Significant Cytopathic Effect Research Articles

The two faces of Blastocystis spp.: is it the cause of colorectal cancer (CRC) or a consequence of it?

IntroductionOver the last few years, there has been an increase in the prevalence of <i>Blastocystis</i> spp. in colorectal cancer (CRC) patients. Moreover, various in vitro and in vivo studies have highlighted that intestinal colonisation of <i>Blastocystis</i> spp. has an influence on host immune responses leading to cellular apoptosis and membrane permeability. It has been suggested that <i>Blastocystis</i> spp. is an important risk factor for the worsening of CRC.AimTo present evidence concerning the association between CRC and <i>Blastocystis</i> spp.Material and methodsA review of the literature was performed by searching Science Direct, PubMed, Scopus and Google Scholar databases up to December, 2023.Results and discussionOut of all in vitro and in vivo studies selected for this review, the majority of them have confirmed a significantly higher prevalence of <i>Blastocystis</i> spp. in colorectal cancer patients in comparison to the control groups. Several in vitro human colorectal carcinoma cell line studies have shown significant cytopathic and immunological effects of <i>Blastocystis</i> spp. Additionally, in vivo experimental animal model studies have shown that <i>Blastocystis</i> spp. infection significantly contributed to large intestinal polyp (colorectal adenoma) formation and the progression of colorectal carcinogenesis.ConclusionsThese studies strongly support suggestions that <i>Blastocystis</i> spp. could be an important factor to existing CRC development by influencing the host immune response and increasing oxidative damage.

Establishment, characterization and application of Largemouth bronze gudgeon (Coreius guichenoti) fin and skin cell lines in disease resistance

Largemouth bronze gudgeon (Coreius guichenoti) is an economically important fish species in the upstream Yangtze River. However, C. guichenoti has been listed in the National Key Wild Animal List given that its wild population dramatically declined under the influence of hydroelectric projects, intensive fishing, and environmental pollution. To protect this endangered species, C. guichenoti fin (CGF) and skin (CGSK) cell lines were established in present study. Both cell types exhibited epithelial morphology, and the optimal culture conditions for CGF and CGSK were cultured at 28 °C in L-15 medium with 20% serum. Currently, CGF and CGSK have been sub-cultured up to 60 passages and 30 passages, respectively. Gene identification results of nd1, 16 s, 12 s, cytb, and coi indicated that these two cells originated from the C. guichenoti, with a chromosomal number of 2n = 50. After cryopreservation in liquid nitrogen for 6 months, cell viability remained above 70%. Subsequently, predominant expressed genes by transcriptome analysis implied that CGSK cells mainly consisted of dermis fibroblast, while CGF cell types primarily comprised myofibroblasts. The successful transfection of siRNA and pEGFP-N1 into CGF and CGSK cells were observed. Furthermore, both cell lines exhibited significant cytopathic effects (CPE) when stimulated with spring viremia of carp virus (SVCV). Therefore, the establishment of CGF and CGSK cell lines provides a solid foundation for the recovery and conservation of genetic resources, as well as immunological and toxicological studies of C. guichenoti.

Replication and Injury Associated With SARS-CoV-2 in Cultured Hepatocytes.

Liver dysfunction is one of the hallmarks of SARS-CoV-2 infection. The mechanism(s) of hepatic injury in SARS-CoV-2 infection remains controversial with some reporting viral replication and cellular injury and others suggesting lack of replication and injury due to non-cytopathogenic etiologies. To investigate this further, we evaluated SARS-CoV-2 replication in immortalized hepatic cell lines and primary hepatocytes, examined whether cell injury was associated with apoptotic pathways, and also determined the effect of the antiviral remdesivir on these processes. Immortalized hepatocyte cell lines (HepG2 and Huh7.5), as well as primary human hepatocytes, were exposed to SARS-CoV-2 at a multiplicity of infection of 0.1 PFU/mL. Viral replication was evaluated by plaque assays, immunohistochemical staining for the viral spike protein, and caspase-3 expression evaluated with and without exposure to remdesivir. All hepatocyte cell lines and primary hepatocytes supported active replication of SARS-CoV-2. Significant cytopathic effect was observed by light microscopy, and caspase-3 staining supported activation of apoptotic pathways. Remdesivir abrogated infection in a dose-dependent fashion and was not independently associated with hepatocyte injury. Hepatocytes appear to be highly permissive of SARS-CoV-2 replication which leads to rapid cell death associated with activation of apoptotic pathways. Viral replication and hepatocytes injury are abrogated with remdesivir. We conclude that active viral replication is most likely a key contributor to liver enzyme abnormalities observed in the setting of acute SARS-CoV-2 infection.

Isolation and identification of Tete virus group (Peribunyaviridae: Orthobunyavirus) from Culicoides biting midges collected in Lichuan County, China.

In July 2018, a virus (JXLC1806-2) was isolated from Culicoides biting midges collected in Lichuan County, Jiangxi Province, China. The virus isolate showed significant cytopathic effects within 48 hours after inoculation with mammalian cells (BHK-21). JXLC1806-2 virus could form plaques in BHK-21 cells, and the virus titer was 1×105.6 pfu/mL. After inoculation with the virus, suckling mice developed disease and died. The nucleotide and amino sequence analysis showed that the JXLC1806-2 virus genome was composed of S, M and L segments. Phylogenetic analysis showed that the S, M and L genes of JXLC1806-2 virus belonged to the Tete serogroup, Orthobunyavirus, but formed an independent evolutionary branch from the other members of the Tete serogroup. The results showed that the JXLC1806-2 virus, which was named as Lichuan virus, is a new member of Tete serogroup, and this is the first time that a Tete serogroup virus has been isolated in China.

Isolation and identification of a megalocytivirus strain (SKIV-TJ) from cultured spotted knifejaw (Oplegnathus punctatus) in China and its pathogenicity analysis

The spotted knifejaw (Oplegnathus punctatus) has recently emerged as a highly economically significant farmed fish in China. However, due to increasing environmental pollution and breeding density, a range of infectious diseases, including the iridovirus pathogen, have begun to spread widely. In this study, we isolated and identified a strain of Megalocytivirus, SKIV-TJ, from cultured spotted knifejaw in Tianjin, China. We observed significant cytopathic effects (CPE) in SKIV-TJ-infected spotted knifejaw brain (SKB) cells, and electron microscopy showed numerous virus particles in the cytoplasm of SKB cells 6 days post-infection. The annotated complete genome of SKIV-TJ (GenBank accession number ON075463) contained 112,489 bp and 132 open reading frames. Based on the multigene association evolutionary tree using 26 iridovirus core genes, SKIV-TJ was found to be most closely related to Rock bream iridovirus (RBIV). Cumulative mortality of spotted knifejaw infected with SKIV-TJ reached 100% by day 9. A transcriptomic analysis were conducted and a total of 5517 differentially expressed genes were identified, including 2757 upregulated genes and 2760 downregulated genes. The upregulated genes were associated with viral infection and immune signaling pathways. Our findings provide a valuable genetic resource and a deeper understanding of the immune response to SKIV infection in spotted knifejaw.

A New Cell Line Derived from the Caudal Fin of the Dwarf Gourami (Trichogaster lalius) and Its Susceptibility to Fish Viruses.

The detection of megalocytiviruses, especially the infectious spleen and kidney necrosis virus (ISKNV), in ornamental fish has increased with the rapid growth of the ornamental fish industry. In this study, dwarf gourami fin (DGF) cells derived from the caudal fin of the dwarf gourami (Trichogaster lalius), which is highly susceptible to red sea bream iridovirus (RSIV) and ISKNV, were established and characterized. The DGF cells were grown at temperatures ranging from 25 °C to 30 °C in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum and were subcultured for more than 100 passages, predominantly with epithelial-like cells. DGF cells had a diploid chromosome number of 2n = 44. Although the initial purpose of this study was to establish a cell line for the causative agents of red sea bream iridoviral disease (RSIV and ISKNV), DGF cells were also susceptible to rhabdoviruses (viral hemorrhagic septicemia virus, hirame rhabdovirus, and spring viraemia of carp virus), exhibiting a significant cytopathic effect characterized by cell rounding and lysis. Additionally, viral replication and virion morphology were confirmed using virus-specific conventional polymerase chain reaction and transmission electron microscopy. Furthermore, both RSIV and ISKNV were replicated at high concentrations in DGF cells compared to other cell lines. Notably, the DGF cells maintained a monolayer during ISKNV infection, indicating the possibility of persistent infection. Thus, DGF can be used for viral diagnosis and may play a critical role in advancing our understanding of ISKNV pathogenesis.

Persistence of Tembusu Virus in Culex tritaeniorhynchus in Yunnan Province, China.

The Tembusu virus (TMUV), a member of the Flaviviridae family, can be transmitted via mosquitoes and cause poultry disease. In 2020, a strain of TMUV (YN2020-20) was isolated from mosquito samples collected in Yunnan province, China. In vitro experiments showed that TMUV-YN2020-20 produced a significant cytopathic effect (CPE) in BHK, DF-1, and VERO cells, while the CPE in C6/36 cells was not significant. Phylogenetic analysis revealed that the strain belonged to Cluster 3.2 and was closely related to the Yunnan mosquito-derived isolates obtained in 2012 and the Shandong avian-derived isolate obtained in 2014. Notably, TMUV-YN2020-20 developed five novel mutations (E-V358I, NS1-Y/F/I113L, NS4A-T/A89V, NS4B-D/E/N/C22S, and NS5-E638G) at loci that were relatively conserved previously. The results of this study demonstrate the continuous circulation and unique evolution of TMUV in mosquitoes in Yunnan province and suggest that appropriate surveillance should be taken.

Establishment of a new intestinal cell line from the grouper (Epinephelus coioides) for aquatic virus pathogenesis

A new cell line (ECGI-21) was developed from the intestine of grouper (Epinephelus coioides). The cell line was routinely grown in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum (FBS) at 28 °C. ECGI-21 cells had an epithelioid morphology. At present, the cell line has been stably passed over 80 generations and has been successfully cryopreserved. The origin of grouper was confirmed by the sequence of 18S ribosomal RNA (18S rRNA). Chromosome analysis indicated that the modal chromosome number was 48. The green fluorescence signals were detected when pEGFP-N3 was transfected into ECGI-21 cells. Moreover, the ECGI-21 cells were susceptible to Singapore grouper iridovirus (SGIV) and Red-spotted grouper nervous necrosis virus (RGNNV), which was demonstrated by the presence of a significant cytopathic effect (CPE), increased expression of viral proteins after viral infection. Meantime, abundant viral particles were observed in the cells infected with SGIV and RGNNV by electron microscopy. Furthermore, virus replication significantly increased the level of interferon (IFN) related signaling molecules and pro-inflammatory cytokine expression in cells. In summary, ECGI-21 cell line was established and become the first stable intestinal cell line from marine fish. It provides a useful tool for studying problems of gastrointestinal health, viral pathogenesis and virus-host interaction in vitro.

A microfluidic cell chip for virus isolation via rapid screening for permissive cells.

Virus identification is a prerequisite not only for the early diagnosis of viral infectious diseases but also for the effective prevention of epidemics. Successful cultivation is the gold standard for identifying a virus, according to the Koch postulates. However, this requires screening for a permissive cell line, which is traditionally time-, reagent- and labor-intensive. Here, a simple and easy-to-operate microfluidic chip, formed by seeding a variety of cell lines and culturing them in parallel, is reported for use in virus cultivation and virus-permissive host-cell screening. The chip was tested by infection with two known viruses, enterovirus 71 (EV71) and influenza virus H1N1. Infection with EV71 and H1N1 caused significant cytopathic effects (CPE) in RD and MDCK cells, respectively, demonstrating that virus cultivation based on this microfluidic cell chip can be used as a substitute for the traditional plate-based culture method and reproduce the typical CPE caused by virus infection. Using this microfluidic cell chip method for virus cultivation could make it possible to identify an emerging virus in a high-throughput, automatic, and unprecedentedly fast way.

Association of Blastocystis hominis with colorectal cancer: A systematic review of in vitro and in vivo evidences.

BACKGROUNDRecently, there have been several findings that showed intestinal colonisation of Blastocystis hominis (Blastocystis) as a risk factor to the worsening of colorectal cancer (CRC). However, studies have shown controversial results in the pathogenicity of Blastocystis.AIMTo review systematically the evidence available on the association between CRC and Blastocystis and the prevalence of Blastocystis in CRC patients and to investigate cytopathic and immunological effects of Blastocystis in in vitro and in vivo studies.METHODSPRISMA guidelines were utilised in conducting this systematic review. Original articles published before February 2, 2020 were included. PubMed, Science Direct, Scopus and Google scholar databases were searched. Manual searching was carried out to find articles missed during the online search.RESULTSOut of 12 studies selected for this systematic review, seven studies confirmed the prevalence of Blastocystis and found it to be between 2%-28% in CRC patients, whereby subtype 1 and subtype 3 were predominantly seen. A total of four studies employing in vitro human colorectal carcinoma cell line study models showed significant cytopathic and immunological effects of Blastocystis. In addition, one in vivo experimental animal model study showed that there was a significant effect of infection with Blastocystis on exacerbation of colorectal carcinogenesis.CONCLUSION Blastocystis is a commonly identified microorganism in CRC patients. These studies have provided supportive data that Blastocystis could exacerbate existing CRC via alteration in host immune response and increased oxidative damage. Future studies of CRC and Blastocystis should attempt to determine the various stages of CRC that are most likely to be associated with Blastocystis and its relationship with other intestinal bacteria.

Investigation on Natural Infection of Covert Mortality Nodavirus in Large Yellow Croaker, Larimichthys crocea

Covert mortality nodavirus (CMNV) recently became more prevalent, severely affecting farmed shrimps, and also other invertebrates and teleost fish, in the coastal areas of China. For better understanding of its prevalence and susceptibility of large yellow croaker Larimichthys crocea to CMNV, an epidemiological survey was carried out in the main producing areas (Ningbo, Zhejiang, China, and Ningde, Fujian, China) and its offshore feeding grounds in the Southern Yellow Sea. The collected cultured L. crocea individuals showed clinical symptoms such as anorexia and abnormal swimming and diagnosed for CMNV infection by using RT-nPCR assay. The positive rates for CMNV in the collected L. crocea samples from Ningbo, Ningde, and the Yellow Sea were 14.29% (4/28), 20.00% (7/35), and 16.67% (1/6), respectively. Furthermore, CMNV diagnosis using TaqMan probe-based reverse transcription quantitative PCR (TaqMan RT-qPCR) yielded positive rates of 25.00% (7/28), 22.86% (8/35), and 16.67% (1/6) respectively in the above-mentioned samples, and with a range of 12.73–3,108.33 of CMNV copy numbers/μg total RNA. Phylogenetic tree analysis based on the RNA-dependent RNA polymerase (RdRp) and capsid protein genes showed that CMNV isolates from L. crocea samples were clustered tightly with the original isolate of CMNV from Penaeus vannamei. The histopathological studies indicated that severe cellular necrosis occurred in the heart, liver, spleen, eye, and gill of naturally infected fish. Stronger positive signals for CMNV-specific probes appeared in the necrotic cells and tissues in the in situ hybridization (ISH) analysis. Transmission electron microscopy (TEM) revealed the presence of numerous 30 nm diameter CMNV-like viral particles. The purified CMNV suspension could cause a significant cytopathic effect (CPE) in epithelioma papilloma cyprini (EPC) cells and resulted in the formation of typical inclusion bodies, and also visible CMNV particles around the lysed EPC cells under the TEM. Taken together, all results of this study demonstrated that L. crocea is susceptible to CMNV. The prevalence of CMNV and its potential impacts on the wild population of L. crocea see the need for further investigations.

Isolation and characterization of a novel strain of duck aviadenovirus B from Muscovy ducklings with acute hepatitis in China.

A new disease designated as Pale liver disease (PLD) has been circulating in Chinese Muscovy duck flocks since 2014, which is characterized by fatigue, diarrhoea, sudden death and acute hepatitis with pale and haemorrhagic liver. In this study, the etiological agents of PLD were isolated, causing a significant cytopathic effect (CPE) by cell rounding. Virus particles were observed by transmission electron microscopic (TEM) observation. The same disease was reproduced by experimental infection with the isolate BG61. The whole genomes of isolates were 43,842nt in length with a GC content of 47.11%, similar to French Muscovy duck adenovirus strain GR with a GC content of 46.08%. The isolates shared 99.71-99.95% and 93.31-93.33% identity with Chinese Muscovy duck adenovirus isolates and GR strain, respectively. The DNA polymerase gene of all Muscovy duck adenovirus strains formed a separate genetic lineage with 99.55-100% amino acid sequence identity. All Chinese Muscovy duck adenovirus isolates contained two fibre genes. In contrast, only one fibre gene was found in GR, the only representative strain in species Duck aviadenovirus B. Anti-DAdV-2serumantibodies had a weakneutralizingactivity against Chinese Muscovy duck adenovirus isolates. The phylogenetic trees of the complete genome, hexon and fibre proteins revealed that all Muscovy duck adenovirus strains formed a major genetic lineage consisting of two clades. Thus, both GR and Chinese Muscovy duck adenovirus strains were proposed to be included in the same species of Duck aviadenovirus B belonging to the genus Aviadenovirus. The species Duck aviadenovirus B included two serotypes or genotypes, such as GR, which represents the strain of serotype 1 or genotype 1 (DAdV B1) and Chinese Muscovy duck adenovirus strains, which belong to serotype 2 or genotype 2 (DAdV B2).

Susceptibility of a cell line derived from the kidney of Chinese rice-field eel, Monopterus albus to the infection of rhabdovirus, CrERV.

Chinese rice-field eels rhabdovirus (CrERV), belonging to the genus Perhabdovirus in the family Rhabdoviridae, is the causative agent of the haemorrhagic disease of Chinese rice-field eels, Monopterus albus. The present study aims to establish a cell line derived from the kidney of Chinese rice-field eel (CrEK) for the further study of the pathogenic virus. CrEK cells were epithelioid-like and grew well in M199 medium supplemented with 10% foetal bovine serum at 28°C, and the cell line has been subcultured for more than 80 times. Karyotyping analysis of CrEK cells at 25th passage indicated a modal chromosome number of 24. Significant cytopathic effect (CPE) was observed in CrEK cells after infection with CrERV, and the virus titre reached 107.8±0.45 TCID50 /mL. The transmission electron microscopy revealed that there were a large number of virus particles in the cytoplasm of cells. The virus infection in cells was also assayed by using indirect immunofluorescence assay (IFA), fluorescence in situ hybridization (FISH), reverse transcription PCR (RT-PCR) and quantitative real-time reverse transcription-PCR (qRT-PCR). In experimental infection, CrERV cultured by cells could cause over 90% mortality in fish. CrEK represents the first kidney cell line originated from Chinese rice-field eels and be a potential material for investigating the mechanism of virus infection in this fish and the control methods for the disease.

Human parainfluenza virus type 1 regulates cholesterol biosynthesis and establishes quiescent infection in human airway cells

Human parainfluenza virus type 1 (hPIV1) and 3 (hPIV3) cause seasonal epidemics, but little is known about their interaction with human airway cells. In this study, we determined cytopathology, replication, and progeny virion release from human airway cells during long-term infection in vitro. Both viruses readily established persistent infection without causing significant cytopathic effects. However, assembly and release of hPIV1 rapidly declined in sharp contrast to hPIV3 due to impaired viral ribonucleocapsid (vRNP) trafficking and virus assembly. Transcriptomic analysis revealed that both viruses induced similar levels of type I and III IFNs. However, hPIV1 induced specific ISGs stronger than hPIV3, such as MX2, which bound to hPIV1 vRNPs in infected cells. In addition, hPIV1 but not hPIV3 suppressed genes involved in lipid biogenesis and hPIV1 infection resulted in ubiquitination and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate limiting enzyme in cholesterol biosynthesis. Consequently, formation of cholesterol-rich lipid rafts was impaired in hPIV1 infected cells. These results indicate that hPIV1 is capable of regulating cholesterol biogenesis, which likely together with ISGs contributes to establishment of a quiescent infection.

Retraction Note: VP2 capsid domain of the H-1 parvovirus determines susceptibility of human cancer cells to H-1 viral infection.

Although H-1 parvovirus is used as an antitumor agent, not much is known about the relationship between its specific tropism and oncolytic activity. We hypothesize that VP2, a major capsid protein of H-1 virus, determines H-1-specific tropism. To assess this, we constructed chimeric H-1 viruses expressing Kilham rat virus (KRV) capsid proteins, in their complete or partial forms. Chimeric H-1 viruses (CH1, CH2 and CH3) containing the whole KRV VP2 domain could not induce cytolysis in HeLa, A549 and Panc-1 cells. However, the other chimeric H-1 viruses (CH4 and CH5) expressing a partial KRV VP2 domain induced cytolysis. Additionally, the significant cytopathic effect caused by CH4 and CH5 infection in HeLa cells resulted from preferential viral amplification via DNA replication, RNA transcription and protein synthesis. Modeling of VP2 capsid protein showed that two variable regions (VRs) (VR0 and VR2) of H-1 VP2 protein protrude outward, because of the insertion of extra amino-acid residues, as compared with those of KRV VP2 protein. This might explain the precedence of H-1 VP2 protein over KRV in determining oncolytic activity in human cancer cells. Taking these results together, we propose that the VP2 protein of oncolytic H-1 parvovirus determines its specific tropism in human cancer cells.

Generation of Combinatorial Lentiviral Vectors Expressing Multiple Anti-Hepatitis C Virus shRNAs and Their Validation on a Novel HCV Replicon Double Reporter Cell Line.

Despite the introduction of directly acting antivirals (DAAs), for the treatment of hepatitis C virus (HCV) infection, their cost, patient compliance, and viral resistance are still important issues to be considered. Here, we describe the generation of a novel JFH1-based HCV subgenomic replicon double reporter cell line suitable for testing different antiviral drugs and therapeutic interventions. This cells line allowed a rapid and accurate quantification of cell growth/viability and HCV RNA replication, thus discriminating specific from unspecific antiviral effects caused by DAAs or cytotoxic compounds, respectively. By correlating cell number and virus replication, we could confirm the inhibitory effect on the latter of cell over confluency and characterize an array of lentiviral vectors expressing single, double, or triple cassettes containing different combinations of short hairpin (sh)RNAs, targeting both highly conserved viral genome sequences and cellular factors crucial for HCV replication. While all vectors were effective in reducing HCV replication, the ones targeting viral sequences displayed a stronger antiviral effect, without significant cytopathic effects. Such combinatorial platforms as well as the developed double reporter cell line might find application both in setting-up anti-HCV gene therapy approaches and in studies aimed at further dissecting the viral biology/pathogenesis of infection.

Identification and characterization of a new strain of nervous necrosis virus isolated from pearl gentian grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) in China

Viral nervous necrosis (VNN), caused by nervous necrosis virus (NNV), is a worldwide disease that severely imperils many kinds of marine and freshwater fish. In this study, a NNV strain CNPgg2018 isolated from pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) juveniles was identified and characterized by histopathology examination, transmission electron microscopic (TEM), reverse transcription-polymerase chain reaction (RT-PCR), virus culture, phylogenetic analysis and experimental infection. The results showed that the diseased fish exhibited symptoms with loss of appetite, darkened body and abnormal swimming behavior. Histopathological examination revealed widespread diffuse vacuolation degeneration appeared in the brain, retina and peripheral nerve tissues. TEM observation showed that numerous virions with a diameter of about 25 nm scattered around the retinal vacuolation. Significant cytopathic effects (CPE) were observed in striped snakehead (SSN-1) and grouper brain (GB) cell lines, not in Chinook salmon embryo (CHSE-214), epithelioma papillosum cyprini (EPC) and fat-head minnow (FHM) cell lines after inoculation of pathological tissue homogenates. RT-PCR and phylogenetic tree analysis indicated that CNPgg2018 was a member of NNV with highly homology to RGNNV genotype. Besides, both monomer and tripolymer forms of capsid protein were detected through structural polypeptide analysis and three-dimensional model prediction. Infection experiment demonstrated that CNPgg2018 was virulent to pearl gentian grouper juvenile with a LD50 of 1.0 × 106.1 TCID50/fish. In conclusion, we investigated a natural disease in pearl gentian grouper and confirmed that the pathogen was NNV. The results of this study provide new insights into the current research on the biology, ecology and host range of NNV.

Human Vaginal Epithelial Cells are Susceptible to the Zika Virus: Implications for Zika Vaginal Transmission

The Zika Virus (ZIKV) is an emerging RNA virus that cause congenital birth defects and neurological compilations among infected individuals. Since the 2015 ZIKV outbreak in Brazil, the Centers for Disease Control, and Prevention (CDC) reports over 35,000 cases ZIKV disease cases within the United States (US) and US territories. Although ZIKV is primarily transmitted through an infected Aedes mosquito, recent studies demonstrate this pathogenic virus can be transmitted via sexual contact with an infected partner. CDC confirmed multiple clinical cases of infected patients acquired ZIKV through sexual transmission within the US. Additionally, mounting evidence from animal and cell studies demonstrate that the female reproductive tract supports ZIKV replication following intravaginal exposure. Despite the growing evidence associating ZIKV to sexual transmission, the underlying molecular mechanism involving vaginal transmission remains elusive. The overarching goal is to examine the role of the vaginal tract in ZIKV sexual transmission on a molecular level. The vaginal tract has multiple layers of epithelial cells that constitutes as the first line of defense against foreign pathogens. Given the capacity of sexual intercourse, we hypothesize that the initial event for ZIKV vaginal transmission is most likely through direct viral uptake at the outermost epithelial layer of the vaginal tract. We aim to test our hypothesis by (1) characterizing the replication kinetics of ZIKV in human vaginal epithelial cells (hVECs) in vitro and (2) determining the functional role of the putative ZIKV entry receptor, AXL, in hVECs. Our recent findings indicate hVECs is a permissive cell target for local ZIKV replication and production. Vaginal infection resulted in de novo replication of the ZIKV RNA genome, active production of the viral envelope protein, and steady release of infectious viral particles. Cell viability studies further indicate that hVECs do not exhibit significant cytopathic effect following ZIKV exposure. Additional studies show that the flaviviral entry receptor AXL is endogenously expressed in hVECs on a protein, mRNA level, and surface level. When expression patterns were compared between Vero and 293T cell lines, our results show similar protein levels of endogenous AXL in both Vero and VK2E6E7 cell lines, which may suggest that ZIKV entry via AXL‐mediated pathway and the dynamics of viral growth act independently of one another during sexual transmission. Our AXL knockdown and overexpression studies validate that this tyrosine receptor kinase is involved in ZIKV vaginal entry. In the future, kinase inhibitory studies will be performed to further validate whether downstream signaling contributes to ZIKV replication and production in hVECs. The outcome of this research will provide further insight into host‐virus interactions on a molecular level, which can lead to devising therapeutic agents aimed at interfering with the pathology caused by the virus.Support or Funding Information• T32 Grant: NIH National Heart, Lung, and Blood Institute, Research Training in Cardiovascular Biology at Meharry

Development and characterization of a continuous cell line (EL) from the liver of European eel Anguilla anguilla.

In the present study, a new hepatic tissue‐origin cell line from European eel Anguilla anguilla has been developed and characterized. This cell line designated EL has been maintained in Leibovitz L‐15 supplemented with 10% fetal bovine serum over 72 months, and subcultured more than 90 times. The EL cell line consisted predominantly of fibroblast‐like cells, which could survive over 100 days in vitro, and could grow at 15–32°C. The optimum temperature for growth was 27°C. The chromosome analysis revealed a modal diploid karyotype of 2n = 38. The origin of this cell line was confirmed by the 18S recombinant (r)RNA sequencing. The susceptibility test indicated significant cytopathic effects in the EL cells with regard to the Rana grylio virus and the Herpesvirus anguillae. The viral replication was confirmed by transmission electron microscopy and polymerase chain reaction analysis. Following poly (I:C) exposure, the expression levels of the immune‐related molecules interferon regulatory factor‐7 (irf7) and transforming growth factor‐β (TGF‐β) were downregulated in EL cells, whereas the expression levels of the rf3 and the cytochrome P450 (CYP450) were upregulated. All four genes were significantly upregulated following inflammation by lipopolysaccharide (LPS). These data suggested the application of EL cell line for viral identification, as well as for immunodiagnosis and pharmacological targeting.

Attenuated measles vaccine strain have potent oncolytic activity against Iraqi patient derived breast cancer cell line.

BackgroundOne of the world's leading causes of death among females is breast cancer. Oncolytic viruses are promising anticancer therapy that can overcome resistance to current conventional therapies. Measles virus replicates in and destroys malignant cells without affecting healthy cells. The study aimed to evaluate the lives attenuated Measles virus vaccine against Iraqi patient derived breast cancer cells that have functional BRCA1/BRCA2 genes and compare its activity against international breast cancer MCF-7 and CAL-51 cell lines.MethodsThe virus was propagated in VERO-hSLAM slam cells. The MTT cytotoxicity assay used to test the virus's ability to kill three human breast cell lines (AMJ13), (MCF-7), and (CAL-51). The cytopathic effect of the measles virus was determined using an H&E stain. Immunocytochemistry assay using specific anti H protein monoclonal antibody for measles virus in the virally infected cells. Finally, apoptosis induction in the infected cells tested using double staining of acridine orange/propidium iodide.ResultsThe result shown that breast cancer cells are effectively infected and destroyed by live attenuated measles virus vaccine, and it caused a significant cytopathic effect in the infected cell lines after 48–72 h of infection with remarkable effect on AMJ13 cells (IC50 was 3.527 for AMJ13, when it was 5.079 and 9.171 for MCF-7 and CAL-51 respectively). Measles virus treatment induces apoptosis significantly in breast cancer cell lines compared with control cells.ConclusionMeV vaccine is useful and safe as anticancer therapy with a notable impact on the local Iraqi breast cancer AMJ13 cells.