Apparent Cytopathic Effect Research Articles

Screening and identification of persistent viruses in cell lines derived from medically important arthropods.

Arthropod-derived cell lines serve as crucial tools for studying arthropod-borne viruses (arboviruses). However, it has recently come to light that certain cell lines harbor persistent infections of arthropod-specific viruses, which do not cause any apparent cytopathic effects. Moreover, some of these persistent viral infections either inhibit or promote the growth of arboviruses. Therefore, it is of utmost importance to identify the presence of such persistent viruses and understand their impact on arboviral infections. In this study, we conducted a comprehensive virome analysis of several arthropod-derived cell lines, including mosquito-derived NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, CCL-126 cells, and tick-derived IDE8 cells, along with flesh fly-derived NIH-Sape-4 cells. The aim was to determine if these cells were infected with persistent viruses. The results revealed the presence of 15 persistent viruses in NIID-CTR, Ar-3, MSQ43, NIAS-AeAl-2, and IDE8 cells. Among these, 11 were already known arthropod-specific viruses, while the remaining 4 were novel viruses belonging to Orthophasmavirus, Rhabdoviridae, Totiviridae, and Bunyavirales. In contrast, CCL-126 and NIH-Sape-4 cells appeared to be free of viral infections. This study provides valuable insights into the diversity and latency of arthropod-specific viruses within arthropod-derived cell lines. Further investigations are required to explore persistent viral infections in other arthropod-derived cell cultures and their effects on arbovirus replication. Understanding these factors will enhance the accuracy and reliability of experimental data obtained using these cell lines.

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

An effective live-attenuated Zika vaccine candidate with a modified 5′ untranslated region

Zika virus (ZIKV) is a mosquito-transmitted flavivirus that has caused devastating congenital Zika syndrome (CZS), including microcephaly, congenital malformation, and fetal demise in human newborns in recent epidemics. ZIKV infection can also cause Guillain-Barré syndrome (GBS) and meningoencephalitis in adults. Despite intensive research in recent years, there are no approved vaccines or antiviral therapeutics against CZS and adult Zika diseases. In this report, we developed a novel live-attenuated ZIKV strain (named Z7) by inserting 50 RNA nucleotides (nt) into the 5′ untranslated region (UTR) of a pre-epidemic ZIKV Cambodian strain, FSS13025. We used this particular ZIKV strain as it is attenuated in neurovirulence, immune antagonism, and mosquito infectivity compared with the American epidemic isolates. Our data demonstrate that Z7 replicates efficiently and produces high titers without causing apparent cytopathic effects (CPE) in Vero cells or losing the insert sequence, even after ten passages. Significantly, Z7 induces robust humoral and cellular immune responses that completely prevent viremia after a challenge with a high dose of an American epidemic ZIKV strain PRVABC59 infection in type I interferon (IFN) receptor A deficient (Ifnar1−/−) mice. Moreover, adoptive transfer of plasma collected from Z7 immunized mice protects Ifnar1−/− mice from ZIKV (strain PRVABC59) infection. These results suggest that modifying the ZIKV 5′ UTR is a novel strategy to develop live-attenuated vaccine candidates for ZIKV and potentially for other flaviviruses.

Virus spread in the liver: mechanisms, commonalities, and unanswered questions.

The liver is home to five known human hepatitis viruses (hepatitis A virus-hepatitis E virus). Despite being phylogenetically unrelated, these viruses replicate and spread in the liver without causing apparent cytopathic effects, and all have evolved strategies to counteract antibody-mediated inhibition of virus spread. In this review, we discuss the current understanding regarding the spread mechanisms for these viruses with an attempt to extract common principles and identify key questions for future studies.

Latent pseudorabies virus infection in medulla oblongata from quarantined pigs.

Pseudorabies virus (PRV) is a major pathogen in pig husbandry and is also a risk to human well-being. Pigs with latent PRV infection carry the virus lifelong, and it can be activated under conducive conditions. This poses a very important challenge to the control of the virus and may even prevent its elimination. To investigate latent infection with wild-type (wt) PRV, and also infection due to the use of live attenuated vaccines on farms, 80 pigs from two large-scale swine operations were traced. At 6months old, the quarantined pigs were slaughtered and brain samples were collected. A PCR assay targeting the gB and gE genes was developed to detect PRV DNA fragments in medulla oblongata. Five of the samples (6.3%) were gB and gE gene fragment double-positive, 60 of the samples (75%) were gB single-positive, and 15 samples (18.7%) showed double-negative. A portion of latency-associated transcripts (LATs), EP0 mRNA, were found to be present in the gB gene fragment positive samples. Furthermore, the five double-positive samples were transmitted blindly, and apparent cytopathic effects were found in three of the five samples in the fourth generation. By means of Western blotting, PCR and sequencing, two of the isolated viruses were found to be related to vaccine strain Bartha-K61. Another was closely related to domestic epidemic strains HN1201 and LA and relatively unrelated to other Asian isolates. These results suggest that the live vaccines are latently present in brains, in a manner similar to wt PRV, and this poses potential safety issues in the pig husbandry industry. Wt PRV and live vaccine viruses were found to co-exist in pigs, demonstrating that the live vaccines were unable to confer complete sterilizing immunity, which may explain outbreaks of pseudorabies on vaccinated farms.

Genome Analysis of a Novel Tembusu Virus in Taiwan.

We identified and isolated a novel Tembusu virus (TMUV) strain TP1906 (TMUV-TP1906) from a Culex annulus mosquito pool collected from the northern part of Taiwan in 2019. The TMUV-TP1906 genome is a 10,990-nucleotide-long, positive-sense, single-stranded RNA, consisting of a single open reading frame (ORF) encoding a polyprotein of 3425 amino acids, with 5′ and 3′ untranslated regions (UTRs) of 94 and 618 nucleotides, respectively. The nucleotide sequence of the TMUV-TP1906 of ORF exhibited 93.71% and 91.27% similarity with Sitiawan virus (STWV) and the TMUV prototype strain MM1775, respectively. The 3′-UTR variable region of TMUV-TP1906 showed nucleotide sequence divergence with other TMUV strains. Phylogenetic analysis of the complete ORF and polyprotein sequences revealed that TMUV-TP1906 is most closely related to STWV which causes encephalitis and retarded growth in chickens. We found that the TMUV-TP1906 caused a cytopathic effect (CPE) in the DF-1 chicken fibroblast cell line, while no apparent CPE was observed in Vero and C6/36 cells. In this study, we first identified and isolated a novel TMUV strain in Taiwan. In addition, to our knowledge, it is the first time that the TMUV strain was isolated from the Cx. annulus mosquitoes. Further study is warranted to investigate the host range and virulence of TMUV-TP1906.

Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virus in vitro

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.

Efficient isolation of human parainfluenza viruses 1 and 3 using MNT-1, a human malignant melanoma cell line system that exhibits an apparent cytopathic effect.

Isolation of human parainfluenza virus (HPIV) serotypes 1 and 3 from clinical specimens is not very efficient because of the lack of a cell culture system capable of inducing CPE. In this study, the utility of a melanoma cell line, MNT-1, that allows HPIV growth and displays CPE was demonstrated. In particularly, the efficiency of isolating HPIV1 and HPIV3 using MNT-1 was greater than for cell lines conventionally used for HPIV isolation. Our demonstrated efficacy of HPIV1 and HPIV3 isolation with apparent CPE using the MNT-1 cell culture system has the potential to improve virus isolation from clinical specimens.

Different modes of herpes simplex virus type 1 spread in brain and skin tissues

Herpes simplex virus type 1 (HSV-1) initially infects the skin and subsequently spreads to the nervous system. To investigate and compare HSV-1 mode of propagation in the two clinically relevant tissues, we have established ex vivo infection models, using native tissues of mouse and human skin, as well as mouse brain, maintained in organ cultures. HSV-1, which is naturally restricted to the human, infects and spreads in the mouse and human skin tissues in a similar fashion, thus validating the mouse model. The spread of HSV-1 in the skin was concentric to form typical plaques of limited size, predominantly of cytopathic cells. By contrast, HSV-1 spread in the brain tissue was directed along specific neuronal networks with no apparent cytopathic effect. Two additional differences were noted following infection of the skin and brain tissues. First, only a negligible amount of extracellular progeny virus was produced of the infected brain tissues, while substantial quantity of infectious progeny virus was released to the media of the infected skin. Second, antibodies against HSV-1, added following the infection, effectively restricted viral spread in the skin but have no effect on viral spread in the brain tissue. Taken together, these results reveal that HSV-1 spread within the brain tissue mostly by direct transfer from cell to cell, while in the skin the progeny extracellular virus predominates, thus facilitating the infection to new individuals.

Infectious pancreatic necrosis virus (IPNV) from salmonid fish enters, but does not replicate in, mammalian cells

BackgroundThe aquatic birnavirus infectious pancreatic necrosis virus (IPNV) causes infectious pancreatic necrosis (IPN), a severe disease in farmed salmonid fish. IPNV has a very broad host range and infects many different species of fish as well as molluscs and crustaceans. Investigation of the host reservoir of a virus may reveal important molecular mechanisms governing the infection processes such as receptors and entry mechanisms. In the present work we have studied whether IPNV is able to infect cells with different mammalian origin.ResultsIPNV bound in a specific manner to a membrane protein of the rabbit kidney cell line RK-13 as shown by the use of a virus overlay protein binding assay (VOPBA). Six different mammalian cell lines were inoculated with IPNV and incubated in parallels at different temperatures. At 7 days post inoculation (dpi), IPNV was detected by indirect immunofluorescent antibody test (IFAT) in all the cell lines. Confocal microscopy confirmed intracellular presence of the virus. No apparent cytopathic effect (cpe) was observed in any of the cultures, and no viral replication was demonstrated with real-time RT-PCR.ConclusionOur results show that IPNV is able to enter into a wide range of mammalian cells, and virus entry is most likely receptor mediated. We found no indication of IPNV replication in any of the mammalian cell lines tested.

Identification of human parechovirus genotype, HPeV-12, in a paralytic child with diarrhea

BackgroundNew genotypes of human parechoviruses have been readily identified after improvement of diverse diagnostic tools. We hereby report the detection of a new genotype, HPeV 12, from a child presented with diarrhea and paralysis. ObjectivesThe genetic variability of human parechoviruses has recently expanded defining 16 genotypes however data available covers only 11 genotypes. The present study was designed to determine the genetic characterization of human parechovirus identified in a child with gastroenteritis and acute flaccid paralysis (AFP). Study designStool samples are referred to Virology Department, NIH-Pakistan for the routine detection of enteroviruses and polioviruses through cell culture and RT-PCR. Five of isolates showing cytopathic effect on L20B cell line but negative for poliovirus were further explored for human parechovirus using multiple cell lines and RT-PCR. ResultsHuman Coxsackie A virus type 2, 3, 6 and 20 were found in four samples whereas the fifth sample contained human parechovirus genotype 12. Efficient growth of human parechovirus was found on L20B cells while Vero and LLC-MK2 cells showed no apparent cytopathic effect. ConclusionsThis study describes the detection of a new human parechovirus genotype (HPeV-12) in a paralytic child with diarrhea. Human parechoviruses are now considered as potential pathogens that may cause a number of serious clinical complications especially in infants and young children. These findings emphasize to conduct large scale epidemiological surveys in the country to understand their association with clinical diseases especially gastroenteritis, respiratory and neurological disorders.

Des-aspartate-angiotensin I exerts antiviral effects and attenuates ICAM-1 formation in rhinovirus-infected epithelial cells

The high frequency of rhinovirus (RV) infection and the lack of an effective treatment, underline the importance of research on novel anti-rhinoviral agents. The present study investigated the effects of des-aspartate-angiotensin I (DAA-I) on the survival of RV14-infected H1HeLa cells; and the early inflammatory processes in RV14-infected A549 lung epithelial cells. The study rationale was based on earlier findings showing that DAA-I is an effective anti-inflammatory agent, and that symptoms and severity of rhinoviral infection are related to the underling inflammation. RV14 concentration dependently caused the death of H1HeLa cells and DAA-I, at concentrations of 10−10 to 10−12M, attenuated the lethal action of RV14 indicating that that DAA-I exerts antiviral action. Unlike its action on H1HeLa cells, RV14 did not cause apparent cytopathic effect on A549 cells, and these cells were used to study the antiviral action of DAA-I. RV14 induced overexpression of ICAM-1, E-selectin and overproduction of superoxide in A549 cells, and DAA-I attenuated the three increases to basal level at concentrations of 10−10 to 10−12M. Losartan, an angiotensin AT1 receptor antagonist, blocked the inhibitory action of DAA-I on superoxide overproduction indicating that the AT1 receptor mediates the action of DAA-I. The present data represent a novel demonstration of the antiviral action of an angiotensin peptide, and a possible involvement of the renin angiotensin system in viral infection. Indeed the angiotensin AT1 receptor has been reported to be obligatory for the development of virus-induced myocardial injury through the proinflammatory action of angiotensin II via the NF-κB/cytokine pathway.

Functional Analysis of Two Cavities in Flavivirus NS5 Polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines

BackgroundsThe aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance.MethodsThe growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST) cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains) and by observing the development of cytopathic effect (CPE) in infected cultures of hamster cell lines.ResultsEleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively.ConclusionThe present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

Isolation of Novel Adenovirus from Fruit Bat (Pteropus dasymallus yayeyamae)

To the Editor: Bats are thought to be one of the most important reservoirs for viruses such as Nipah virus, severe acute respiratory syndrome (SARS) coronavirus, and Ebola virus (1). These pathogens became known after extensive surveys of bats following outbreaks. As a first step in investigating unidentified pathogens in bats and to help forecast the potential threat of emerging infectious diseases, we tried to isolate and characterize viruses that persistently infect bats. In the process, we isolated a novel adenovirus from a fruit bat in Japan. Pteropus dasymallus yayeyamae, or Ryukyu flying fox, is a fruit bat of Japan. With the permission of the governor of Okinawa, we caught 1 adult male bat of this species and used its spleen and kidneys to establish primary cell cultures. On the 4th passage of the primary adherent cells derived from the spleen, a cytopathic effect (CPE) appeared without any visible microbe, indicating that the cell culture contained a virus. The virus, tentatively named Ryukyu virus 1 (RV1), caused apparent CPE on primary kidney cells derived from a Ryukyu flying fox and on our established bat kidney T1 (BKT1) cells, which were derived from the kidney of a horseshoe bat (Rhinolophus ferrumequinum) and transformed with expression plasmid DNA encoding the large T antigen of replication origin-defective simian virus 40. To identify the virus, RV1, we applied the rapid determination of viral RNA (RDV) system version 1.0 (2). However, no viral nucleic acid sequence was detected from an RNA sample in the RV1-infected BKT1 cells. For detection of viral DNA, we developed a system for rapid determination of viral DNA sequences (RDV-D) by minor modification to the RDV system for RNA viruses (2–4). The results indicated that 2 of the fragments were homologous to the gene encoding the precursor of terminal protein (pTP) of adenoviruses. Further RDV-D analysis showed that 6 fragments (139 bp, DDBJ/EMBL/GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB302970,term_id:159576533,term_text:AB302970}}AB302970) were homologous to the pTP gene and that another 6 fragments (316bp, DDBJ/EMBL/GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB302971,term_id:159576535,term_text:AB302971}}AB302971) were homologous to the gene encoding the precursor of protein VI (pVI) of adenoviruses. These results indicated that RV1 must belong to the family Adenoviridae. To further confirm that RV1 isolate was an adenovirus, we used PCR and sequencing. We performed the first reaction with the outer primer pair (polFouter and polRouter) of a nested PCR method, targeting the viral DNA polymerase gene with highly degenerate consensus primers that have been described recently (5). A fragment of ≈550 bp was amplified from RV1 as well as from human adenoviruses-1, -3, -4, and -7 (data not shown). Sequence analysis of the amplified product (DDBJ/EMBL/GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB303301,term_id:159885613,term_text:AB303301}}AB303301) showed that RV1 was homologous to tree shrew adenovirus 1 (70.0% amino acid sequence identity), porcine adenovirus 5 (69.2%), canine adenovirus 1 (68.9%), human adenoviruses-3, -16, -21 and -50 (68.9%), and other viruses (>64.8%) in genus Mastadenovirus, but less homologous (46.7%–57.8%) to viruses in other genuses, Siadenovirus, Aviadenovirus, and Atadenovirus. In addition, a phylogenic tree based on amino acid sequences indicated that RV1 belongs to family Adenoviridae, genus Mastadenovirus (Figure). Figure Phylogeny of adenoviruses based on analysis of partial amino acid sequences of DNA polymerase protein. Trees were estimated by using the neighbor-joining method based on the amino acid pairwise distance and MEGA 4.0 software (www.megasoftware.net). Numbers ... Electron microscopy of RV1-infected BKT1 cells indicated that RV1 accumulated in the nucleus and that the size of capsids was 60–70 nm (data not shown). Restriction endonuclease analysis of the RV1 genome indicated that the genome was ≈20–30 kbp (data not shown). These features are consistent with RV1 being an adenovirus. Until now, a number of RNA viruses have been isolated from bats, but isolation of DNA virus is rare (1). The isolation of the novel adenovirus seems to be possible because of usage of the primary cells originated from the host; DNA viruses might have more restricted host range than RNA viruses and require host-originated cells for the growth. In addition, our success in DNA virus isolation might have resulted from usage of the adult animal latently and persistently infected with DNA viruses such as adenovirus and herpesvirus. In conclusion, we isolated a novel virus from a fruit bat. This virus was isolated from a healthy bat, which suggests that the virus may persistently infect fruit bats. Although its pathogenicity for humans is still unknown, knowledge of RV1 will be useful in epidemiologic studies of infectious diseases emerging from bats because persistently infecting viruses might be isolated together with primary pathogens. We are planning to establish cell lines from bats and isolate more viruses from persistently infected bats.

Intracellular Restriction of a Productive Noncytopathic Coronavirus Infection

Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 10(7) to 10(8) PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition.

Susceptibility of human and rat neural cell lines to infection by SARS-coronavirus

Pathological characterization of autopsied tissues from patients with SARS revealed severe damage in restricted tissues, such as lung, with no apparent cell damage in other tissues, such as intestine and brain. Here, we examined the susceptibility of neural cell lines of human (OL) and rat (C6) origins to SARS-associated coronavirus. Both of the neural cell lines showed no apparent cytopathic effects (CPE) by infection but produced virus with infectivity of 10 2–5 per ml, in sharp contrast to the production by infected Vero E6 cells of >10 9 per ml that showed a lytic infection with characteristic rounding CPE. Interestingly, the infection of intestinal cell line CaCo-2 also induced no apparent CPE, with production of the virus at a slightly lower level as that of the Vero E6 cell culture. Notably, the cellular receptor for the virus, angiotensin-converting enzyme 2 was expressed at similar levels on Vero E6 and CaCo-2 cells, but at undetectable levels on OL and C6 cells.

The simultaneous detection of both enteroviruses and adenoviruses in environmental water samples including tap water with an integrated cell culture-multiplex-nested PCR procedure

To evaluate an integrated cell culture (ICC)-multiplex-nested PCR using the buffalo green monkey kidney (BGMK) cells for the simultaneous detection of both enteroviruses and adenoviruses in surface water and tap water samples and optimize the procedure for more sensitive detection of virus showing no apparent cytopathic effect (CPE). A total of 69 surface water and 50 tap water samples were analysed by the ICC-multiplex-nested PCR. All the PCRs were performed five times with a cell lysate from each flask after at least 2 weeks incubation. Forty-six surface water samples (66.7%) and 23 tap water samples (46.0%) exhibited CPE by the cell culture method. By using an ICC-multiplex-nested PCR, 53 surface water samples (76.8%) and 29 tap water samples (58.0%) were determined as containing infectious enteric viral particles. An ICC-PCR method with a long incubation time using BGMK cells enables the simultaneous detection of enteroviruses and adenoviruses from environmental water samples, including tap water, even with low numbers of viruses. A method capable of detecting small numbers of viral particles is necessary.

Use of Cell Culture-PCR Assay Based on Combination of A549 and BGMK Cell Lines and Molecular Identification as a Tool To Monitor Infectious Adenoviruses and Enteroviruses in River Water

Viral contamination in environmental samples can be underestimated because a single cell line might reproduce only some enteric viruses and some enteric viruses do not exhibit apparent cytopathic effects in cell culture. To overcome this problem, we evaluated a cell culture-PCR assay based on a combination of A549 and Buffalo green monkey kidney (BGMK) cell lines as a tool to monitor infectious adenoviruses and enteroviruses in river water. Water samples were collected 10 times at each of four rivers located in Gyeonggi Province, South Korea, and then cultured on group 1 cells (BGMK cells alone) and group 2 cells (BGMK and A549 cells). Reverse transcription and multiplex PCR were performed, followed by phylogenetic analysis of the amplicons. Thirty (75.0%) of the 40 samples were positive for viruses based on cell culture, and the frequency of positive samples grown on group 2 cells (65.0%) was higher than the frequency of positive samples grown on group 1 cells (50.0%). The number of samples positive for adenoviruses was higher with A549 cells (13 samples) than with BGMK cells (one sample); the numbers of samples positive for enteroviruses were similar with both types of cells. By using phylogenetic analysis, adenoviral amplicons were grouped into subgenera A, C, D, and F, and enteroviral amplicons were grouped into coxsackieviruses B3 and B4 and echoviruses 6, 7, and 30, indicating that A549 and BGMK cells were suitable for recovering a wide range of adenoviral and enteroviral types. The cell culture-PCR assay with a combination of A549 and BGMK cells and molecular identification could be a useful tool for monitoring infectious adenoviruses and enteroviruses in aquatic environments.

Differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses.

Hantaviruses represent important human pathogens and can induce hemorrhagic fever with renal syndrome (HFRS), which is characterized by endothelial dysfunction. Both pathogenic and nonpathogenic hantaviruses replicate without causing any apparent cytopathic effect, suggesting that immunopathological mechanisms play an important role in pathogenesis. We compared the antiviral responses triggered by Hantaan virus (HTNV), a pathogenic hantavirus associated with HFRS, and Tula virus (TULV), a rather nonpathogenic hantavirus, in human umbilical vein endothelial cells (HUVECs). Both HTNV- and TULV-infected cells showed increased levels of molecules involved in antigen presentation. However, TULV-infected HUVECs upregulated HLA class I molecules more rapidly. Interestingly, HTNV clearly induced the production of beta interferon (IFN-beta), whereas expression of this cytokine was barely detectable in the supernatant or in extracts from TULV-infected HUVECs. Nevertheless, the upregulation of HLA class I on both TULV- and HTNV-infected cells could be blocked by neutralizing anti-IFN-beta antibodies. Most strikingly, the antiviral MxA protein, which interferes with hantavirus replication, was already induced 16 h after infection with TULV. In contrast, HTNV-infected HUVECs showed no expression of MxA until 48 h postinfection. In accordance with the kinetics of MxA expression, TULV replicated only inefficiently in HUVECs, whereas HTNV-infected cells produced high titers of virus particles that decreased after 48 h postinfection. Both hantavirus species, however, could replicate equally well in Vero E6 cells, which lack an IFN-induced MxA response. Thus, delayed induction of antiviral MxA in endothelial cells after infection with HTNV could allow viral dissemination and contribute to the pathogenesis leading to HFRS.