory cytokine array.The result (Figure 3)demonstrated that PRV7S-infected NP460 cells had downregulated expression of cytokines involved in cell growth and proliferation,which are vascular endothelial growth factor (VEGF-A),epidermal growth factor (EGF),tissue inhibitor of metalloproteinase 1 (TIMP1) and TIMP2,compared to the non-infected NP460 cells.The finding indicates that the proliferative activities of PRVinfected cells are exhausted by viral replication,and this might be a result of PRV7S infection whereby PRV7S uses host cell machinery to replicate and therefore manipulates host cell gene expression to favour its replication[7].The cell viability was observed to be decreased with reduced cell proliferation as described.Upon PRV7S infection,the expression of pro-inflammatory cytokines was affected as well.For instance,IL-6,chemokine C-X-C motif ligand 1 (CXCL1 or GRO),chemokine C-C motif ligand 5 (CCL5) was downregulated,while IL-8 was slightly upregulated.Furthermore,CCL2 expression drastically increased nearly 12-fold higher in PRV7Sinfected NP460 cells compared to non-infected NP460 cells.CCL2 is a chemoattractant that promotes the recruitment of monocytes and macrophages to the site of infection.In the context of reovirus infection,CCL2 upregulation has been demonstrated in the brain and lung of mice models during reovirus (especially PRV) infection,together with some other proinflammatory cytokines and chemokines[8].Additionally,a study in human melanoma cells found that reovirus infection can lead to the upregulation of CCL2 and other chemokines[9].Therefore,these findings suggest that CCL2 plays a significant role in PRV7S infection and host inflammatory response.Besides,PRV7Sinfected NP460 cells reduced IL-6 cytokine expression,while IL-8 was slightly increased.IL-6 and IL-8 are both pro-inflammatory cytokines responsible for the regulation of immune responses,inflammation,and hematopoiesis;while IL-8 is also a chemoattractant that recruits immune cells,especially neutrophils,to the site of infection to suppress viral replication.Studies of reovirus infection have shown that the virus can induce the upregulation of several pro-inflammatory cytokines,including IL-6 and IL-8,as part of the innate immune response to control viral replication[9].However,the specific mechanism for the upregulation of IL-8 and downregulation of IL-6 in nasopharyngeal cells upon PRV7S infection may not be fully understood.Both CCL2 and IL-8 are chemoattractants and were observed to be upregulated in NP460 cells during PRV7S infection.Probably,these cytokines were more prominently and primarily to be released by epithelial cells,in this context,NP460 during an infection.It could be possible that PRV7S may interfere with host signalling pathways,transcription factors,specific host-virus interactions and innate immune response of the host cell which then influence the expression of these cytokines.

Figure 3.Membrane-based cytokine antibody array.(A) Membrane blot of cytokine expression of noninfected NP460 cells versus PRV7S-infected NP460 cells at 35 dpi.Red arrows indicate cytokines with changes in intensity.(B) Quantification of cytokine expression of non-infected NP460 cells versus PRV7Sinfected NP460 cells at 35 dpi based on signal intensity.

GRO (CXCL1) and RANTES (CCL5) were slightly downregulated in PRV-infected NP460 cells compared to non-infected cells.CXCL1 and CCL5 are very likely to be upregulated in different viral infections[10].It is well-established that CCL5 is an antiviral and proinflammatory cytokine which recruits immune cells,and CXCL1 is an immune cell chemoattractant (granulocytes recruitment) that induces shape change and adhesion of the granulocytes.Hence,we hypothesize that the downregulation of CXCL1 and CCL5 might be related to the reovirus replication mechanism which allows the infected-host cell (cancer cell) to migrate and fuse with other cells in order to spread and propagate the virus.Above that,the downregulation of both CXCL1 and CCL5 may prevent or delay the inflammatory response and immune cell recruitment which ideally aids in the PRV7S infection.As mentioned by Alcami,the downregulation of certain cytokines might be one of the viral evasion strategies,also known as viral anti-cytokine strategies[11].However,there are some exceptions.CCL5 was upregulated in dendritic cells loaded with reovirus-infected human melanoma Mel888 cells(DC-MelReo) which may induce chemotaxis of the natural killer cell,but CXCL1 was secreted at basal level or even undetected[12].Lastly,according to Xiao et al.[13],another possibility is that the downregulation of CXCL1,CCL5 and some other cytokines might be a sign of immunological tolerance from cells without RAS mutation towards PRV infection.

This study underscores the importance of investigating the immune response of noncarcinoma cells to oncolytic virus infection and its potential safety concerns as an oncolytic virus.However,the limitation of our study is that the cytokine array blot was only performed once and required further study.Thus,more in-depth studies are needed to discover the complete immune modulation of non-carcinoma cells during PRV infection and further investigation is necessary to determine if aberrant viral replication occurs in PRV7S infection in NP460 cells.

No potential conflicts of interest were disclosed.

&These authors contributed equally to this work.

#Correspondence should be addressed to VOON Kenny,E-mail: kenny.voon@nottingham.edu.my Tel:+6012-2730539

Biographical notes of the first authors: LOH Mun Khin Alson,male,born in 1996,MSc,majoring in virology and cell biology;SIEW Zhen Yun,male,born in 2000,BSc(Hons),majoring in virology,cell biology and biochemistry.Received:March 26,2023;Accepted:June 15,2023