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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
Influenza is one of the most ancient and intriguing diseases that has been accompanying our civilization for millennia. While mankind has successfully defeated many dangerous influenza infections in the last couple of centuries, influenza control remains a serious problem for public health. A number of influenza vaccines and antiviral compounds have been licensed in recent times. However, the influenza virus is still ahead of us, as it continues to persistently infect humans to this day. Influenza: A Century of Research shows how influenza virology has developed historically and the tremendous knowledge that has been uncovered in the study of influenza. In this monograph, the authors present a historical perspective on influenza, chronologically, with an emphasis on its virology. Chapters cover information about the isolation of the first influenza viruses, substrates, and models for studying influenza, structure, and life cycle of the influenza virus, mechanisms of attenuation and virulence. Chapters progress into the multidisciplinary aspects of influenza research such as influenza virus ecology and the evolutionary origin of epidemic and pandemic influenza viruses. A significant part of the book also covers the description of the prevention and treatment of influenza and reasons that have contributed to insufficient control for influenza. The questions of how the COVID-19 pandemic affects the circulation of seasonal respiratory viruses, and if we can eliminate this virus are also addressed.
Influenza: A Century of Research is an informative source of information for a broad range of readers, academic or otherwise, who are interested in knowing more about the disease.
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FOREWORD
Almost 100 years have passed since the discovery of the first influenza virus. Intensive research and advances in scientific methods have largely filled the information gap and blank spots in virology, but not fully. “Influenza: A Century of Research” is a comprehensive Book covering in condensed form a success achieved over a century of studying the virus from the discovery of the first influenza virus through to the main aspects of influenza and the development of vaccines and antivirals.
Clearly written by two experts in the field, the book is easy to read. Illustrations in full color are simple for understanding and suggestive and the bibliography is quite comprehensive – the Book is filled with 855 references from the era of discovery of the first viruses until the last findings in the influenza research. The Book summarizes and systematizes available information on this regard and aims to contribute to emphasize the need for better control for influenza.
The book is divided into seven key chapters covering historical aspects, models, and substrates for studying influenza virus, replication, ecology and evolution of influenza virus, prophylaxis and treatment, the impact of influenza and COVID-19 pandemics on the circulation of seasonal respiratory viruses. As an expert in influenza chemotherapy, I am most impressed by the sections examined types of influenza vaccines and anti-influenza drugs. “Influenza: A Century of Research” offers a high level of consistency of chapters. Their flow constitutes a continuous narrative coupled with the text’s clear, enables readers from professionals to students to grasp key principles of influenza virology with ease.
The discussion of the reasons contributed to insufficient control for influenza is of particular interest. We cannot predict the future but we can try to protect ourselves from the influenza burden. The contents of this book are intended for all those working or studying in the area of influenza. I have read this Book with great interest and can recommend it to a wide range of readers, who will have to evaluate how the authors coped with their task.
PREFACE
Influenza is one of the most mysterious and ancient diseases that have been accompanying our civilization for centuries. The first written description of influenza-like disease belongs to Hippocrates (5th century BC). However, the exact age of the influenza virus is unknown.
Humankind has successfully defeated many dangerous infections that caused harm to human health and safety. Plaque, polio, smallpox, tuberculosis, measles, and many other infectious diseases were completely eradicated or controlled. In contrast, influenza control remains a serious problem for public health.
According to the World Health Organization, influenza is responsible for 3-5 million severe illnesses worldwide and up to 650 thousand respiratory deaths. The unpredictable character of the influenza virus spread is a significant threat to humans. In the last eight decades after the first influenza virus was isolated, huge progress has been made in studying, preventing, and treating influenza. Several influenza vaccines and antiviral compounds have been licensed. However, the influenza virus is still ahead of us. Therefore, this eBook aims to show how influenza virology has developed historically. We briefly demonstrate the tremendous success that has been achieved in the study of influenza over the past 100 years and discuss if there is a way to control this infection.
A lot has been written about influenza already. PubMed and other online resources supporting the search and retrieval of peer-reviewed biomedical and life sciences literature comprise over 200,000 articles, reviews, and books. In this eBook, we have tried to illuminate knowledge on influenza from a historical perspective, chronologically, with an emphasis on the virological part of the studies.
Chapter 1 describes a history of isolation of the first influenza viruses and dwells on the historical aspect of the development of appropriate substrates and models for studying influenza. Chapter 2 addresses the genome and capsid structure, molecular mechanisms of replication, and life cycle of the influenza virus. Mechanisms involved in the attenuation and virulence of the influenza virus are also discussed. Chapter 3 presents a comprehensive review of the influenza virus ecology and evolution. The origin of epidemic and pandemic influenza viruses is discussed as well. Chapter 4 focuses on influenza prophylaxis and treatment. Historical aspects of current achievements in this field were reviewed. Reasons contributed to insufficient control for influenza are highlighted in Chapter 5. The main properties of the influenza virus that may influence control for influenza are described. The COVID-19 pandemic affected both all aspects of our lives and the circulation of seasonal respiratory viruses. Chapter 6 describes some issues arising with the spread of pandemic viruses in general and SARS-CoV-2 infection in particular.
The short final Chapter 7 has been written in the form of conclusion; it is devoted to briefly summarize the 100-year study of influenza. Despite the huge number of research and years or rather centuries of research, a cure for the virus has not yet been found. The possibility of defeating influenza in the nearest future is discussed.
The intended audience for this eBook includes students of biological and medical colleges, Ph.D. students, post-docs, a wide range of virologists who are specialized in the field of influenza, and everyone interested in this infection.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The author declares no conflict of interest, financial or otherwise.
ACKNOWLEDGEMENTS
Finally, the authors would like to thank Dr. Vladimir Zarubaev for writing the foreword, Bentham Science Publishers for the continuous support throughout the process of writing this book, and the Russian Science Foundation (grant 21-75-30003) for the financial support.
Historical Aspects
Irina Kiseleva,Natalie LarionovaAbstract
The first animal influenza A virus was isolated in 1931 by Richard Shope. The virus caused a highly contagious, influenza-like disease in pigs. Two years later, in 1933, the first human influenza A virus was isolated by Wilson Smith and colleagues. Soon after, in 1940, a representative of influenza virus type B was discovered by Thomas Francis, Jr. Being obligate intracellular parasites, viruses can be cultivated only within sensitive substrates. Three main substrates for the cultivation of influenza viruses are known: sensitive animals, embryonated chicken eggs, and tissue cultures. Today, in the twenties of the 21st century, sensitive animals are not often used for the isolation of the infectious virus. However, they are widely used to study and model a number of infectious diseases, including influenza. A list of these animals used for influenza research is very long, starting from ferrets and mice and ended with exotic zebrafish.
INTRODUCTION
Influenza is one of the most mysterious and ancient diseases known for centuries. The exact age of the influenza virus is unknown. Humankind has successfully eradicated many deadly infections like plaque, polio, etc. While acknowledging the measures of the World Health Organization (WHO), Centers for Disease Control and Prevention (CDC), and other specialized agencies responsible for international and national public health, have taken to control influenza, it remains a serious cross-border problem. According to WHO, influenza is responsible for million cases of severe illnesses worldwide and up to 650 thousand respiratory deaths [1]. The unpredictable character of the influenza virus spread is a significant threat to humans.
Isolation of the first influenza viruses resulted in a rapid flowering of influenza research. The right choice of adequate animal models and substrates for virus cultivation is very important. Therefore, in this chapter, the historical aspect of the development of appropriate substrates and models for studying influenza is discussed.
THE FIRST INFLUENZA VIRUSES
Influenza viruses belong to the Orthomyxoviridae family. Members of this family, which include seven genera [2, 3], are characterized by similarities in the structure of the viral particle and the way of reproduction. These are enveloped RNA viruses with a single-stranded segmented minus-stranded (negative-stranded) genome [4, 5]. Looking back in history, it is remarkable that the first influenza virus was isolated in Italy 119 years ago but classified as “fowl plaque” or “fowl pox” virus. Fifty years later, it was found that this is one of the highly pathogenic avian influenza A viruses.
In 1931, Richard Shope isolated a filterable agent that caused a highly contagious, influenza-like disease in pigs [6-8]. Soon after, during the 1933 epidemic, the first human influenza A virus was isolated by Smith et al. [9]. After several unsuccessful attempts to infect different species such as guinea pigs, mice, snakes, and hedgehogs, it was found that only ferrets exhibited catarrhal, nasal, and temperature symptoms of respiratory disease similar to humans. In 1940, the first human influenza B virus was discovered [10], and in 1968, the new influenza A virus subtype H3N2 was identified in tissue culture [11]. In 1947, in the United States, type C influenza virus C/Taylor/1233/1947 was identified in a human with upper respiratory symptoms [12].
In the early 1950s, the type D influenza virus (Sendai strain) that typically affects rodents and is not pathogenic for humans was isolated. However, later it was discovered that the Sendai virus belongs to the family Paramyxoviridae. The real influenza D virus belongs to the recently characterized new genus in the Orthomyxoviridae family. This virus was originally detected in pigs in the US [13]; however, cattle are now believed to be the main reservoir of the type D influenza viruses [13-15].
The taxonomic division of influenza viruses is based on their antigenic characteristics. According to the antigenic specificity of the internal structural components of the virion: the nucleoprotein (NP) and the matrix protein (M1), they are subdivided into four genera A, B, C and D [2, 3]. Type C and D have 7 RNA segments and encode 9 proteins, while types A and B have 8 RNA segments. Viruses belonging to different genera do not have common antigens, differ in epidemiological features and the severity of the disease they cause. Genetic reassortment between viruses of different genera does not occur. Comparative characteristics of influenza viruses A, B, C and D are presented in Table 1.
Influenza A virus causes the most severe diseases and may infect a wide range of host species. There are 18 hemagglutinin (HA) subtypes and 11 neuraminidase (NA) subtypes, respectively [16]. Among 198 potential influenza A subtype combinations, 131 subtypes have been detected in wild nature [30]. Serotypes of HA, in turn, form two phylogenetic groups that differ significantly from each other, but within each group, the domain encoding the HA stalk is antigenically similar: group 1 includes hemagglutinins H1, H2, H5, H6, H8, H9, H11 – H13, H16 and H17, group 2 - hemagglutinins H3, H4, H7, H10, H14 and H15 [31]. Two subtypes of influenza A viruses are routinely circulating in humans: A (H1N1)pdm09 and A (H3N2). Influenza virus ecology and evolution will be discussed in detail in Chapter 3.
Human influenza A viruses cause annual epidemics and occasionally pandemics. In addition, viruses of zoonotic origin sporadically infect humans, causing severe respiratory infections and high mortality. Most zoonotic viruses are incapable of sustained human-to-human transmission, but mutations or reassortment with human influenza viruses in extremely rare cases leads to the emergence of a new virus with pandemic potential that can be transmitted by airborne droplets [32]. Only three serotypes of HA (H1, H2, and H3) and two serotypes of NA (N1 and N2) were integral parts of the pandemics pathogens and circulated widely among humans during the era of influenza studies [33]. The annual (seasonal) epidemics due to influenza A viruses are caused by the high rate of antigenic mutations that allow the virus to escape the immune defenses. Influenza viruses of genera B and C are not subdivided into serotypes, and no pandemics occur with their participation [34].
Influenza B viruses cause seasonal epidemics with the typical pattern of influenza infection. They are subject to antigenic variability too, although the rate of appearance mutations is 3-5 times lower compared to influenza A viruses [34, 35]. Since the early 1980s, two branches (lines) of the influenza B virus have been circulating alternately and jointly, differing significantly in antigenic characteristics of HA and NA and other properties [19, 35]. Influenza B virus causes less severe diseases than influenza A virus but can cause outbreaks and the influenza C virus causes acute respiratory illness most commonly in infants and young children, usually only associated with mild upper respiratory illness.
Influenza D virus has been identified recently. Its primary reservoir is cattle. Sheep, goats, pigs, horses, and camels are also susceptible to infection [3, 17, 36]. It is still unknown whether the influenza D virus causes disease in humans, but 97% of people in contact with cattle in Florida have antibodies to the influenza D virus [29].
MODELS FOR STUDYING INFLUENZA
Laboratory animals are widely used to study and model a number of diseases of an infectious and non-infectious nature, as well as for isolation of infectious agents. Animals, especially vertebrates, have repeatedly been used throughout the history of medical and biological discoveries and to this day, play a key role in biomedical researches. The first references to animal studies could be found in the writings of Greek philosophers and physicians Aristotle and Erasistratus, who most likely were the first to perform experiments on living animals. Louis Pasteur was the pioneer in the use of animals to prove the infectious nature of some diseases, such as anthrax [37, 38] or rabies [39].
Fig. (1)) Use of laboratory animals, %. The areas of animal models’ use are wide.Since the 18th century, the use of laboratory animals in experimentation became more common and the numbers of laboratory animal procedures conducted continue to rise progressively [40-42]. The areas of animal models’ use can be seen in Fig. (1). The majority of animals are used in biomedical research (testing vaccines and biologicals, cancer research, heart diseases, and circulation research, etc.), basic research including military, space, etc., drug research (toxicity tests, cosmetics, new antiviral compounds, etc.) and in education.
Animal research is now becoming even more prevalent - it is estimated that the number of mice and rats used in research has been increasing every year. Over 50 years ago, the principles of the 3Rs (Replacement, Reduction, and Refinement) providing a framework for performing more humane animal research were developed (Fig. 2). 3Rs are guiding principles for the more ethical use of animals in preclinical studies. Replacement methods which avoid/replace the use of animals; reduction methods which minimize the number of animals used per experiment; refinement methods which minimize animal suffering and improve welfare. The questions of how to refine to lessen pain, reduce the number of animals used, and replace with non-animal methods and how many animals should be included in the group are under discussion [43, 44].
Fig. (2)) Three guiding principles for the more ethical use of animals in preclinical studies (3Rs principles).Ferrets were the first animals successfully used for the isolation of the first human influenza virus (see section “The first influenza viruses” above). Currently, ferrets are widely used to study the pathogenesis of influenza infection (Fig. 3) [45-51] and transmissibility of influenza viruses [45, 47, 50]. Moreover, according to WHO recommendations [52], ferrets are used for preclinical characterization of potentially pandemic influenza vaccines [53-60].
Fig. (3)) Nasal symptoms and gross examination of the lungs of ferrets infected with A/South Africa/3626/2013 (H1N1)pdm09 influenza virus [61]. (a, b) The ferrets were inoculated with the virus. (a) Severe lung lesions. (b) Nasal discharge on the external nares (red arrow). (c) The lungs of the ferret inoculated with phosphate-buffered saline.A wide range of other experimental animals (mice, poultry, guinea pigs, cotton rats, pigs, hamsters, macaques) are used to study the various aspects of the manifestation of influenza infection and the selection of therapeutic and prophylactic drugs to treat influenza infection (Table 2) [47, 50, 62]. The most common models are mice (over 70% of all models used). A zebrafish has been actively used in the past few years to study the immune response to influenza and the selection of anti-influenza chemotherapy drugs [63-65]. Another exotic animal, the tree shrew, was shown to be physiologically and genetically related to primates, which make it a potential animal model of human diseases [66, 67]. Proper modeling of various aspects of the manifestation of influenza infection in the relevant sensitive animals is the key to scientific success.
The difference between a sensitive animal model as a substrate for virus isolation or research and a natural reservoir (host) of the influenza virus should be clearly understood (Fig. 4). Mice, ferrets, guinea pigs, etc., are very sensitive experimental animal models, but they never meet the influenza virus in their natural habitats. Between animals-substrates and animals-hosts, an additional group may be distinguished – animals who are not natural hosts but maybe occasionally infected. An accidental host accidentally harbors an organism that is not ordinarily parasitic in the particular species and usually does not infect it. Influenza A viruses can infect species other than the natural hosts in which they normally circulate on rare occasions.
Fig. (4)) Natural reservoirs and laboratory models for influenza A viruses.For instance, humans could become accidental hosts for the avian influenza A (H5N1) virus. While avian influenza A viruses are highly species-specific, they may occasionally cross the species barrier to infect other species causing a disease of high lethality (Fig. 5).
Fig. (5)) Laboratory-confirmed human cases of avian influenza A (H5N1) reported to WHO in 2003-2020 and mortality rate (reprinted with permission from Book Publisher International) [98]. 1The number of deaths (%) from the total number of laboratory-confirmed H5N1 cases. 2Data from January 2020 through 28 February 2020.This disease should not be confused with seasonal human influenza, generally caused by human H1N1pdm09, H3N2, or B viruses. Episodic transmission of avian influenza viruses to humans occurs when there is close contact with infected birds. Due to the high lethality and virulence, the avian influenza virus of subtype H5N1 is one of the world's largest pandemic threats.
Ferrets being a highly experimental model that is sensitive to the influenza virus, could occasionally become the accidental hosts. During the influenza season, laboratory breeding ferrets can catch influenza from sick lab technicians or ferret's farmworkers. Influenza disease in ferrets can be fatal (Fig. 3).
For obtaining adequate results and their correct assessment, three important conditions must be met:
The right choice of anesthetic for animal study.Standardization of the infectious dose.The right choice of the virus as a model for research.The Right Choice of Anesthetic for Animal Study
Virological and histopathological examinations occupy an important role in preclinical animal studies of influenza. A whole range of manipulations with animals is carried out under anesthesia to minimize their suffering. The refinement of anesthetic choice is an important part of experimental procedures. Ketamine is widely used in veterinary practice as a surgical anesthetic for general anesthesia. However, in some countries, ketamine is included in the list of narcotic drugs, psychotropic substances, and their precursors, which are subject to control, and its use is strictly limited. In veterinary practice, isoflurane is also broadly used as an anesthetic agent. Efficacy and safety of isoflurane have been shown in a number of nonclinical [99, 100] and clinical [101] trials. However, isoflurane may induce airway irritation [102] and should be used with caution in studies of the pathogenesis of respiratory viruses. It was shown that a five-fold inhalation of isoflurane might dramatically affect lung tissue and cause injuries in the form of hemorrhagic lung edema. Gross necropsy examination revealed gross lung lesions in a mock-inoculated group similar to animals infected with wild-type (WT) influenza virus [103]. This finding corresponded to a previous study [104]. The authors reported that general anesthesia with volatile agents, including isoflurane, provoked lung injury, acute inflammatory response, and leukocytic infiltration in rats. In contrast, the absence of any non-specific lung damages in ferrets caused by the injection of zoletil 100 was reported in studies of influenza virus [103, 105, 106] (Fig. 6).
Fig. (6)) Macroscopic view of the lungs of ferrets after multiple rounds of anesthesia (reprinted with permission from Vaccine Research) [103]. Yellow arrows – pulmonary hemorrhages. Ferrets received five applications of anesthesia. Isoflurane maintained inhalation anesthesia; intramuscular anesthesia was induced by injection with zoletil 100. At the end of the experiment, ferrets of all groups were humanely euthanized with a combination of Zoletil 100 and xylazine. (a) The ferret was anesthetized with isoflurane and received only PBS (placebo); (b) The ferret was anesthetized with isoflurane and inoculated with WT influenza B virus; (c) The ferret was anesthetized with zoletil 100 and received only PBS (placebo); (d) The ferret were anesthetized with zoletil 100 and inoculated with WT influenza B virus.Standardization of the Infectious Dose
Standardization of the infectious dose is very important for study design and subsequent interpretation of the results. The literature describes three options for standardizing the dose of influenza virus to be used for infect experimental animals: (i) the 50% minimum infectious dose (MID50) [107, 108]. Depending on the objectives, animals were usually infected with 10-100 MID50. However, a preliminary determination of the MID50 is often a very expensive procedure, especially when it comes to animals such as monkeys or ferrets. As the option, the infectious dose to be inoculated to animals can be established (ii) by the measure of infectious virus titer in embryonated chicken eggs (50% embryo infectious dose, EID50) or in tissue culture (50% tissue culture infectious dose, TCID50; plaque-forming units, PFU); the animals are infected with one dose in the range of 5.0-7.0 log10 EID50 [95, 109-113] or 3.0-8.0 log10 TCID50/PFU [105, 114-120]. (iii). If the virus to be tested is lung-adapted and may cause animals’ death, the 50% lethal dose (LD50) can be used [47, 107, 116].
The Right Choice of the Virus as a Model for Research
The susceptibility of the animal model to the influenza virus depends not only on the animal species but also on the virus strain to be tested in this model.
Historically, the pathogenesis of influenza infection has been studied in mice using lab lung-adapted (mouse-adapted) strains [47]. The number of classical mouse-adapted viruses is limited, for instance, A/Puerto Rico/8/34 (H1N1), B/Lee/40, A/Victoria/35/72 (H3N2), A/Aichi/2/68 (H3N2) and a few more. They have been used for pathogenicity studies for decades. However, today, in the twenties of the 21st century, all of them are antigenically distinct from circulating viruses.
The process of influenza virus adaptation to a new host is rather difficult. Fortunately, certain influenza viruses of the avian origin [108, 121], reconstructed 1918 Spanish influenza pandemic virus [122], and some H1N1pdm strains of the 2009 pandemic [123, 124] may cause disease in mice without prior adaptation. As it can be seen in Fig. (7), the non-mouse-adapted A/South Africa/3626/2013 (H1N1)pdm09 virus displayed severe lung lesions comparable to that of the mouse-adapted A/Puerto Rico/8/34 virus [61].
It allows making the overall conclusion that circulating non-mouse-adapted influenza viruses may be used for preclinical assessments of antiviral compounds or vaccine candidates. The advantage of non-adapted viruses is that (i) there is no need for additional lab work for their adaptation to model animals; (ii) no additional mutations occur in the genome of the virus during the long adaptation to another host.
Fig. (7)) Gross examination of the lungs of mice infected with A/South Africa/3626/2013 (H1N1)pdm09 and A/Puerto Rico /8/34 (H1N1) viruses (adapted from [61]). 1A/Puerto Rico/8/34 influenza virus. 2A/South Africa/3626/2013 influenza virus.SUBSTRATES FOR INFLUENZA VIRUS CULTIVATION
Being obligate intracellular parasites, viruses cannot be grown on any culture medium as bacteria can. Viruses can be cultivated only within suitable living hosts (substrates). Three main substrates for the cultivation of influenza viruses are known: (i) sensitive animal models; (ii) embryonated chicken eggs (herein simply referred to as “eggs”); (iii) tissue (cell) culture (Fig. 8). The primary objectives of the cultivation of the influenza virus include isolation and identification of viruses in clinical specimens, the development of vaccines, and extensive research on influenza.
Experimental Animals
In the past, animals played an important role both as substrates for influenza virus isolation and as experimental models in influenza studies. As of today, in the twenties of the 21st century, the role of animals as a substrate became less prominent.
By the time the influenza virus was first isolated, laboratory animals were the only known substrate. In 1933, Smith et al. [9] isolated in ferrets the human influenza A strain. In 1937, another human influenza A virus was localized in mice by Smorodintsev et al. [125-127].
Embryonated Chicken Eggs
After chicken embryos were discovered as an alternative system for isolation of different viruses [128], a new era of virus isolation, including the influenza virus began.
Fig. (8)) Primary objectives of cultivation of influenza virus. Three main substrates for the cultivation of influenza viruses (sensitive animals, embryonated chicken eggs, and tissue culture) are known.Tissue Culture
For decades, influenza viruses were propagated from clinical specimens in eggs or cultures of rhesus monkey kidney (Vero cell line) [129]. An alternative tissue culture system for the primary isolation and cultivation of influenza viruses was described in 1968 [130]. The authors found MDCK cells to be susceptible to a number of influenza virus strains.
In 1978, WHO officially suggested that continuous MDCK cells provide a satisfactory alternative to eggs and monkey kidney cultures for the isolation of influenza viruses from clinical specimens [131].
Laboratory Methods for Diagnosing Influenza
Influenza can be difficult to diagnose through clinical signs alone because so many of the symptoms are similar to other acute respiratory diseases [132]. Laboratory methods are potentially more reliable and provide a much clearer picture of the nature of the disease. In 2011, a new edition of the “WHO manual for the laboratory diagnosis and virological surveillance of influenza” [133] was published, in which, on 153 pages, current methods used for the laboratory diagnosis of influenza are described in detail. In 2018 it was supplemented with “WHO information for the molecular detection of influenza viruses” [134] and the next year, WHO published revised guidance for National Influenza Centres which contained a description of the next-generation sequencing (NGS) of influenza viruses [135]. This document provides information on molecular detection and diagnostic protocols for the surveillance of influenza viruses in humans. NGS, a novel DNA sequencing technique, provides high speed and throughput that can be used in research and diagnostic virology [136].
The two main strategies in influenza diagnosis are to isolate the virus or its fragments directly from respiratory secretions (collection of specimens) or to measure the body’s immune response to infection (retrospective analysis) (Fig. 9). Laboratory diagnostic procedures for testing influenza include three groups of methods: (i) virus isolation by virological methods, (ii) serological diagnosis, and (iii) molecular identification (Fig. 9).
Fig. (9)) Current strategies and laboratory procedures for diagnosing influenza.Virus Culturing by Virological Methods
There are no dedicated methods in the diagnosis of influenza. The HA (hemagglutination) and HAI (inhibition of hemagglutination) tests described by Hirst [137-139] in the early 1940s of the XX century are successfully used to this day. The conventional gold standard for influenza diagnosis is cell-derived or egg-derived viral culture [140]. Viral culture tests are accurate and conclusive methods for diagnosing influenza and continue to provide a conclusive diagnosis. While the long turnaround times make this less useful in clinical settings, it is still a valid method for research. However, results can take 48-72 hours, up to 10 days.
Serological Diagnosis
Antibodies to influenza virus appear after 2 weeks and peak 4 to 7 weeks after infection. A variety of serological tests, including hemagglutination inhibition (HAI) assays and enzyme immunoassays (immunofluorescence antibody staining, IFA, enzyme-linked immunosorbent assay, ELISA), exist [140]. A ≥ 4-fold increase in influenza virus antibody titers in paired samples obtained at least 2 weeks apart establishes the serologic diagnosis of influenza. The requirement for paired samples obtained weeks apart renders this method not useful for urgent clinical diagnostic testing, but it can be helpful for retrospective analysis [133, 140].
Molecular Identification
Nowadays, viral culture has been somewhat superseded by tests based on reverse transcription polymerase chain reaction (RT-PCR), which produces results in hours rather than days and is considered the most sensitive and specific test for influenza [134, 135, 140]. Molecular tests afford the detection of influenza genetic material.
Rapid Influenza Diagnostic Tests (RIDTs)
RIDTs are immunoassays that can identify the presence of nucleoprotein of influenza virus in respiratory specimens, and display the result as positive vs. negative. RIDTs are the most common testing methods used to diagnose influenza [141, 142]. These point-of-care tests help in appropriate specimen collection and provide near-instantaneous results. However, not all RIDTs can distinguish between influenza A and B; and no RIDTs can distinguish between the varying strains of influenza A. Besides, if compared with other testing methods, RIDTs have the lowest sensitivities and the highest rate of false-negatives.
CONCLUDING REMARKS
The history of the detection of the influenza A virus dates back to the 1930s of the last century when the first swine influenza and human influenza viruses were isolated in animal models. From that moment, the number of animal models has grown substantially, and today is approaching 20 different species. Intensive study of sensitive substrates allowed us to discover many cell cultures highly sensitive to a variety of influenza viruses. Nevertheless, embryonated chicken eggs remain a widely used substrate for the isolation of influenza viruses and the production of influenza vaccines. Several hundred animal and human influenza viruses were isolated in embryonated chicken eggs and sensitive cell cultures, and their sequences were deposited in different databases. As of 16 March 2021, according to only one of the influenza databases, GISAID [143], a total of 1,448,836 sequences of 338,310 influenza virus strains of different types were deposited.
