Primary Rhesus Monkey Kidney Cells Research Articles

Molecular Investigation of an Ontario Mumps Outbreak using Whole Genome Sequencing

BackgroundIn early 2017 an outbreak of Mumps virus affected over 100 individuals in the province of Ontario, concurrent with multiple mumps virus outbreaks across North America. Traditional genotyping of mumps outbreaks relies on sequencing a portion of the small hydrophobic (SH) gene, but has limited capability to distinguish between strains of the same genotype. Most mumps cases in Ontario in recent years are of genotype G. We used a novel whole genome sequencing (WGS) protocol to perform a molecular epidemiological investigation of the outbreak.MethodsThroat (n = 5) and buccal (n = 15) swabs positive by RT-PCR for SH or Fusion (F) gene targets were cultured in primary Rhesus monkey kidney cells. Cell free viral extract underwent RT-PCR and subsequent PCR amplification using overlapping primer pairs to cover the entire 15 kilobase (kb) genome. The first 8 samples were amplified with 18 pairs of overlapping primers, which was reduced to 9 sets (average fragment size 1.9 kb, range 1.6–2.8 kb) for the final 12 samples. Mumps cDNA libraries were prepared with Nextera XT kit and WGS of the indexed fragments was performed with V2 reagent kits on the Illumina MiSeq instrument. Reference based genome assembly was performed using samtools version 1.4. Phylogenetic analysis was performed by maximum likelihood method in MEGA7.ResultsWe identified two distinct genotype G lineages comprised of 9 patients each and closely related to a 2009–2010 outbreak in Ontario and New York (Figure 1). Inter-lineage single nucleotide polymorphism (SNP) differences ranged from 25 to 31, whereas intra-lineage SNPs ranged from 0 to 8 SNPs. Two outlying sequences, of genotype C and G respectively, may represent sporadic introduction of virus from other areas. Time from virus isolation to SNP based analysis was approximately 4 days.ConclusionWGS of Mumps virus culture isolates using the PCR fragment method identified two distinct genotype G lineages in a large provincial outbreak. This method may aid public health authorities identify separate transmission chains in the case of large outbreaks.Disclosures All authors: No reported disclosures.

Identification of Endogenous Coccidioides posadasii Contamination of Commercial Primary Rhesus Monkey Kidney Cells

Here we describe the identification of endogenous Coccidioides posadasii contamination in commercial rhesus monkey kidney (RhMK) cells and the subsequent nationwide alert that reduced the risk of laboratory exposure. This extraordinary event highlights the necessity for laboratories to remain vigilant in the use of appropriate biosafety procedures, particularly when working with unknown pathogens.

Comparative Analysis of the Full-Length Genome Sequence of a Clinical Isolate ofHuman Parainfluenza Virus 4B

We are engaged in airborne transmission and epidemiology studies of respiratory pathogens, with particular interest in human parainfluenza virus type 4 (hPIV-4) and other lesser studied viruses. In this paper, hPIV-4 was detected in primary rhesus monkey kidney (PRMK) cells that had been inoculated with nasopharyngeal swab material obtained from a child with a mild upper respiratory tract illness. Attempts to isolate the virus in pure culture were hampered by the presence of a fast-growing simian spumavirus that was a contaminant of the PRMK cells. Total RNA was extracted from the PRMK cell culture, and PCR followed by sequencing of a subgenomic section of the fusion protein gene suggested the hPIV-4 was subtype 4B. At the time of this work, two complete but dissimilar hPIV-4B genomes had been deposited by others in GenBank. To gain better insights on hPIV-4B, and to test methods that we are developing for viral forensics, the entire genomic sequence of our virus was determined from archived RNA. The hPIV-4B genomic sequence that we determined conforms to the paramyxovirus “rule of six.” Here, we compare and contrast the genetic features of the three completely sequenced hPIV-4B genomes currently present in GenBank.

Flow cytometric detection and serotyping of enterovirus for the clinical laboratory

Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1–4 after inoculation. Flow cytometry results for echovirus 6, 9, 11, and 30 and coxsackievirus B1 correlated with IFA in all cases. Coxsackievirus B1 and echovirus 30 infections were detected 1 day earlier by flow cytometry than IFA. Flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems.

Calu-3/A-549 mixed cells as a replacement for primary rhesus monkey kidney cells for virus detection

Background Primary kidney cells derived from rhesus macaques (pRhMK) are heavily relied upon for the detection and culture of a wide range of clinically relevant viruses. The use of these cultures is problematic due to the possible presence of endogenous viruses, the need to sacrifice a primate, and the inherent variance found in primary cultures. Objective To develop a continuous cell line or mixed cell co-culture that could replace dependence on pRhMK cells. Study design Cells from the Calu-3 and A-549 cell lines were used to prepare mixed cell monolayers that were compared to pRhMK cells for their ability to detect respiratory viruses, measles, mumps, enteroviruses, and herpes viruses. Clinically derived and laboratory virus strains were used for these comparisons in culture plates or 16 mm tubes. Results Calu-3/A-549 cells are more sensitive than pRhMK for the detection of adenovirus, enteroviruses and herpes simplex virus and are about equally sensitive for the detection of other respiratory viruses, measles, mumps and varicella-zoster virus. Conclusions Calu-3/A-549 cells are an equivalent or better alternative to pRhMK cells for the detection of many clinically relevant viruses.

Comparison of SmartCycler real-time reverse transcription-PCR assay in a public health laboratory with direct immunofluorescence and cell culture assays in a medical center for detection of influenza A virus.

A single-tube real-time (fluorogenic) reverse transcription (RT)-PCR with the SmartCycler instrument (SmartCycler RT-PCR) for influenza A virus detection was evaluated with 238 respiratory specimens. Direct immunofluorescence antibody staining (DFA) and primary rhesus monkey kidney cell culture were performed on-site at Yale-New Haven Hospital. Specimens were transported to the Connecticut Department of Public Health Laboratory for real-time RT-PCR. Cell culture detected influenza A virus in all 150 influenza A virus-positive specimens, DFA detected the virus in 148 influenza A virus-positive specimens, and SmartCycler RT-PCR detected the virus 143 influenza A virus-positive specimens. The sensitivity and specificity of RT-PCR were 95.3 and 100%, respectively. The high sensitivity and specificity and the rapid turnaround time made the SmartCycler RT-PCR valuable for the rapid diagnosis of influenza A, especially in a public health laboratory. The closed real-time RT-PCR system avoided cross-contamination possible with RT-PCR and the excessive manipulations required for conventional RT-PCR analysis and saved time and labor as well. In a medical center, rapid diagnosis by DFA was labor intensive but was 98.7% sensitive and 100% specific compared to the results of culture and provided results within 2 h throughout operating hours, helping with bed allocation on admission and patient management.

Rapid virological surveillance of community influenza infection in general practice

The annual outbreak of influenza in Scotland is monitored by sentinel general practices, which report influenza-like illness. We piloted real time virological surveillance to investigate whether polymerase chain reaction (PCR)...

A comparison of virus isolation, indirect immunofluorescence and nested multiplex polymerase chain reaction for the diagnosis of primary and recurrent herpes simplex type 1 and type 2 infections

134 swabs in viral transport medium were received from 126 patients with suspected clinical HSV-1 and HSV-2 infections. They were tested by (i) nested multiplex polymerase chain reaction NMPCR (strongly positive specimens had visible bands on both rounds of PCR) without prior extraction, (ii) culture in primary rhesus monkey kidney, E6-Vero, RD and HEp-2 cells and (iii) antigen detection by immunofluorescence (IF). Antigen detection employed four novel pools (A–D) of monoclonal antibodies (Mab): A was HSV-1 specific, B was HSV-2 specific while C and D were generic. In comparison to NMPCR the sensitivity and specificity of (i) culture was 59% (22/37) and 100% (134/134), (ii) IF by Pool A was 59% (16/27) and 100% (117/117), (iii) IF by Pool B was 40% (4/10) and 100% (130/130) and (iv) IF by Pools C and D were 60% (18/30) and 100% (96/96). Specimens positive by culture were more likely to be strongly positive by NMPCR ( χ 2 P=0.004). Typing by each method concurred on all occasions. NMPCR was cost effective, easier to perform and was the most sensitive method for HSV detection. It should become the method of choice for HSV diagnosis.

Comparison of detection methods for adenovirus from enteric clinical specimens

Fecal samples submitted for virus examination over July 1990 to June 1991 from children <3 years of age were examined by electron microscopy (EM), virus culture (VC), and enzyme immunoassay [EIA, group-reactive and adenovirus (Ad) 40/41 specific; Cambridge BioScience] to compare the detection rate of adenovirus from pediatric fecal specimens. Ad isolates of serotypes 1–7 grown in HEp-2 or primary rhesus monkey kidney cells were identified by neutralization. Graham 293 cell cultures were used only when specimens were found to be positive for Ad by EM, type-specific Ad40/41 EIA, and for isolates not identified by neutralization. Ads grown in 293 cells were identified by DNA restriction endonuclease analysis. Of the 1187 specimens examined, 105 (9%) were found to be positive for Ad. VC detected 93, while 12 additional positives were detected by EM or EIA. The relative sensitivity of VC, EIA, and EM for the 105 specimens was 89% (93), 45% (47), and 35% (37), respectively. Among the 105 positive specimens, enteric Ad, nonenteric Ad, and untypeable Ad were 28% (29), 65% (68), and 7% (8), respectively. Of 37 EM positives, 62% (23) were enteric Ad; 27% (10) were nonenteric including serotypes 2, 3, 4, 5, 12, and 31, with 4, 1, 1, 2, 1, and 1 isolates of each type positive, respectively; and 11% (4) were detectable only by EM. Five isolates were identified as variant of Ad 2(3), Ad 3(1) and Ad 31(1). Over a 1-year period, a single Ad41 variant strain was the most frequently detected enteric Ad in Winnipeg, Manitoba, Canada. For maximum detection rates of Ad viruses in pediatric fecal specimens, a combination of EM, VC, and EIA is required, but group-reactive EIA, or EM followed by Ad40/41-specific EIA of initial positives, are the most direct and efficient methods for enteric Ad detection.

Sensitivity of NCI-H292 human lung mucoepidermoid cells for respiratory and other human viruses

NCI-H292 mucoepidermoid carcinoma cells from human lungs were shown in an earlier report to be a fully adequate substitute for primary rhesus monkey kidney (MK) cells for the isolation and propagation of the human paramyxoviruses. Although sensitivity for ortho- and paramyxoviruses was the principal reason for using MK cells, the cells were also sensitive to many other viruses, which constituted another important value of MK cells. That MK cells supported the initial isolation and growth of so many respiratory viruses made it a mandatory cell type for any clinical laboratory. We therefore felt it was imperative to evaluate the virus spectrum of NCI-H292 cells, which are being used as a substitute for MK cells in many laboratories. In the present report, we show that NCI-H292 cells are sensitive for vaccinia virus, herpes simplex virus, adenoviruses, BK polyomavirus, reoviruses, measles virus, respiratory syncytial virus, some strains of influenza virus type A, most enteroviruses, and rhinoviruses, in addition to the parainfluenza and mumps viruses originally reported. Furthermore, these viruses replicate in NCI-H292 cells to the same virus and antigen titers and at the same speed of replication as they do in their usually preferred cells. The NCI-H292 cells are therefore an excellent substitute for MK cells in terms of laboratory safety, ease of availability, paramyxovirus isolation, and broad virus spectrum but cannot substitute for MK cells for the isolation of influenza viruses.

Comparison of hemagglutination and hemadsorption tests for influenza detection

Hemagglutination (HA) testing of the supernatant of primary rhesus monkey kidney cell culture tubes inoculated with clinical specimens was compared with hemadsorption (HAd) of the same tube for identifying the presence of hemagglutinin activity due to influenza. A total of 476 respiratory specimens were screened for influenza by this method. The 127 influenza or parainfluenza positive cultures showed 100% concordance between the two tests. All negative cultures were both also negative by HA and HAd. In contrast to the HAd method, HA testing was performed with less tube manipulation, less hands-on time, and less potential for cell culture tube contamination.

A novel approach to enterovirus typing.

A novel method was developed for typing enteroviruses producing cytopathic effect. Monolayers of primary or secondary rhesus monkey kidney cells were prepared and covered with an agarose overlay. Wells were formed in the agarose, the well bottom being optimally determined to be 3 mm from the monolayer and homotypic enterovirus antiserum, intersecting antiserum pool or antiserum-diluent as control was added. After 2 h at 37 degrees C, the test virus isolate was added to each well. Cultures were incubated at 37 degrees C and were examined daily until cytopathic effect was readily observed in the control well. Monolayers were fixed and stained for macroscopic reading. Enterovirus identity based on the inhibition of cytopathic effect was confirmed with a conventional micro-neutralization method. In all, 51 enterovirus isolates were typed. Included were 21 polioviruses, 9 coxsackieviruses and 21 echoviruses, all of which were correctly identified. This method takes advantage of the ability of enterovirus and antibody to diffuse through agarose. It is simple to perform. It does not require preliminary titration of the test virus isolate and tolerates 1,000 fold fluctuations in virus concentration, thereby offering laboratories a more rapid and efficient means of typing enteroviruses.

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.

Department of Laboratory Medicine, Bellin Memorial Hospital, Green Bay, Wisconsin

The isolation of measles virus in primary Rhesus monkey kidney cells (PRMK) in patients with documented giant-cell pneumonia who have presented without a rash is limited. The diagnosis usually is made by cytologic examination of nasal or bronchial secretions in which characteristic multinucleated giant cells with intranuclear and intracytoplasmic inclusion bodies are observed. The diagnosis of giant-cell pneumonia has been associated with measles virus but not exclusively. Canine distemper, herpes group viruses, and parainfluenza infections have been associated with these cells. In addition, vitamin A deficiency also has been cytologically associated with multinucleated giant cells. The authors describe the isolation of measles virus from bronchial washing and sputum in PRMK cells at 4 days from an 11-year-old child with acute interstitial pneumonia who was in remission for acute lymphocytic leukemia. Classic cytopathologic effect (CPE) consisting of syncytial and hole formation on the PRMK monolayer was apparent. In addition, a foamy appearance of the monolayer was noted in an otherwise clean lot of monkey cells. Confirmatory testing with measles antibody of the infected areas of the monolayer by indirect immunofluorescence (IFA) was positive for measles antigen and negative for mumps, parainfluenza (types I, II, and III) and influenza A and B virus. Serologic studies for measles antibody revealed an IFA IgG titer of greater than 1:10,240, and an IgM titer of 1:128. Cytologic examination of the same bronchial fluid revealed the typical giant cells with characteristic inclusions associated with measles virus. Because this disease usually is severe, and often fatal, prompt recognition of this virus is essential, not only to the patient, who can be treated with immunoglobulin and/or antiviral therapy, but also to prevent the spread of the virus to other patients and medical personnel. These findings also support direct evidence for the etiologic role of measles virus in giant-cell pneumonia that has been detected either histologically or cytologically and in tissue culture at autopsy.

NCI-H292 as an alternative cell line for the isolation and propagation of the human paramyxoviruses

SummaryPrimary rhesus monkey kidney (MK) cells have long been the cells of choice for isolation and propagation of the human paramyxoviruses (parainfluenza 1, 2, 3, 4A, 4B, and mumps). However, problems with the supply and cost of MK cells and the presence of endogenous viruses, including herpes B virus and SV-5, necessitated a search for an alternative cell line. Continuous cell cultures of human origin (L132, A-549, HuT-292, HEK, G-293, G-401, A-498, A-704, CAKI-1, RD) and simian origin (LLC-MK2, BSC-1, MA-104, Vero) were evaluated for their capacity to support the growth of the human paramyxoviruses, as followed by cytopathic effect, hemadsorption, hemagglutination, and EIA. NCI-H292 (HuT-292) human lung mucoepidermoid carcinoma cells (ATCC # CRL-1848) proved to be the most sensitive line for cultivating all serotypes and strains of the paramyxoviruses. These cells were also shown to be a suitable substitute for MK in primary isolation of paramyxoviruses from clinical specimens. RPMI-1640 with 1.5µg/ml trypsin was the preferred maintenance medium; alternatively, Eagle's MEM supplemented with 1.5µg/ml trypsin and 0.1% ITS was satisfactory. NCI-H292 cells are a continuous line with excellent growth characteristics, although the genetic polyploidy of the cells may limit the number of passages of usable cells.

Anti-influenza virus activity of the compound LY253963

The compound LY253963 (1,3,4-thiadiazol-2-ylcyanamide) inhibited the in vitro replication of representative influenza A and B viruses in Madin-Darby canine kidney (MDCK) cells at concentrations of 1–3.2 μg/ml. The yield of an influenza A (H3N2) virus in primary rhesus monkey kidney (RMK) cells was inhibited at 0.1–0.3 μg/ml. However, similar concentrations were inhibitory for the growth of uninfected MCDK or RMK cells. Combination drug studies generally found indifferent interactions between LY253963 and ribavirin or rimantadine. In timing of additional studies, hemagglutinin expression was inhibited to the greatest extent when LY253963 exposure was begun at least 8 h before viral infection, which suggested either slow uptake or intracellular metabolism of LY253963 to an active form. Virus-specific protein synthesis was inhibited to a greater extent by ribavirin 10 μg/ml or rimantadine 1 μg/ml than by LY253963 10 μg/ml. No drug-resistant mutants were detected during serial passage of an influenza A (H3N2) virus in the presence of LY253963 1–16 μg/ml. In summary, we found that LY253963 inhibited influenza A and B virus replication in several cell types, but that it was associated with cytostatic effects at low concentrations. These studies failed to identify a selective anti-influenza action.

Adenovirus transformed monkey cell lines permissive to enteric adenovirus type 40

Plasmids containing the E1 regions of adenovirus serotypes 3 and 5 were transfected into primary Rhesus monkey kidney cells. The presence of viral DNA sequences was detected in transformed cell lines. All these cell lines expressed the E1A proteins. In addition, Ad5 transformed cells, have the E1B 21 kDa protein located in the nuclear membrane. These cell lines were permissive to the enteric adenovirus serotype 40 but not to serotype 41.

Presumptive identification of enteroviruses with RD, HEp-2, and RMK cell lines

The limited supply of the Lim Benyesh-Melnick antiserum pools for the typing of enteroviruses has made this test inappropriate for routine use in most clinical laboratories. We studied the correlation between the enterovirus groups and the cell lines on which they displayed cytopathic effect in order to make identifications without using the neutralization test. This study indicated that a presumptive identification of the enterovirus group could be made on the basis of characteristic cytopathic effect displayed after passage into rhabdomyosarcoma (RD), HEp-2, and primary rhesus monkey kidney (RMK) cells. Echoviruses and group A coxsackieviruses could be isolated in RD and RMK cells but not HEp-2 cells. Group B coxsackievirus could be isolated in RMK and HEp-2 cells but not RD cells. Poliovirus could be isolated in all three cell lines. We recommend the use of these cell lines to make presumptive enterovirus group identifications for routine viral isolates.

Herpesvirus simiae Contamination of primary rhesus monkey kidney cell cultures

Herpesvirus simiae Contamination of primary rhesus monkey kidney cell cultures

Rapid 24-well plate centrifugation assay for detection of influenza A virus in clinical specimens

Two methods for detection of influenza virus in 234 clinical respiratory specimens were compared: (i) a 24-well plate-centrifugation assay using Madin Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza A and B (Centers for Disease Control, Atlanta, GA) after incubation for 16 h and 40 h, and (ii) conventional tube cell culture using MDCK cells and primary rhesus monkey kidney cells. Influenza A was identified in 23 specimens (10%). No influenza B was recovered. The rapid centrifugation and tissue culture methods were positive for influenza A in 21 (91%) and 16 (70%) of the 23 specimens, respectively. Fourteen specimens were positive by both methods, 2 were positive by tissue culture alone, and 7 were positive by rapid centrifugation only. Of the 21 specimens positive by rapid centrifugation, 16 (76%) were detected after overnight incubation, and 5 (24%) were positive only after incubation for 40 h. Cytopathic effect was observed in 13 (81%) of the 16 isolates identified by tissue culture after an average of 6 days, and 3 (19%) were identified only by hemadsorption and staining with monoclonal antibodies at day 10. Compared with conventional tissue culture, the 24-well plate centrifugation assay is a more rapid and more sensitive method for detecting influenza virus in clinical specimens.