key: cord-275719-ru33ubss authors: Roingeard, Philippe; Raynal, Pierre‐Ivan; Eymieux, Sébastien; Blanchard, Emmanuelle title: Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety date: 2018-11-09 journal: Rev Med Virol DOI: 10.1002/rmv.2019 sha: doc_id: 275719 cord_uid: ru33ubss Transmission electron microscopy (TEM) is the only imaging technique allowing the direct visualization of viruses, due to its nanometer‐scale resolution. Between the 1960s and 1990s, TEM contributed to the discovery of many types of viruses and served as a diagnostic tool for identifying viruses directly in biological samples, either in suspension or in sections of tissues or mammalian cells grown in vitro in contact with clinical samples. The diagnosis of viral infections improved considerably during the 1990s, with the advent of highly sensitive techniques, such as enzyme‐linked immunosorbent assay (ELISA) and PCR, rendering TEM obsolete for this purpose. However, the last 20 years have demonstrated the utility of this technique in particular situations, due to its “catch‐all” nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. Thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, TEM provided the answer. TEM is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. It is also used to check the microbiological safety of biological products. Many biopharmaceuticals are produced in animal cells that might contain little‐known, difficult‐to‐detect viruses. In this context, the “catch‐all” properties of TEM make it possible to document the presence of viruses or virus‐like particles in these products. However, the last 20 years have demonstrated the utility of this technique in particular situations, due to its "catch-all" nature, making diagnosis possible through visualization of the virus, without the need of prior assumptions about the infectious agent sought. Thus, in several major outbreaks in which molecular techniques failed to identify the infectious agent, TEM provided the answer. TEM is also still occasionally used in routine diagnosis to characterize infections not diagnosed by molecular assays. It is also used to check the microbiological safety of biological products. Many biopharmaceuticals are produced in animal cells that might contain little-known, difficult-to-detect viruses. In this context, the "catch-all" properties of TEM make it possible to document the presence of viruses or virus-like particles in these products. microscope had a much higher resolution than any of the light microscopes available at the time and promised to revolutionize many aspects of science, including cell biology and virology. Ernst Ruska was a physicist (1986 Nobel prize winner in physics), but his younger brother, Helmut Ruska, who had trained in medicine, rapidly recognized the potential of this microscope for investigating the nature of viruses. 3 In the early 1940s, viruses were classified according to their hosts and the clinical symptoms they caused. Despite the lack of established methods of biological sample preparation for transmission electron microscopy (TEM) at this time, Helmut Ruska was able to characterize the morphology of several viruses and he developed a rough viral classification based on the size and shape of the viral particles. 4 TEM was rapidly adopted for its first major use in clinical virology: the differential diagnosis of smallpox, caused by the variola virus Abbreviations: ELISA, enzyme-linked immunosorbent assay; EM, electron microscopy; EMEA, European Medicines Agency; FDA, Food and Drug Administration; FPERT, fluorescent product-enhanced reverse transcription; LCMV, lymphocytic choriomeningitis virus; PCR, polymerase chain reaction; SARS, severe acute respiratory syndrome; SFTS, severe fever with thrombocytopenia syndrome; TEM, transmission electron microscopy from the poxvirus family, and chickenpox, caused by the varicellazoster virus of the herpes family, based on investigations of fluid samples from the vesicles on the patients' skin. 5 The chickenpox virus appeared to be spherical and 140 to 150 nm in diameter, with a central body, a structure clearly different from that of the much larger, brick-shaped smallpox virus. "dirty" clinical samples, such as plasma, urine and feces in the 1970s constituted a major breakthrough for studies of these viruses. 8 The etiologic agents of hepatitis B 9 and A 10 were detected in plasma and stool samples, respectively. The BK virus, a polyomavirus, was identified for the first time in the urine of patients undergoing renal transplantation. 11 Rotaviruses were also identified by TEM as the main cause of epidemic gastroenteritis in humans and animals. 12, 13 However, many other viruses were also found to cause gastroenteritis. The first of these viruses was the Norwalk virus, identified during an outbreak of gastroenteritis in Norwalk, Ohio, USA. 14,15 Viruses with a similar morphology were subsequently discovered elsewhere and called "Norwalk-like" viruses, to reflect the similarity of their appearance on TEM, 16 before being officially renamed "noroviruses." 17 Other viruses from the adenovirus, 18 astrovirus, 19, 20 and calicivirus 21 families were also identified in the stool samples of children with gastroenteritis. TEM was, thus, widely used on negatively stained samples for routine diagnosis, as a rapid, "catch-all" method for distinguishing between the diverse viruses potentially implicated in human gastroenteritis, providing a diagnosis within 15 minutes of the arrival of the sample in the laboratory. Although more time-consuming, due to the need to embed a sample in resin and cut ultrathin sections with an ultramicrotome, the TEM has also proved useful in medical virology, in searches for viruses in tissues. Panels E and F in Figure 1 illustrate a parapoxvirus (Orf virus) visualized in a skin biopsy specimen from a patient with a severe finger ulcer 22 This was the case, in particular, for the diagnosis of viral gastroenteritis, for which molecular techniques capable of identifying most of the virus families involved in human gastroenteritis have been established. [24] [25] [26] [27] A similar shift in practice occurred in veterinary medicine, with ELISAs and PCR progressively replacing TEM for the routine diagnosis of viral infections. [28] [29] [30] [31] In human medicine, the use of TEM to differentiate between smallpox virus and the other viruses present in the fluids of cutaneous vesicles is no longer required, since the successful eradication of the variola virus in 1980 thanks to a worldwide vaccination program. 32 It has been argued that TEM remains potentially useful for this application in a context of bioterrorism. 33, 34 However, the risk of smallpox reappearing is extremely small, and even in the unlikely event of this happening, molecular techniques would undoubtedly outperform TEM for this diagnosis. Consequently, the number of laboratories making use of TEM for diagnostic purposes has decreased considerably. sometimes revealing unexpected infectious agents, while molecular methods require previous knowledge of the virus to be tested. TEM has also the advantage of being able to potentially identify double or multiple infections caused by more than one virus, which could be missed by molecular or antigen tests. Moreover, the nature of the samples to be analyzed can be diverse, from body fluids or biopsies analyzed directly or after cell culture. In some cases, TEM has also been used to confirm a diagnosis previously established with molecular techniques. [51] [52] [53] [54] Figure 3 illustrates 71 Most of the viral particles produced by these cells such as intracytoplasmic or intracisternal A-type particles are defective and noninfectious ( Figure 4) . However, other particles such as C-type particles bud at the cell surface and may infect nonrodent cells 72 ( Figure 5 ). Some murine retroviruses have been shown to be tumorigenic in primates, 73 and cases of leukemia have been reported in children with severe combined immunodeficiency treated by gene therapy involving the use of murine retroviral vectors. 74 All these elements demonstrate the relevance of tracking the presence of retroviruses in biological products derived from rodent cells. Reverse transcriptase assays performed on bulk harvests are often hampered by high background levels due to cell-derived DNA polymerases. 75 TEM may, therefore, help to document the presence of retrovirus-like particles in these bulk harvests ( Figure 6 ). TEM can also be used to gauge the concentration of viral particles to validate the clearance of retroviruses or any other virus suspected to be present in the master cell bank. Fortunately, the endogenous retroviruses present in the Chinese hamster ovary (CHO) cell line, the main rodent cell line used to produce biological products in the biotech industry, have been shown to be noninfectious. 76 Testing requirements are now lower for this well-characterized cell line than for other cell lines with which experience is more limited. Nevertheless, Detection of retrovirus-like particles by transmission electron microscopy (TEM) with negative staining in bulk harvests of rodent cells used for the production of biological products. Scale bars represent 200 nm in both panels novel cell substrates, including insect cell lines in particular, are now being introduced into the biotech industry. 77 Their use will carry new concerns about unknown viruses for which there is a potential risk of contamination, and TEM will undoubtedly be useful for documenting the presence of viruses or virus-like particles in these cells and the products derived from them. In conclusion, although TEM is sometimes seen as a somewhat "oldfashioned" technique, it still has an important role to play in virus detection. It is particularly useful for identifying unknown agents involved in particular outbreaks or transmission clusters. In routine diagnosis, it may be useful to confirm or even, in some cases, to guide the diagnosis of a viral infection. TEM can also be used to check the viral safety of biopharmaceutical products. This technique has several disadvantages, such as the cost of electron microscopes and their maintenance, the need for well-trained microscopists, and timeconsuming analysis, particularly if the samples must be embedded in resin for the cutting of ultrathin sections. However, all the techniques available have benefits and disadvantages, and their complementary natures mean that there are advantages to be gained by using them in combination. In this respect, the principal advantage of TEM is its ability to provide an image of the virus, providing additional confidence in the result. Nobel lecture: the development of the electron microscope and of electron microscopy Die bedeutung der ubermikroskopie fur die virusforschung Versuch zu einer ordnung der virusarten The use of the electron microscope in diagnosis of variola, vaccinia, and varicella A negative staining method for high resolution electron microscopy of viruses Direct electron-microscopy of organ culture for the detection and characterization of viruses Viruses in the stools Virus-like particles in serum of patients with Australia antigen-associated hepatitis Hepatitis A: detection by immune electron microscopy of a virus-like antigen associated with acute illness New human papovavirus (B.K.) isolated from urine after renal transplantation Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis Virus particles in gastroenteritis Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis The discovery of the 27-nm Norwalk virus: an historic perspective The electron microscopical and physical characteristics of small round human fecal viruses: an interim scheme for classification A summary of taxonomic changes recently approved by ICTV Epidemic viral enteritis in a long-stay children's ward Viruses and gastroenteritis in infants 28 nm particles in faeces in infantile gastroenteritis Caliciviruses in man Orf skin ulcer BK virus infection in a renal transplant recipient Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples Real-time reverse transcription-PCR for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis Development of a ssRNA internal control template reagent for a multiplex RT-PCR to detect turkey astroviruses An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces Epidemiology of Norwalk-like virus infections in cattle in The Netherlands Detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink Countering the posteradication threat of smallpox and polio Bioterrorism and electron microscopic differentiation of poxviruses from herpesviruses: dos and don'ts Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future A morbillivirus that caused fatal disease in horses and humans Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia Role of electron microscopy in Nipah virus outbreak investigation and control A novel coronavirus associated with severe acute respiratory syndrome Ultrastructural characterization of SARS coronavirus The detection of monkeypox in humans in the western hemisphere Fever with thrombocytopenia associated with a novel bunyavirus in China Transmission of lymphocytic choriomeningitis virus by organ transplantation Fatal hemorrhagic fever caused by West Nile virus in the United States Life-threatening Cache Valley virus infection A new phlebovirus associated with severe febrile illness in Missouri Nosocomial outbreak of parechovirus 3 infection among newborns Diagnosis of progressive multifocal leukoencephalopathy in the absence of DNA from the JC virus in the cerebrospinal fluid New adenovirus species found in a patient presenting with gastroenteritis Discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin Complete genome sequence of a Sapporo virus GV.2 variant from a 2016 outbreak of gastroenteritis in Sweden Clinicopathologic analysis of coxsackievirus a6 new variant induced widespread mucocutaneous bullous reactions mimicking severe cutaneous adverse reactions Novel poxvirus infection in an immune suppressed patient Novel poxvirus infection in 2 patients from the United States Novel orthopoxvirus infection in an Alaska resident Novel human reovirus isolated from children with acute necrotizing encephalopathy Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia Lethal outbreak of disease associated with feline calicivirus infection in cats An outbreak of the polyomavirus infection in budgerigars and cockatiels in Slovakia, including a genome analysis of an avian polyomavirus isolate Fatal poxvirus outbreak in a colony of New World monkeys Identification and phylogenetic analysis of orf virus from goats in Taiwan An outbreak of teat papillomatosis in cattle caused by bovine papilloma virus (BPV) type 6 and unclassified BPVs Poxvirus infection in a great tit (Parus major) Outbreak of cutaneous form of poxvirus on a commercial turkey farm caused by the species fowlpox An outbreak of pseudocowpox in fattening calves in southern Brazil Fatal outbreak in Tonkean macaques caused by possibly novel orthopoxvirus, Italy The use of convalescent sera in immuneelectron microscopy to detect non-suspected/new viral agents Guidance for industry: A viral safety evaluation of biotechnology products derived from cell lines of human or animal origin Adventitious agent issues One-step fluorescent probe productenhanced reverse transcriptase assay Retroviruses produced by hybridomas Genomic organization and expression of endogenous retrovirus-like elements in cultured rodent cells Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 Evaluation of a quantitative productenhanced reverse transcriptase assay to monitor retrovirus in mAb cell-culture Characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals Virus detection by transmission electron microscopy: Still useful for diagnosis and a plus for biosafety We wish to thank Fabienne Arcanger, Sonia Georgeault, Christine The authors have no competing interest.ORCID Philippe Roingeard http://orcid.org/0000-0001-9131-3341