Use of A-549 cells in a clinical virology laboratory.

A-549 cells were compared with other cell lines for virus recovery, except from specimens submitted specifically for detection of cytomegalovirus. Of 589 specimens submitted specifically for detection of herpes simplex virus (HSV), 163 (28%) were positive for HSV--159 (97.5%) in A-549 cells and 156 (96%) in primary rabbit kidney cells. HSV cytopathic effect was identified an average of 0.6 day earlier in A-549 cells. Virus was recovered from 194 (11%) of 1,790 specimens submitted for general virus isolation. Of 40 HSV isolates, 85% were positive in A-549 cells, 72.5% were positive in MRC-5/WI-38 cells, and 42.5% were positive in HEp-2 cells. With adenovirus, 96% of 45 isolates were detected in A-549 cells, 62% were detected in HEp-2 cells, 38% were detected in MRC-5/WI-38 cells, and 31% were detected in PMK cells. Of the 76 enterovirus-positive specimens, 71% were positive in PMK cells, 62% were positive in A-549 cells, and 62% were positive in MRC-5/WI-38 cells. None of 12 respiratory syncytial virus, 14 rhinovirus, or 7 influenza A virus isolates were detected in A-549 cells. Of the cell lines examined, A-549 cells performed optimally for recovery of HSV and adenovirus, they allowed good growth of many of the enterovirus isolates, but they did not allow recovery of any of the respiratory syncytial virus, rhinovirus, or influenza A virus isolates.     

Advantages of multiple cell culture systems for detection of mixed-virus infections.

Simultaneous infections by two or more viruses occur frequently, especially in immunosuppressed patients. In order to detect more than one viral agent in a single specimen, multiple cell systems have been employed in our laboratory. Specimens are routinely inoculated into four different cell cultures, namely: MRC-5, a human diploid lung fibroblast cell strain; A549, a human continuous cell line; primary guinea pig embryo (GPE) cell culture, and primary rhesus monkey kidney (RhMK) cell culture. For rapid detection of cytomegalovirus (CMV) antigen, MRC-5 cells grown in shell vials containing coverslips are also inoculated with the same specimens followed by centrifugation. During 1989, nine cases of multiple-virus isolations were obtained in this laboratory. In all nine patients, CMV was detected in MRC-5 cells. Five of the nine cases were co-infected with HSV-1, three were co-infected with adenovirus, and one was co-infected with both HSV-1 and adenovirus. All four adenovirus isolates were obtained in A549 cells. Of the six HSV-1 isolates, one was detected in all three cell cultures, e.g. MRC-5, A549 and GPE; one was detected in both MRC-5 and A549 cells, and four were isolated in a single-cell type only. For nine CMV-positive cases, five were obtained by both conventional and centrifugation cultures, two each were detected by centrifugation or conventional culture only. Thus for a maximum detection of viruses present in a single specimen, it is suggested that multiple-cell-culture systems, together with more than one technique, should be employed.     

A-549 is a suitable cell line for primary isolation of coxsackie B viruses

A common receptor for coxsackie B virus and adenovirus has been described recently in cells of human and murine origin. Since the established cell line A-549 is suitable for adenoviruses, the potential use of A-549 cells for the isolation of coxsackie B viruses from clinical samples was investigated. All throat swabs sent to the laboratory between April 1998 and June 1999 were inoculated onto monolayers of MRC-5 and A-549 cells in tubes, and the enterovirus isolates obtained were typed. From April to June 1999, A-549 cells were compared prospectively to Buffalo green monkey (BGM) cells, considered as the most susceptible cell line for isolating coxsackie B viruses. Fifty-six out of 171 enterovirus isolates (33%) displayed a cytopathic effect (CPE) in the A-549 monolayer only, 48 isolates (28%) in the MRC-5 monolayer only, and 67 isolates (39%) in both cell lines. Most isolates that showed CPE in A-549 cells only (48 out of 56, 86%) were coxsackie B viruses, belonging to four different serotypes (B1, B2, B4, and B6). When BGM and A-549 cells were inoculated in parallel, both recovered the same number of coxsackie B isolates (n = 20), and the CPE was noted on approximately the same day. In conclusion, growth in A-549 but not MRC-5 cells identified coxsackie B viruses in most cases. A-549 was comparable to BGM for primary isolation of coxsackie B viruses.     

Use of serum substitutes for the growth of four cell lines commonly used for virus isolation

Four serum extenders (Opti-MEM I, BioRich, Excyte I and Excyte III) and two serum substitutes (Omni Serum and Serum Plus) were evaluated for the reduction or replacement of FBS for the growth and maintenance of four representative cell lines used for virus isolation. MRC-5, human fetal foreskin fibroblast (HF), A549 and Hep-2 cells were seeded into culture flasks containing MEM with 10% FBS or with the serum extenders or substitutes and subcultured into 16 x 125 mm glass tubes and 1-dram shell vials. Only cells cultured with Opti-MEM I and Omni Serum grew consistently in tubes and vials and these reagents were compared to FBS for viral isolation and detection. Laboratory stocks of CMV, HSV, VZV, adenovirus (Ad) and RSV were tested. In HF and MRC-5 cells, CMV was isolated in cells cultured in either Opti-MEM I or Omni Serum before CPE appeared in cultures containing FBS. Ad and VZV CPE were observed first in HF and MRC-5 cells containing Omni Serum. HSV CPE was seen at the same time in HF and MRC-5 cells with all 3 media. HSV and Ad CPE in A549 cells were also seen at the same time in all 3 media. RSV and Ad CPE in Hep-2 cells were observed first in FBS containing media. Both Opti-MEM I and Omni Serum performed well as substitutes for FBS for growth of stock viruses without loss of cell sensitivity.     

Comparison of cell cultures for rapid isolation of enteroviruses.

Cell culture isolation is still the most reliable method for the detection of enteroviruses from clinical specimens. Rapid diagnosis of enterovirus infection affects patient management. To increase yield and enhance the rapidity of enterovirus isolation in cell cultures, we used Buffalo green monkey kidney (BGM) cells and subpassages of primary human embryonic kidney (HEK) cells in addition to the human diploid fibroblast (MRC-5) cells and primary cynomolgus or rhesus monkey kidney (MK) cells routinely used for enterovirus culturing. Growth characteristics of enteroviruses from 421 specimens were studied. All specimens were cultured in MRC-5, MK, and BGM cells, and 204 of these specimens were also cultured in HEK cells. Forty-two percent of the enteroviruses became positive within 3 days, and 85% did so within 7 days. MRC-5 cells provided the highest yield of enteroviruses overall and were the best cell type for the recovery of poliovirus and echovirus. MK cells provided the second best yield but were more useful than MRC-5 cells for coxsackievirus. BGM cells supported the growth of additional isolates of coxsackievirus and enhanced the speed of isolation. HEK cells supported the growth of additional isolates of both coxsackievirus and echovirus, but subculturing was always required for definite enterovirus cytopathic effects. The recovery rate increased 11% when two additional cell lines were used. The use of two tubes of MK cells significantly increased the yield of all enterovirus types. We conclude that the use of multiple appropriate cell lines increases yield and enhances the rapidity of enterovirus isolation.     

Rapid enterovirus RNA detection in clinical specimens by using nucleic acid sequence-based amplification

Enterovirus (EV) detection by nucleic acid sequence-based amplification was compared with EV isolation in cell culture. The NucliSens Basic kit (bioMerieux) was utilized for RNA detection. For virus isolation, samples were inoculated into MRC-5, primary rhesus monkey kidney, A549, rhabdomyosarcoma, and/or Buffalo green monkey kidney cells.     

CV-1 and MRC-5 mixed cells for simultaneous detection of herpes simplex viruses and varicella zoster virus in skin lesions.

BACKGROUND: Culture for varicella zoster virus (VZV) is relatively insensitive. Herpes simplex viruses (HSV) culture methods, which rely on primary rabbit kidney (pRK), mink lung (Mv1Lu) or the ELVIS HSV culture system fail to detect VZV. Culture of atypical vesicular skin lesions should be able to detect both HSV and VZV. OBJECTIVES: In this study, we evaluated the sensitivity of a newly developed mixture of CV-1/MRC-5 cells for the concurrent detection of both HSV and VZV. STUDY DESIGN: The CV-1/MRC-5 mixed cells were compared with pRK cells and Mv1Lu cells for the detection of HSV and to MRC-5 and A-549 cells for the detection of VZV. Fresh clinical samples submitted for HSV culture, VZV culture, and/or direct immunofluorescent assay (DFA) as well as frozen clinical samples previously positive for VZV were used for these comparisons. RESULTS: This preliminary study suggest that CV-1/MRC-5 mixed cells are as sensitive as pRK and Mv1Lu cells for the detection of HSV and appear to be more sensitive than MRC-5 and A-549 cells for the detection of VZV. Although the sample size is small, pre-CPE staining with VZV specific monoclonal antibody (Mab) at day 2 post-inoculation may provide a rapid detection of VZV with these mixed cells, but not with MRC-5 or A549 cells. In addition, culture of VZV in mixed cells from fresh clinical specimens appears to be as sensitive as antigen detection by DFA. Finally, 1% of specimens from skin lesions submitted for HSV culture grew VZV, highlighting the importance of culturing for both VZV and HSV, particularly in the case of atypical lesions. CONCLUSION: CV-1/MRC-5 mixed cells are highly sensitive for the simultaneous culture of HSV and VZV. The ability to detect either HSV or VZV from skin lesions is important for patient management.     

Development of plaque assays for adenoviruses 40 and 41

Enteric adenoviruses, important agents of infantile gastroenteritis, are difficult to culture with low titers and limited CPE. Consequently, few plaque assays have been reported and none are used routinely by investigators who may need reproducible quantitative assays for these viruses. CPE in A549 cells (an epithelial lung carcinoma cell line) was induced by isolates of human adenovirus (HAdV) serotypes 40 or 41 that were obtained by prior limited passage in primary cynmolgous monkey kidney (pCMK), human embryonic kidney (HEK), and Graham 293 cells. CPE with HAdV 40 (Dugan strain) and HAdV 41 (Tak strain) inoculated in A549 cells was also observed. Monolayers of A549 cells were inoculated with a low multiplicity of infection (MOI) of the archived stock isolates and harvested at days 10-14 with full CPE. Subsequent passages were harvested in as few as 7 days with 100% CPE to prepare virus stocks for plaque assay. Large individual plaques under agarose overlay were picked prior to staining and clonal stocks prepared. Titers of final stock preparations after six to eight passages in A549 cells were in the range of 5 x 10(7)-1 x 10(8)PFU/ml, which provides adequate virus for quantitative recovery studies. The particle to infectivity (P:I) ratios of the early passages of virus stocks were in the range reported previously. The ratio of non-infectious to infectious particles decreased with successive passages of HAdVs 40 and 41 in A549 cells. The specificity of the assay was confirmed by neutralization of plaques with type-specific antisera. Furthermore, sequence analysis of the HAdVs 40 and 41 plaque forming stocks ruled out contamination with any other HAdVs. The plaque assay developed will be useful for evaluation of virus recovery methods from water, food or other environmental matrices, as well as determination of the efficacy of water treatment techniques for inactivation of these viruses.     

Comparison of the continuous cell line 293 with human embryo kidney cells and human embryo fibroblast cells for the cultivation of ocular viruses.

The continuous cell line 293 was evaluated as a replacement for primary human embryo kidney (HEK) cells in the cultivation of ocular viruses. The 293 cells were found to be as sensitive as HEK cells and human embryo fibroblast (HEF) cells for the cultivation of adenoviruses and herpes simplex virus (HSV) respectively. As a continuous cell line, 293 cells are preferable to HEK and HEF cells for the routine isolation of ophthalmic viruses.     

Conventional tube cell culture compared with centrifugal inoculation of MRC-5 cells and staining with monoclonal antibodies for detection of herpes simplex virus in clinical specimens.

During a 15-month period, two methods for detection of herpes simplex virus (HSV) in 699 clinical specimens were compared: (i) 24-well-plate centrifugation (24WPC) with MRC-5 cells and staining with type-specific monoclonal antibodies (Syva Co., Palo Alto, Calif.) after incubation for 16 to 18 h and (ii) conventional tube cell culture with primary rabbit kidney and A549 cells. HSV was identified by conventional tube cell culture in 165 (24%) of 699 specimens and by the 24WPC method in 116 (17%) of 699 specimens. One specimen was positive for HSV by the 24WPC method alone, compared with 50 specimens positive only by conventional cell culture (P less than 0.0001). The sensitivity, specificity, and positive and negative predictive values of the 24WPC technique with MRC-5 cells for detection of HSV in clinical specimens were 70, 99.8, 99, and 91%, respectively. Centrifugal inoculation of MRC-5 cells in 24-well plates and staining with monoclonal antibodies after incubation for 16 to 18 h is an insensitive means of detecting HSV in clinical specimens and should not replace conventional tube cell culture with primary rabbit kidney cells.     

Foamy virus serotypes 1 and 2 in rhesus monkey tissues

Summary The isolation and serologic identification by cross neutralization tests showed the presence of simian foamy virus serotypes 1 and 2 in primary rhesus monkey brain, spleen, and kidney cell cultures. The frequency of isolation of foamy viruses from brain and spleen cell cultures was 86% and from kidney cell cultures it was 36%. In primary spleen cell cultures 50% of the foamy virus isolates were type 2. Of the 14 animals from which foamy virus was isolated, only 4 demonstrated serum neutralizing antibody to the homologous serotype. The cytopathology of foamy viruses in the primary cell cultures is described.     

Comparison of the sensitivity of human embryo kidney cells, HeLa cells, and WI38 cells for the primary isolation of viruses from the eye.

A comparison has been made of the efficiency of human embryo kidney (HEK) cills, HeLa cells, and WI38 cells for the isolation of viruses from the eyes of patients suffering from acute conjunctivitis or keratoconjunctivitis. From a total of 99 specimens 21 adenoviruses (serotypes 3, 4, 7, 8, and 13) were isolated in HEK cells, eight (serotypes 3 and 8) in HeLa cells, and four (serotype 3) in WI38 cells. Of the ten herpes simplex viruses isolated nine were recovered in HEK cells, seven in WI38 cells, and none in HeLa cells. The combination of HeLa cells and WI38 cells is not considered an adequate alternative to the difficult-to-obtain HEK cells for the isolation of viruses from the eye.     

Utility of direct immunofluorescence and virus culture for detection of varicella-zoster virus in skin lesions.

A direct immunofluorescence assay (DFA) with a monoclonal antibody from Ortho Diagnostic Systems was compared with conventional cell culture for the rapid detection of varicella-zoster virus (VZV) in 140 dermal lesions from 133 patients. A total of 79 (56%) specimens were positive for VZV: 40 (51%) by DFA alone, 2 (3%) by culture only, and 37 (47%) by both culture and DFA. After discordant analysis, the sensitivities and negative predictive values, respectively, were 97.5% (77 of 79) and 96.8% (61 of 63) for DFA and 49.4% (39 of 79) and 60.4% (61 of 101) for viral culture. Of the 39 positive viral cultures, VZV was isolated from 38 (97%) cultures in A549 cells, 23 (59%) in primary rhesus monkey kidney cells, and only 16 (41%) in MRC-5 cells. We conclude that DFA is the optimal method for rapid identification of VZV. In addition, better recovery of VZV in culture may be achieved by using A549 cells.     

Comparative sensitivities of viruses to cell cultures and transport media

The relative sensitivities and temporal permissivenesses of human embryonic kidney, baboon kidney, and human fibroblast (WI-38) cells for primary isolation of viruses were compared. Of 405 viruses isolated, 83% (335) were cultivated in human embryonic kidney cells, and 49% (198) in baboon kidney cells. WI-38 cells supported the growth of 34% of 199 viral agents. Of the 405 isolates, 70% manifested a cytopathic effect first in human embryonic kidney cells. Daily cumulative rates for primary virus isolation from 1960 to 1974 were examined. Of 558 virus isolates, 58% showed cytopathic effects within seven days after inoculation of the specimen. Comparison of two transport media demonstrated that the total number of isolates from tryptic soy broth approximated twice the number detected with charcoal viral transport medium.     

Studies on the defectiveness of adeno-associated virus (AAV). II. Effect of growth in CV-1 cells on the replication of adeno-associated virus.

The mechanisms by which helper viruses can promote AAV macromolecular synthesis have been investigated. The third system used was a continuous line of African green monkey kidney cells (CV-1) which is permissive for AAV when a simian adenovirus helper is used. When HSV was used as a helper, no AAV DNA, structural proteins, or IF antigens were detected, indicating the absence of HSV helper activity. HSV production and plaqueing in CV-1 cells were found to be comparable to those seen in Hep-2 cells (an epidermoid cancer line) and Vero cells (another line of African green monkey kidney cells). In both of these cell lines, HSV expresses its AAV helper functions. It was also observed that AAV had no effect on HSV replication in either Vero or CV-1 cells. Comparison of HSV protein production in CV-1 and Hep-2 cells demonstrated the absence of HSV proteins ICPO and ICP46 in the CV-1 line following the release of a cycloheximide block.     

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of herpes simplex virus antigen.

A commercial enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus (HSV) antigen in clinical specimens and culture lysates was evaluated. A total of 1,155 specimens and an additional 335 cell culture lysates were tested by ELISA, and results were compared to those obtained by cell culture in primary rabbit kidney, human foreskin, and MRC-5. The sensitivity and specificity of the direct test were 52.5 and 96.9% and those of the culture lysate test were 98.7 and 96.9%, respectively. The sensitivity of the direct ELISA correlated with early cytopathic effect in cell culture and varied with specimen source, ranging from 100% with skin lesions to 40.9% with cervical swabs. Of 60 cervical specimens from asymptomatic individuals, 22 (36.6%) yielded false-positives, which may be due to noninfectious HSV. No reproducible cross-reactions were found with other viruses isolated. The Ortho HSV ELISA was found to be rapid, sensitive, and specific for detection of HSV from cell culture lysates, but it needs reevaluation for direct specimen testing, in particular for screening of asymptomatic obstetrical patients.     

Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells

Over two winters in Newcastle upon Tyne, respiratory secretions, negative by immunofluorescence staining for other respiratory viruses, were tested for the presence of human metapneumovirus (HMPV) by RT-PCR. In the second winter, specimens were also tested by immunofluorescence staining with an anti-HMPV polyclonal rabbit antiserum and immunofluorescence positive specimens were inoculated into a line of human bronchiolar cells, 16HBE140. Overall, 55 of 549 (10%) specimens tested were positive for HMPV by RT-PCR. Of 162 specimens tested by both RT/PCR and immunofluorescence staining, 23 were positive by both techniques. Of five specimens positive by RT-PCR alone, only one was confirmed with a second set of primers. Of three specimens positive by immunofluorescence alone, only one was confirmed by virus culture. All four previously recognized sub-genotypes of the virus were identified by both RT-PCR and immunofluorescence staining. Sub-genotype A1 was prevalent in the first winter and B1 prevalent in the second. HMPV replication and virus isolation rates were higher in 16HBE140 cells than in monkey kidney cells and did not require exogenous trypsin. Low passage isolates of both sub-genotypes A2 and B1 replicated slowly reaching peak titers only 12 days after inoculation. In summary, single round RT/PCR and immunofluorescence staining with a polyclonal rabbit antiserum proved of equal sensitivity in the diagnosis of HMPV infection in respiratory secretions both detecting 96% of confirmed positive specimens. 16HBE40 cells provided a significant improvement on monkey kidney cells for the isolation and propagation of the virus.     

Comparison of primary rhesus and cynomolgus monkey kidney cell cultures for viral isolation from clinical specimens.

Rhesus monkey kidney and cynomolgus monkey kidney cell cultures were compared for viral isolation by using clinical specimens that yielded 203 viral isolates. Cynomolgus and rhesus monkey kidney cells were comparable for the isolation of 22 adenoviruses, 12 coxsackieviruses, and one poliovirus. Four of 50 echoviruses and seven of ten herpesviruses were detected only in cynomolgus monkey kidney cells. Influenza virus was isolated in 84 instances, of which eight were detected only in rhesus and four only in cynomolgus monkey kidney cells. Rhesus monkey kidney cells yielded six more parainfluenza virus isolates. Except possibly for parainfluenza virus, cynomolgus monkey kidney cells appear to be as sensitive as rhesus monkey kidney cells for viral isolation from clinical specimens.     

Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation.

Monoclonal antibodies (Syva Co., Palo Alto, Calif.) were used for the detection and serotyping of herpes simplex virus (HSV) isolates by immunofluorescence 16 h after inoculation of MRC-5 monolayers in 3.7-ml shell vials and after low-speed centrifugation. A total of 119 specimens were inoculated into conventional tube cell cultures and shell vials. Of 98 specimens inoculated on the same day of receipt in the laboratory (fresh specimens), all 23 (23.5%) HSV-positive specimens were identified by serotype in 16 h in shell vials by immunofluorescence, whereas only 8 of 23 HSV-positive specimens (34.8%) produced cytopathic effects in conventional tube cell cultures in this time period. Similarly, of 21 original specimen extracts previously determined to be culture positive for HSV (stored specimens), all were detected and serotyped by the immunofluorescence test with monoclonal antibodies 16 h postinoculation compared with the recognition of only 8 of these isolates (38.1%) by cytopathic effect that soon. This technique of centrifugal inoculation of HSV in shell vials containing MRC-5 cells permitted detection of this virus in all positive specimens with serotype determination within 16 h postinoculation.     

Isolation of adeno-associated virus type 4 from a culture of green monkey kidney cells

In the process of biological control of uninfected green monkey kidney (GMK) cell cultures a thermostable hemagglutinating agent designated No. 5056 was isolated alongside with adenovirus-SV. By its antigenic properties the 5056 strain was identified as adeno-associated virus type 4 (AAV-4). In control of 574 specimens of GMK culture batches, 40 AAV-4 strains were isolated in the presence of a helper adenovirus. Some biological properties of the isolates and their resistance to certain physical and chemical treatments were studied. Two isolates of the satellite virus were examined in the electron microscope. A correlation between the rate of AAV-4 isolation from GMK cultures and the presence of complement-fixing antibody to AAV-4 in monkey sera was observed.