Article

Studies on the mechanism of adaptation of influenza virus to mice

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Abstract

1. When strains of influenza A virus which have been isolated in chick embryos are introduced into the mouse lung, the virus multiplies readily and achieves initially a titer which is as high as is even obtained, even after repeated passage. The high initial titer of virus may be unaccompanied by any lethal or visible pathogenic effects; but with four or five mouse passages the agent becomes lethal in high titer and causes extensive pulmonary consolidation, though its capacity to multiply in the lung has not increased. In one example the adaptation to mouse lung was accompanied by increasing capacity to agglutinate guinea pig red cells without a corresponding increase in agglutinating power for chicken cells. Influenza B virus, in preliminary tests, did not behave in a similar fashion. 2. The adaptation of influenza A virus to mice is accompanied by changes in antigenic pattern, as detected by cross-tests with the agglutination inhibition method. Two strains, initially similar, with passage, changed in pattern along divergent paths so that they became not only unlike the parent strains but unlike each other. This finding has important implications for the interpretation of the strain difference problem in human influenza.

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... Detailed studies of Type B strains from isolated outbreaks have not been reported. Changes in the antigenic composition of single strains of influenza virus on passage in laboratory animals or in tissue cultures have been reported (13,35,64,92). Unfortunately these latter studies were largely incidental and thus have contributed little to our knowledge of the mechanism of antigenic change. ...
... The recent studies of Hirst (92), Friedewald and Hook (72), and of Wang (148) are interesting because of the picture they enable us to form concerning pathogenicity as an inherent property of the virus particle. Hirst (92) demonstrated that the ALA-41, KIL41, and NY-43 strains of Type A multiply readily on initial mouse passage achieving a maximum egg infectivity titer, but unaccompanied by any lethal or visible pathogenic effects. With 4 or 5 mouse passages these virus strains become lethal, causing extensive pulmonary consolidation (92). ...
... Hirst (92) demonstrated that the ALA-41, KIL41, and NY-43 strains of Type A multiply readily on initial mouse passage achieving a maximum egg infectivity titer, but unaccompanied by any lethal or visible pathogenic effects. With 4 or 5 mouse passages these virus strains become lethal, causing extensive pulmonary consolidation (92). With the NY43, the only strain examined in this way, Hirst (92) observed that the process of adaptation was accompanied by increasing capacity to agglutinate guinea pig erythrocytes without a corresponding increase in agglutinating power for chicken erythrocytes. ...
... While some human seasonal influenza A and B viruses can replicate in the murine respiratory tract, mice are not a natural host for influenza viruses, and adaptation through repeated passage is usually required to increase the virulence of the human-origin viruses for this species (Andrewes et al. 1934;Beaudette et al. 1957;Francis 1934;Francis and Magill 1935;Hirst 1947;Shope 1935). Importantly, even non-mouse-adapted influenza viruses that cause minimal or no disease in mice are often capable of some replication in the respiratory tract (Hirst 1947), since otherwise mouse passage would not allow adaptation to occur. ...
... While some human seasonal influenza A and B viruses can replicate in the murine respiratory tract, mice are not a natural host for influenza viruses, and adaptation through repeated passage is usually required to increase the virulence of the human-origin viruses for this species (Andrewes et al. 1934;Beaudette et al. 1957;Francis 1934;Francis and Magill 1935;Hirst 1947;Shope 1935). Importantly, even non-mouse-adapted influenza viruses that cause minimal or no disease in mice are often capable of some replication in the respiratory tract (Hirst 1947), since otherwise mouse passage would not allow adaptation to occur. The adaptation process increases the ability to replicate and may also increase virulence, either directly as a result of increased replication or through other factors. ...
... However, several lines of evidence suggest that this is not the sole reason for mouse adaptation. As noted above, even viruses that are avirulent in mice may replicate in the lungs (Hirst 1947). Some viruses that preferentially bind α2,6-linked SA, such as the 1918 pandemic H1N1, replicate efficiently and cause significant disease in mice (Qi et al. 2009;Tumpey et al. 2005), while many LPAIV that preferentially bind α2,3-linked SA cause only minimal morbidity in this species (Driskell et al. 2010). ...
Article
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Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account.
... Intranasal instillation of influenza virus freshly isolated from humans into mice does not produce overt disease, although virus may multiply in the lungs, bronchioles, and trachea as well as in nasal tissue (94,103,161). Passage of virus through mice produces strains which cause MICROBIOL. REV. ...
... In ferrets, although low titers of virus were rapidly inactivated by ferret blood in vitro (240), inhibitors present in nasal washings from ferrets infected with either a virulent or an attenuated clone of influenza virus had a similar action on both clones (Husseini, Sweet, and Smith, unpublished data). Adaptation to mice was accompanied by viral resistance to fi inhibitors in one study (29) but not in others (11,94,179). Hence, for influenza of humans, ferrets, and mice, it seems unlikely that resistance to nonspecific inhibitors is important in virulence. ...
... However, mouse phagocytes appear effective against influenza virus; liver parenchyma cells, which could be infected via the bile duct, were not infected by blood-borne virus due to phagocytosis by the Kupffer cells (153). Virus association with erythrocytes (87) may be a mechanism for avoiding phagocytosis, but mouse-adapted strains are no more resistant to serum inhibitors than are nonadapted strains (11,94,179). Hence, we have no valid explanation for the more easily demonstrable viremia which occurs in mice compared with that in humans and ferrets. ...
... However, IBVs are not consistently found infecting species outside humans [2,3], which then requires adaptation of the viruses to develop a disease model for in vivo research. Scientists have used serial lung passaging, termed mouse-adapting, of virus to study viral characteristics or to increase pathogenicity of IBVs [22][23][24][25][26] and IAVs [27][28][29] in mice. Mouse-adapted strains are valuable for vaccine and therapeutic research by causing severe infection, which can highlight differences in protection after vaccination or therapeutic delivery. ...
... However, this process is very time consuming and resource intensive. Some studies reporting the mouse-adaptation of IBVs expanded the virus to high titer in eggs [22,23]. Alternatively, many more recent reports of mouse-adapted IBVs opt to grow the viruses in MDCK cell culture after passage in mice [24][25][26]. ...
Article
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Despite the yearly global impact of influenza B viruses (IBVs), limited host range has been a hurdle to developing a readily accessible small animal disease model for vaccine studies. Mouse-adapting IBV can produce highly pathogenic viruses through serial lung passaging in mice. Previous studies have highlighted amino acid changes throughout the viral genome correlating with increased pathogenicity, but no consensus mutations have been determined. We aimed to show that growth system can play a role in mouse-adapted IBV lethality. Two Yamagata-lineage IBVs were serially passaged 10 times in mouse lungs before expansion in embryonated eggs or Madin–Darby canine kidney cells (London line) for use in challenge studies. We observed that virus grown in embryonated eggs was significantly more lethal in mice than the same virus grown in cell culture. Ten additional serial lung passages of one strain again showed virus grown in eggs was more lethal than virus grown in cells. Additionally, no mutations in the surface glycoprotein amino acid sequences correlated to differences in lethality. Our results suggest growth system can influence lethality of mouse-adapted IBVs after serial lung passaging. Further research can highlight improved mechanisms for developing animal disease models for IBV vaccine research.
... Notably, mice are naturally insusceptible and insensitive to infection with influenza viruses and mice infected with newly isolated human influenza A viruses usually become asymptomatic. Many strains of mice can be infected experimentally with influenza viruses, particularly with mouse lung-adapted viruses [6], and allow the infected viruses to replicate in their lungs [5]. Following infection with influenza A virus, the virus induced humoral immunity can clear the viruses in the lungs around five days post infection. ...
... Adaption is thought to be the driving force in evolution, during which organisms are selected in nature because of increased fitness conferred by beneficial mutations. Although mice are not naturally infected with seasonal H1N1 influenza viruses, many strains of mice can be infected with human strains of influenza viruses, which can be adapted to the mouse by serial lung passage [6]. In our study, a virulent strain of mouse adapted virus with virulence was produced following eight serial lung-to-lung passages in mice. ...
Article
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The experimental infection of a mouse lung with influenza A virus has proven to be an invaluable model for studying the mechanisms of viral adaptation and virulence. The mouse adaption of human influenza A virus can result in mutations in the HA and other proteins, which is associated with increased virulence in mouse lungs. In this study, a mouse-adapted seasonal H1N1 virus was obtained through serial lung-to-lung passages and had significantly increased virulence and pathogenicity in mice. Genetic analysis indicated that the increased virulence of the mouse-adapted virus was attributed to incremental acquisition of three mutations in the HA protein (T89I, N125T, and D221G). However, the mouse adaption of influenza A virus did not change the specificity and affinity of receptor binding and the pH-dependent membrane fusion of HA, as well as the in vitro replication in MDCK cells. Notably, infection with the mouse adapted virus induced severe lymphopenia and modulated cytokine and chemokine responses in mice. Apparently, mouse adaption of human influenza A virus may change the ability to replicate in mouse lungs, which induces strong immune responses and inflammation in mice. Therefore, our findings may provide new insights into understanding the mechanisms underlying the mouse adaption and pathogenicity of highly virulent influenza viruses.
... The basis of this phenomenon is that inbred mice possess an interferon-inducible restriction factor known as Mx1 3 . However, most influenza strains can be experimentally adapted for mouse virulence by serial lung-to-lung passages 4,5 . Mouse adaptation results in the acquisition of functions that are critical determinants of virulence with increased viral titers in the lungs and increased pathogenesis and mortality. ...
... Mouse adaptation results in the acquisition of functions that are critical determinants of virulence with increased viral titers in the lungs and increased pathogenesis and mortality. Mouse-adapted influenza mutants usually induce pathologic changes in the bronchi or lungs, possess an increased ability to infect alveolar cells and can cause lethal pneumonitis [4][5][6] . The main advantage of using mice is that the pulmonary pathology is similar to that seen in the cases of viral pneumonia in humans 7 . ...
Article
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The mouse is the most widely used animal model for influenza virus research. However, the susceptibility of mice to seasonal influenza virus depends on the strain of mouse and on the strain of the influenza virus. Seasonal A/H3N2 influenza viruses do not replicate well in mice and therefore they need to be adapted to this animal model. In this study, we generated a mouse-adapted A/H3N2 virus (A/Switzerland/9715293/2013 [MA-H3N2]) by serial passaging in mouse lungs that exhibited greater virulence compared to the wild-type virus (P0-H3N2). Seven mutations were found in the genome of MA-H3N2: PA(K615E), NP(G384R), NA(G320E) and HA(N122D, N144E, N246K, and A304T). Using reverse genetics, two synergistically acting genes were found as determinants of the pathogenicity in mice. First, the HA substitutions were shown to enhanced viral replication in vitro and, second, the PA-K615E substitution increased polymerase activity, although did not alter virus replication in vitro or in mice. Notably, single mutations had only limited effects on virulence in vitro. In conclusion, a co-contribution of HA and PA mutations resulted in a lethal mouse model of seasonal A/H3N2 virus. Such adapted virus is an excellent tool for evaluation of novel drugs or vaccines and for study of influenza pathogenesis.
... In later studies, human influenza A viruses were cultivated in embryonated chicken eggs prior to infection in mouse models. In these cases, the viruses replicated well but caused asymptomatic infections with little or no pathology, even when given at very high titers (Hirst 1947b;Novak et al. 1993). Murine infection with nonadapted influenza viruses has revealed that infection in mice is variable, but once established, replicating virus can be isolated from the lung, trachea, and nares for at least 5-6 days (Novak et al. 1993). ...
... Repeated passage of human influenza viruses in mouse lungs can quickly adapt the virus to the mouse and result in virulent mouse-adapted viruses (Hirst 1947b;Novak et al. 1993;Smeenk and Brown 1994). Mouse-adapted viruses can cause severe pathology, morbidity and mortality, and lethal pneumonia caused by mouseadapted influenza virus infection is similar to the pathology seen in human lower respiratory tract infections (Smeenk and Brown 1994). ...
Article
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Influenza viruses are emerging and re-emerging viruses that cause worldwide epidemics and pandemics. Despite substantial knowledge of the mechanisms of infection and immunity, only modest progress has been made in human influenza vaccine development. The rational basis for influenza vaccine development originates in animal models that have helped us to understand influenza species barriers, virus-host interactions, factors that affect transmission, disease pathogenesis, and disease intervention strategies. As influenza evolution can surmount species barriers and disease intervention strategies that include vaccines, our need for appropriate animal models and potentially new host species will evolve to meet these adaptive challenges. This chapter discusses animal models for evaluating vaccines and discusses the challenges and strengths of these models.
... On the one hand, adaptation allows the virus to cross species boarders, evade immune or therapeutic pressures and optimize its replication in a given host system [1]. On the other hand, it challenges manufacturers to adapt emerging strains to existing egg-based or cell-culture-based system processes to obtain maximum yields for formulation of potent vaccines [2,3]. Escape from immune pressure, balancing host cell receptor binding avidity of input virus with the release of progeny virus as well as adjustment to altered endosomal pH-values or to different, specific sialic acid containing host cell receptors have been described as driving forces for adaptation processes in virus evolution [2,[4][5][6]. ...
Article
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The genome of influenza A viruses is constantly changing (genetic drift) resulting in small, gradual changes in viral proteins. Alterations within antibody recognition sites of the viral membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) result in an antigenetic drift, which requires the seasonal update of human influenza virus vaccines. Generally, virus adaptation is necessary to obtain sufficiently high virus yields in cell culture-derived vaccine manufacturing. In this study detailed HA N-glycosylation pattern analysis was combined with in-depth pyrosequencing analysis of the virus genomic RNA. Forward and backward adaptation from Madin-Darby Canine Kidney (MDCK) cells to African green monkey kidney (Vero) cells was investigated for two closely related influenza A virus PR/8/34 (H1N1) strains: from the National Institute for Biological Standards and Control (NIBSC) or the Robert Koch Institute (RKI). Furthermore, stability of HA N-glycosylation patterns over ten consecutive passages and different harvest time points is demonstrated. Adaptation to Vero cells finally allowed efficient influenza A virus replication in Vero cells. In contrast, during back-adaptation the virus replicated well from the very beginning. HA N-glycosylation patterns were cell line dependent and stabilized fast within one (NIBSC-derived virus) or two (RKI-derived virus) successive passages during adaptation processes. However, during adaptation new virus variants were detected. These variants carried "rescue" mutations on the genomic level within the HA stem region, which result in amino acid substitutions. These substitutions finally allowed sufficient virus replication in the new host system. According to adaptation pressure the composition of the virus populations varied. In Vero cells a selection for "rescue" variants was characteristic. After back-adaptation to MDCK cells some variants persisted at indifferent frequencies, others slowly diminished and even dropped below the detection limit.
... On the basis of these results, we wanted to know whether the pathogenicity of a 2009 H1N1v isolate could be enhanced during passaging of the virus in a mammalian host system. The assumption that lung-to-lung passaging would increase pathogenicity was driven by findings of several previous reports [12][13][14]. Numerous studies have identified mutations within the HA [15][16][17][18] and the polymerase subunits as mediators of increased pathogenicity upon passaging in mice [6,16,[18][19][20]. ...
Article
Influenza impressively reflects the paradigm of a viral disease in which continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics in humans. Because of the continuous threat of novel influenza outbreaks, it is essential to gather further knowledge about viral pathogenicity determinants. Here, we explored the adaptive potential of the influenza A virus subtype H1N1 variant isolate A/Hamburg/04/09 (HH/04) by sequential passaging in mice lungs. Three passages in mice lungs were sufficient to dramatically enhance pathogenicity of HH/04. Sequence analysis identified 4 nonsynonymous mutations in the third passage virus. Using reverse genetics, 3 synergistically acting mutations were defined as pathogenicity determinants, comprising 2 mutations in the hemagglutinin (HA[D222G] and HA[K163E]), whereby the HA(D222G) mutation was shown to determine receptor binding specificity and the polymerase acidic (PA) protein F35L mutation increasing polymerase activity. In conclusion, synergistic action of all 3 mutations results in a mice lethal pandemic H1N1 virus.
... In preliminary experiments, MDCK culture-derived WSN/33 (H1N1, IAV) was only moderately lethal to mice, so we adapted the parent WSN/33 to mice using serial passages to enhance its virulence [37][38][39], as described in the Section 2. Mouseadapted WSN/33 was able to kill more than 80% of mice within 10 days when the mice inoculated daily for 3 to 4 days. The anti-influenza activities of PWE, PEE, 3,4-diCQA, chlorogenic acid, and oseltamivir phosphate were evaluated using the mouse-adapted WSN/33 strains. ...
Article
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Brazilian green propolis water extract (PWE) and its chemical components, caffeoylquinic acids, such as 3,4-dicaffeoylquinic acid (3,4-diCQA), act against the influenza A virus (IAV) without influencing the viral components. Here, we evaluated the anti-IAV activities of these compounds in vivo. PWE or PEE (Brazilian green propolis ethanol extract) at a dose of 200 mg/kg was orally administered to Balb/c mice that had been inoculated with IAV strain A/WSN/33. The lifetimes of the PWE-treated mice were significantly extended compared to the untreated mice. Moreover, oral administration of 3,4-diCQA, a constituent of PWE, at a dose of 50 mg/kg had a stronger effect than PWE itself. We found that the amount of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA in the mice that were administered 3,4-diCQA was significantly increased compared to the control group, while H1N1 hemagglutinin (HA) mRNA was slightly decreased. These data indicate that PWE, PEE or 3,4-diCQA possesses a novel and unique mechanism of anti-influenza viral activity, that is, enhancing viral clearance by increasing TRAIL.
... To study the pathogenesis of influenza B virus infections and to test the immunogenicity and protective efficacy of vaccines, experimental animal models that mimic human disease after infection with influenza B viruses are crucial. Most studies with influenza B viruses have been performed in mice (Mus musculus) and ferrets (Mustela putorius furo), while also cotton rats (Sigmodon hispidus), dogs, guinea pigs, pigs, Syrian golden hamsters (Mesocricetus auratus) and cynomolgus macaques (Macaca fascicularis) were experimentally inoculated with various influenza B virus strains (Table 2) [68,85,88,[93][94][95][96][97][98][99][100][101][102][103][104][105][106]. Except for a study performed in dogs, virus replication was observed in all experimental animal models, and a study in pigs actually showed pigto-pig transmission of a B/Victoria strain [68]. ...
Article
In contrast to influenza A viruses, which have been investigated extensively, influenza B viruses have attracted relatively little attention. However, influenza B viruses are an important cause of morbidity and mortality in the human population and full understanding of their biological and epidemiological properties is imperative to better control this important pathogen. However, some of its characteristics are still elusive and warrant investigation. Here, we review evolution, epidemiology, pathogenesis and immunity and identify gaps in our knowledge of influenza B viruses. The divergence of two antigenically distinct influenza B viruses is highlighted. The co-circulation of viruses of these two lineages necessitated the development of quadrivalent influenza vaccines, which is discussed in addition to possibilities to develop universal vaccination strategies.
... Transmission of influenza viruses between mice is also inefficient compared to transmission between humans [60], and mice to human transmission of influenza virus has not been reported. Consequently, influenza viruses must be adapted if they are to infect mice, but undergoing serial passages can lead to mutations that alter the virulence and growth kinetics of influenza viruses [61][62][63]. These changes can lead to non-predictive results from viral challenge studies and live-attenuated influenza vaccination studies, which use weakened strains of influenza viruses. ...
Article
The intranasal route of vaccination presents an attractive alternative to parenteral routes and offers numerous advantages, such as the induction of both mucosal and systemic immunity, needle-free delivery, and increased patient compliance. Despite demonstrating promising results in preclinical studies, however, few intranasal vaccine candidates progress beyond early clinical trials. This discrepancy likely stems in part from the limited predictive value of rodent models, which are used frequently in intranasal vaccine research. In this review, we explored the factors that limit the translatability of rodent-based intranasal vaccine research to humans, focusing on the differences in anatomy, immunology, and disease pathology between rodents and humans. We also discussed approaches that minimize these differences and examined alternative animal models that would produce more clinically relevant research.
... Mice are ideal animal models for investigating pathogenic mechanisms and host range determinants of influenza A viruses [31], and can be used to generate mouse-adapted variants by serial lung-to-lung passages [17,32,33]. Mouse adapted viruses have acquired virulence determining functions, and usually induced pathology in bronchi or lungs of infected mice [34,35,36], that is similar to human influenza pneumonia [37]. In the present study, we generated a mouse-adapted H9N2 virus with significantly higher virulence than wide-type virus. ...
Article
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H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.
... Mouse is the most widely used mammalian model for IAv studies. However, experimental infection of mice with the newly isolated human isolates can only result in replication in the respiratory tract without further signs of pathology [92]. This is because of the predominant presence of α2,3-linked sialic acid (SA) receptors in the respiratory tract (Fig. 1). ...
Article
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The PA protein is the third subunit of the polymerase complex of influenza A virus. Compared with the other two polymerase subunits (PB2 and PB1), its precise functions are less defined. However, in recent years, advances in protein expression and crystallization technologies and also the reverse genetics, greatly accelerate our understanding of the essential role of PA in virus infection. Here, we first review the current literature on this remarkably multifunctional viral protein regarding virus life cycle, including viral RNA transcription and replication, viral genome packaging and assembly. We then discuss the various roles of PA in host adaption in avian species and mammals, general virus-host interaction, and host protein synthesis shutoff. We also review the recent findings about the novel proteins derived from PA. Finally, we discuss the prospects of PA as a target for the development of new antiviral approaches and drugs.
... Mice are the preferred animal model of IAV infection due to their relatively low cost, small size, and ease of handling [32]. Adaptation to mice is required for human-IAV infection of new hosts and acquisition of pathogenicity [33,34], thereby making the mouse model suitable for analyzing the factors responsible for virulence and crossspecies adaptability. We generated a mouse-adapted H3N2 virus [maSW293B8 (MA_SW)] derived from A/Switzerland/9715293/2013 by serial lung-cell-lung passages in Balb/c and Effect of viral mutations on mouse adaptation DBA/1J mice, which resulted in higher mortality rates in mice (Table 4 and S1 Table). ...
Article
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Elucidating the genetic basis of influenza A viruses (IAVs) is important to understand which mutations will determine the virulence and the host range of mammals. Here, seasonal H3N2 influenza was adapted in mice by serial passage and four mutants, each carrying amino acid substitutions related to mouse adaptation in either the PB2, HA, NP, or NA protein, were generated. To confirm the contribution of each gene to enhanced pathogenicity and mouse adaptation, mice were inoculated with the respective variants, and virulence, replication, histopathology, and infectivity were examined. The virus harboring HA mutations displayed increased infection efficiency and replication competence, resulting in higher mortality in mice relative to those infected with wild-type virus. By contrast, the NP D34N mutation caused rapid and widespread infection in multiple organs without presenting virulent symptoms. Additionally, the PB2 F323L mutation presented delayed but elevated replication competence in the respiratory tract, whereas the S331R mutation in NA showed no considerable effects on mouse adaptation. These results suggested that mouse-adapted changes in HA are major factors in increased pathogenicity and that mutations in NP and PB2 also contribute to cross-species adaptability. Our findings offer a better understanding of the molecular basis for IAV pathogenicity and adaptation in a new host.
... Mice are not a natural host for IAVs but have long been used as an animal model to study the replication, pathogenesis, and immune responses of many different viruses from avian and mammalian hosts (15)(16)(17)(18). While some IAV strains appear to infect and replicate to high levels in the lungs or other respiratory tissues of mice, many show relatively limited replication, most do not spread naturally from mouse to mouse, and many cause little disease unless they are adapted by serial passage (19)(20)(21). ...
Article
Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts, we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and that the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza virus evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza virus pathology, host passage evolution will vary depending on the specific virus tested.
... A549) and air-liquid interface culture systems 60 have been used to study the tissue responses to infection and to monitor viral life cycles (Slepushkin et al., 2001;Wu et al., 2016), but these systems cannot fully represent the complexity of infection. Similarly, animal models have been widely used in influenza virus research, yet interpretation of these results can be confounded by dissimilarities between these models and humans, and between lab-adapted and natural viruses (Hemmink et al., 2018;Hirst, 1947;Kim et al., 2015;Radigan et al., 2015;Shin et al., 2015). For 65 example, laboratory mouse models such as C57BL/6J are not natural hosts to influenza virus, and human influenza virus inoculated in their nasal tract cannot progress to the lung (Ivinson et al., 2017). ...
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Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.
... A549) and air-liquid interface culture systems 60 have been used to study the tissue responses to infection and to monitor viral life cycles (Slepushkin et al., 2001;Wu et al., 2016), but these systems cannot fully represent the complexity of infection. Similarly, animal models have been widely used in influenza virus research, yet interpretation of these results can be confounded by dissimilarities between these models and humans, and between lab-adapted and natural viruses (Hemmink et al., 2018;Hirst, 1947;Kim et al., 2015;Radigan et al., 2015;Shin et al., 2015). For 65 example, laboratory mouse models such as C57BL/6J are not natural hosts to influenza virus, and human influenza virus inoculated in their nasal tract cannot progress to the lung (Ivinson et al., 2017). ...
Article
Full-text available
Influenza virus infections are major causes of morbidity and mortality. Research using cultured cells, bulk tissue, and animal models cannot fully capture human disease dynamics. Many aspects of virus-host interactions in a natural setting remain unclear, including the specific cell types that are infected and how they and neighboring bystander cells contribute to the overall antiviral response. To address these questions, we performed single-cell RNA sequencing (scRNA-Seq) on cells from freshly collected nasal washes from healthy human donors and donors diagnosed with acute influenza during the 2017-18 season. We describe a previously uncharacterized goblet cell population, specific to infected individuals, with high expression of MHC class II genes. Furthermore, leveraging scRNA-Seq reads, we obtained deep viral genome coverage and developed a model to rigorously identify infected cells that detected influenza infection in all epithelial cell types and even some immune cells. Our data revealed that each donor was infected by a unique influenza variant and that each variant was separated by at least one unique non-synonymous difference. Our results demonstrate the power of massively-parallel scRNA-Seq to study viral variation, as well as host and viral transcriptional activity during human infection.
... Some respiratory viruses, including influenza, SARS, and MERS, have been serially passaged in mice to generate mouse-adapted virus strains, which have provided important tools for studying viral and host determinants of disease and tropism [39][40][41][42][43]. We initially sought to generate mouse-adapted HMPV by serially passaging infected lung homogenate directly into a recipient mouse. ...
Article
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The host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. Many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. STAT2 is divergent between species and therefore has a role in species restriction of some viruses. To understand the role of STAT2 in human metapneumovirus (HMPV) infection of human and murine tissues, we first infected STAT2−/− mice and found that HMPV could be serially passaged in STAT2−/−, but not WT, mice. We then used in vitro methods to show that HMPV inhibits expression of both STAT1 and STAT2 in human and primate cells, but not in mouse cells. Transfection of the murine form of STAT2 into STAT2-deficient human cells conferred resistance to STAT2 inhibition. Finally, we sought to understand the in vivo role of STAT2 by infecting hSTAT2 knock-in mice with HMPV, and found that mice had increased weight loss, inhibition of type I interferon signaling, and a Th2-polarized cytokine profile compared to WT mice. These results indicate that STAT2 is a target of HMPV in human infection, while the murine version of STAT2 restricts tropism of HMPV for murine cells and tissue.
... [11][12][13][14][15]. However, repeated passaging of human-derived IAV in mice can select for HA variants with affinity for SAα-2, 3-Gal receptor thereby permitting the use of mice to characterize immune responses and pathogenicity during IAV infection [16][17]. ...
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Despite effective vaccines, influenza remains a major global health threat due to the morbidity and mortality caused by seasonal epidemics, as well as the 2009 pandemic. Also of profound concern are the rare but potentially catastrophic transmissions of avian influenza to humans, highlighted by a recent H7N9 influenza outbreak. Murine and human studies reveal that the clinical course of influenza is the result of a combination of both host and viral genetic determinants. While viral pathogenicity has long been the subject of intensive efforts, research to elucidate host genetic determinants, particularly human, is now in the ascendant, and the goal of this review is to highlight these recent insights.
... Finally, these advantages of B6 mice are complemented by the use of recent human isolates of IAV and IBV, as we report here. These viruses provide an advantage to much older mouse-adapted strains of virus because the genetic features of the influenza virus strains have not been extensively perturbed by extended passage in mice as mouse-adapted viruses often are [21,38]. ...
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The adaptive T cell response to influenza B virus is understudied, relative to influenza A virus, for which there has been considerable attention and progress for many decades. Here, we have developed and utilized the C57BL/6 mouse model of intranasal infection with influenza B (B/Brisbane/60/2008) virus and, using an iterative peptide discovery strategy, have identified a series of robustly elicited individual CD4 T cell peptide specificities. The CD4 T cell repertoire encompassed at least eleven major epitopes distributed across hemagglutinin, nucleoprotein, neuraminidase, and non-structural protein 1 and are readily detected in the draining lymph node, spleen, and lung. Within the lung, the CD4 T cells are localized to both lung vasculature and tissue but are highly enriched in the lung tissue after infection. When studied by flow cytometry and MHC class II: peptide tetramers, CD4 T cells express prototypical markers of tissue residency including CD69, CD103, and high surface levels of CD11a. Collectively, our studies will enable more sophisticated analyses of influenza B virus infection, where the fate and function of the influenza B-specific CD4 T cells elicited by infection and vaccination can be studied as well as the impact of anti-viral reagents and candidate vaccines on the abundance, functionality, and localization of the elicited CD4 T cells.
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The molecular mechanism by which pandemic 2009 influenza A viruses were able to sufficiently adapt to humans is largely unknown. Subsequent human infections with novel H1N1 influenza viruses prompted an investigation of the molecular determinants of the host range and pathogenicity of pandemic influenza viruses in mammals. To address this problem, we assessed the genetic basis for increased virulence of A/CA/04/09 (H1N1) and A/TN/1-560/09 (H1N1) isolates, which are not lethal for mice, in a new mammalian host by promoting their mouse adaptation. The resulting mouse lung-adapted variants showed significantly enhanced growth characteristics in eggs, extended extrapulmonary tissue tropism, and pathogenicity in mice. All mouse-adapted viruses except A/TN/1-560/09-MA2 grew faster and to higher titers in cells than the original strains. We found that 10 amino acid changes in the ribonucleoprotein (RNP) complex (PB2 E158G/A, PA L295P, NP D101G, and NP H289Y) and hemagglutinin (HA) glycoprotein (K119N, G155E, S183P, R221K, and D222G) controlled enhanced mouse virulence of pandemic isolates. HA mutations acquired during adaptation affected viral receptor specificity by enhancing binding to alpha2,3 together with decreasing binding to alpha2,6 sialyl receptors. PB2 E158G/A and PA L295P amino acid substitutions were responsible for the significant enhancement of transcription and replication activity of the mouse-adapted H1N1 variants. Taken together, our findings suggest that changes optimizing receptor specificity and interaction of viral polymerase components with host cellular factors are the major mechanisms that contribute to the optimal competitive advantage of pandemic influenza viruses in mice. These modulators of virulence, therefore, may have been the driving components of early evolution, which paved the way for novel 2009 viruses in mammals.
Article
Some of the peculiarities of strains of influenza A and B virus from two epidemics have been described. The influenza B virus of 1945-46, when compared with influenza A virus, proved to be much more difficult to isolate from human sources by any known means. Its adaptation to the chick embryo (by any route) or to mice was much slower than that of A virus. It did not keep nearly as well on storage at -72 degrees C. either in throat garglings or as passage material. Its adaptation to amniotic growth was usually much better than to allantoic growth even after repeated allantoic passages. It failed to show primary evidence of occurring in the O form, although many of the secondary O characteristics were present and persisted. Its titer in throat washings was not demonstrably high as compared with certain strains of A virus, which were demonstrated in garglings at dilutions of 10(-5) and 10(-6). The antigenic patterns of influenza A strains from two epidemics were compared. No antigenic differences of significant degree were found among the strains of either epidemic and the difference between the strains of the two epidemics was very slight. A similar study was made of the influenza B strains of the epidemic of 1945-46. This also showed complete lack of significant strain differences. The implications of these findings for influenza prophylaxis are discussed.
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During passage in mice which had been vaccinated with the homologous, and with closely related strains of influenza virus, the passage strain developed a lessened susceptibility to the deleterious effects of the "immune" environment, concommittant with which was a developed capacity to evoke antibodies which reacted with earlier strains of virus—a capacity which was inapparent in the parent strain. However, the parent strain exhibited a relatively broad range of surface reactivity which was not apparent in the derived strain. The data are interpreted to mean that the hereditary change resulted from spatial rearrangement and quantitative redistribution of antigens in the virus particle (in which the surface is viewed as being distinct from the inner bulk), and are viewed as enhancing the idea that influenza virus variation (i.e., "mutation") may result from a rearrangement of existing hereditary elements.
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The non-pathogenic (unadapted) line of the Rhodes strain of influenza virus multiplied readily in the mouse lung, but its rate of multiplication was slower and its hemagglutination titer attained at the peak of growth was much lower than that of the adapted line of the same virus. The implication of these findings to mouse adaptability of the virus is discussed.
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The rates of elution from RBC of the Lee and PR8 strains of influenza virus were studied by means of a step-wise elution technique. By means of a single treatment with lanthanum acetate or irradiation with ultraviolet and subsequent passage in chick embryos, it was possible to alter the elution rate of the Lee strain so that it was similar to that of the PR8 strain. This alteration proved to be persistent on serial passage in the absence of the agent which caused it. As far as was determined, the elution rate of the virus appeared to be the only property which was altered. The phenomenon can be most readily understood on the assumption that the difference in elution rates of the two strains is due to a heterogeneous population of virus particles in the Lee strain with respect to elution rate.
Article
A STUDY OF INFLUENZA VIRUS INFECTION IN THE HAMSTER HAS YIELDED THE FOLLOWING RESULTS: 1. Two influenza A strains (Ga. 47 and PR8) multiplied readily in the hamster lung, although no lung lesions were produced during six serial passages. On further passage both viruses abruptly acquired the capacity to produce pulmonary consolidation and death of the animals. 2. Extracts of the lungs during the early passages contained complement-fixing antigen and infectious virus, as revealed by titration in mice and embryonated eggs. Agglutinins for chicken, human, and guinea pig red cells, however, were not demonstrable at this time. With further passage a close correlation was observed between the capacity of the virus to produce lung lesions in the hamster and to agglutinate mammalian types of red cells. In addition, quantitative changes in the virus population were demonstrated in the lung extracts by complement fixation tests and titrations in mice and eggs. 3. Incubation at 37 degrees C. was effective in bringing out agglutinins in high titer for chicken red cells in lung extracts, which originally failed to agglutinate chicken cells but agglutinated mammalian red cells. This method did not increase the titers of mammalian cell agglutinins. 4. The body temperature of the hamster was found to decrease within 1 to 4 days after inoculation of influenza virus. In the early passages the temperature returned to normal within 24 hours, but with the development of the pathogenic strain of virus the temperature remained at subnormal levels until death. 5. The Lee strain of influenza B virus produced pulmonary lesions in the hamster on the first passage and no increase in pathogenicity of the virus occurred during eleven serial passages. Virus was demonstrable in extracts of the lungs by all the methods used and no difference was observed in its capacity to agglutinate fowl and mammalian types of red cells. The implications of these findings are considered briefly in the discussion.
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Das Schicksal von nicht mausadaptiertem Influenzavirus in der Muselunge wurde unter Anwendung der Immunofluoreszenztechnik untersucht. Die Virusreplikation, die ebenso wie beim adaptierten Virus in den Alveolarepithelzellen im bergangsbereich vom Bronchulus zum Saccus alveolaris beginnt, setzt wesentlich spter ein als nach Infektion mit adaptiertem Virus, erreicht etwa 10 Stunden post infectionem ihr Maximum und klingt dann wieder ab. Die Virusausschleusung aus der Zelle erfolgt wesentlich langsamer. Weder die Bronchiolen noch die Bronchien und die Trachea werden befallen. Noch 8 Tage post infectionem finden sich einzelne infizierte Alveolarepithelzellen, die offenbar Virus abgeben.The fate of not mouse adapted influenza virus in the mouse lung was investigated by immunofluorescence methods. The replication of the non adapted virus, which starts just like that of the adapted virus in the alveolar epithelia at the transition from the bronchioles to the alveolar sacs, begins considerably later than after infection with adapted virus. It is at a maximum at about 10 hours p.i. and then subsides. The virus release by the cells is much slower. Neither the bronchioles nor the bronchi or the trachea are involved. Even after 8 days p.i. some isolated alveolar cells show some fluorescence indicating virus release.
Chapter
There are two aspects to any discussion of variation amongst the influenza viruses. On the one hand there are the directly observable changes in character that occur in the course of experimental manipulation and on the other there are the differences that are observed in the epidemiology of different outbreaks of virus influenza and the differences which are observed in strains as isolated. It is natural and probably legitimate to infer that the different antigenic types of influenza virus A that were isolated in Britain during the 1936–7 epidemic were variants from a common ancestral stock. But until a great deal more is known of the natural history of influenza it will be difficult to guess how far back that common ancestry might be. The related problems of how the influenza virus persists in interepidemic years and what is responsible for the genesis of a new epidemic, have not yet been solved. It is a possibility but no more than a possibility that as suggested by Andrewes (1942) each new influenza epidemic represents the emergence of a more virulent mutant from the trickle of low grade infection that persists through interepidemic periods. Provided such a more active strain finds a population with low specific immunity, it will find opportunities both for spread and for the appearance of further mutants. An equally conceivable alternative is that epidemics arise by the simultaneous development into overt activity of many strains which have persisted in carriers or otherwise during the interepidemic period. On this view the epidemic is determined only by (a) the existence of a large enough susceptible population, and (b) conditions, social and meteorological, that are apt for its spread. Preliminary mutation would not be required nor would the existence of multiple strains within a single epidemic be valid evidence for the occurrence of contemporary variation.
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Genetic analyses indicated that the pandemic H1N1/2009 influenza virus originated from a swine influenza virus (SIV). However, SIVs bearing the same constellation of genetic features as H1N1/2009 have not been isolated. Understanding the adaptation of SIVs with such genotypes in a new host may provide clues regarding the emergence of pandemic strains such as H1N1/2009. In this study, an artificial SIV with the H1N1/2009 genotype (rH1N1) was sequentially passaged in mice through two independent series, yielding multiple mouse-adapted mutants with high genetic diversity and increased virulence. These experiments were meant to mimic genetic bottlenecks during adaptation of wild viruses with rH1N1 genotypes in a new host. Molecular substitutions in the mouse-adapted variants mainly occurred in genes encoding surface proteins (hemagglutinin [HA] and neuraminidase [NA]) and polymerase proteins (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acid [PA] proteins and nucleoprotein [NP]). The PB2D309N and HAL425M substitutions were detected at high frequencies in both passage lines and enhanced the replication and pathogenicity of rH1N1 in mice. Moreover, these substitutions also enabled direct transmission of rH1N1 in other mammals such as guinea pigs. PB2D309N showed enhanced polymerase activity and HAL425M showed increased stability compared with the wild-type proteins. Our findings indicate that if SIVs with H1N1/2009 genotypes emerge in pigs, they could undergo rapid adaptive changes during infection of a new host, especially in the PB2 and HA genes. These changes may facilitate the emergence of pandemic strains such as H1N1/2009.
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Emerging and re-emerging viral diseases occur with regularity within the human population. The conventional ‘one drug, one virus’ paradigm for antivirals does not adequately allow for proper preparedness in the face of unknown future epidemics. In addition, drug developers lack the financial incentives to work on antiviral drug discovery, with most pharmaceutical companies choosing to focus on more profitable disease areas. Safe-in-man broad spectrum antiviral agents (BSAAs) can help meet the need for antiviral development by already having passed phase I clinical trials, requiring less time and money to develop, and having the capacity to work against many viruses, allowing for a speedy response when unforeseen epidemics arise. In this chapter, we discuss the benefits of repurposing existing drugs as BSAAs, describe the major steps in safe-in-man BSAA drug development from discovery through clinical trials, and list several database resources that are useful tools for antiviral drug repositioning.
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In human and murine studies, IFN-γ is a critical mediator immunity to influenza. IFN-γ production is critical for viral clearance and the development of adaptive immune responses, yet excessive production of IFN-γ and other cytokines as part of a cytokine storm is associated with poor outcomes of influenza infection in humans. As NK cells are the main population of lung innate immune cells capable of producing IFN-γ early in infection, we set out to identify the drivers of the human NK cell IFN-γ response to influenza A viruses. We found that influenza triggers NK cells to secrete IFN-γ in the absence of T cells and in a manner dependent upon signaling from both cytokines and receptor-ligand interactions. Further, we discovered that the pandemic A/California/07/2009 (H1N1) strain elicits a seven-fold greater IFN-γ response than other strains tested, including a seasonal A/Victoria/361/2011 (H3N2) strain. These differential responses were independent of memory NK cells. Instead, we discovered that the A/Victoria/361/2011 influenza strain suppresses the NK cell IFN-γ response by downregulating NK-activating ligands CD112 and CD54 and by repressing the type I IFN response in a viral replication-dependent manner. In contrast, the A/California/07/2009 strain fails to repress the type I IFN response or to downregulate CD54 and CD112 to the same extent, which leads to the enhanced NK cell IFN-γ response. Our results indicate that influenza implements a strain-specific mechanism governing NK cell production of IFN-γ and identifies a previously unrecognized influenza innate immune evasion strategy.
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This chapter discusses cell's role in controlling virus populations that has led to the use of cultured cells to investigate host effects on virus structure, function, and synthesis. Mice and hamsters were also used for early isolations of influenza virus from human sources but in these hosts, unapparent infections occurred following the initial inoculation. The isolation and growth of influenza viruses in embryonated chicken eggs, first reported only two years after the initial isolation of an influenza virus from a human source, revolutionized influenza virus research. The growth of the influenza viruses in chick embryos also made it possible for earlier investigators to identify another kind of genetic variability, which enables host cells to impose selective pressures on influenza virus populations. The first successful attempts at growing the influenza viruses in cultured tissues of chick embryos were reported shortly after the first isolation of human influenza viruses in ferrets. The use of plaque morphology to identify viral strains or to select viral mutants has in general not been of great value for the study of influenza viruses.
Chapter
Influenza virus genetics comprises the genetics of a highly mutable RNA virus and the unusual genetics of a segmented-genome virus capable of genetic interaction with homologous strains. Genetic reassortment occurs in nature and has been used in the laboratory in the fabrication of vaccine and reagent viruses with desirable characteristics.
Chapter
In diesem und den folgenden Abschnitten werden Probleme der Immunitätsforschung berührt, die den Rahmen rein serologischer Forschung überschreiten. Aber zum Verständnis der mit der Entstehung der Antikörper zusammenhängenden Fragen ist es notwendig, das Wesentliche dieser Probleme anzudeuten.
Chapter
Influenza ist eine akute Infektionskrankheit des Menschen, welche durch einem der vershiedenen Stämme des epitheliotropen Influenzavirus verursacht wird und ein charakteristisches epidemiologisches Verhalten zeight: Sie führte häufig in der kalten Jahreszeit zu epidemischer, in Abständen von 25–40 Jahren zu pandemischer Ausbreitung mit sehr groβer Mortalität.
Article
Summary The author describes the adaptation onto mice of an influenza A strain, isolated byMulder from the tracheal mucosa of a man, who died from an acute fulminant staphylococcal-pneumonia and discusses the nature of the adaptation phenomenon.
Chapter
Virus infections are well-known causes of central nervous system–related symptoms in both animals and humans. Such symptoms can range from those associated with severe and persistent developmental defects following congenital infections to transient changes in sleep regulation associated with acute respiratory infections. An increasing number of studies report associations between signs of exposure to infections and the later diagnosis of schizophrenia and other nonaffective psychoses. These include associations with serological signs of exposure to chronic and acute maternal infections during pregnancy and with hospitalizations resulting from acute infections during childhood or early adolescence. It is not known if these exposures are causally related to the later development of psychosis and the mechanisms explaining these associations remain to be established. In this chapter, experimental viral infections of potential relevance for the etiology, pathogenesis, and modeling of persistent neuropsychiatric disorders will be reviewed.
Chapter
Die Krankheiten der Myxovirusgruppe umfassen solche des Menschen und der Tiere. Zum Teil sind es reine Menschen- oder Tierkrankheiten, zum Teil Tierkrankheiten, die auch auf den Menschen übertragbar sind (Zoo-Anthroponosen). Bei den Menschenkrankheiten handelt es sich vorwiegend um respiratorische Syndrome.
Chapter
Genetics is concerned with the manner in which the characteristics of an organism are transmitted to its descendants. In view of the near universality of sexual reproduction in organisms of human interest, it has come by convention to be interested primarily in the way in which differences between individuals of the same species can be analysed by controlled breeding experiments. An elaborate structure has now been erected in which a series of genetic conventions, gene, allele, linkage group, cross-over values etc. have been correlated in outline, in many instances in detail, with cytological appearances in germ and somatic cells. There is general agreement that desoxyribosenucleic acid (DNA) is an essential part of the nuclear mechanism of inheritance. Largely as a result of the general acceptance of the Watson-Crick formulation of the structure of DNA, it is widely held that the physical basis of genetic “codes” is the arrangements of pairs of bases in the essential DNA molecules of the chromosomes.
Chapter
Bei der Gewinnung von Influenzavirus aus befruchteten Hühnereiern machten Hirst (191) sowie Mc Clelland und Hare (246) erstmalig und gleichzeitig (1941) die Beobachtung, daß embryonales Hühnerblut, welches aus zufällig verletzten Gefäßen in die virushaltige Allantoisflüssigkeit einströmte, nahezu augenblicklich agglutiniert wurde. Wie diese Autoren zeigten, kann dieser Vorgang auch in vitro nachgewiesen werden, wobei die Möglichkeit gegeben ist, das haemagglutinierende Prinzip zu titrieren. Daß die haemagglutinierende Wirkung als eine spezifische Virusfunktion zu betrachten ist, ergab sich nicht nur aus der Beobachtung, daß auch ein Influenzavirus anderer Provenienz (Mäuselunge) dieselbe haemagglutinierende Eigenschaft aufweist, sondern vor allem aus der Feststellung, daß zwischen der Infektiosität und der agglutinierenden Aktivität eine weitgehende quantitative Parallelität besteht, und daß die Haemagglutination von Influenza-virus A und B durch korrespondierende Immunseren in typspezifischer Weise und nach Maßgabe des Antikörpergehaltes aufgehoben bzw. gehemmt werden kann. Der Haemagglutinationstiter kann demnach — zumindest unter Verwendung von frischen, bzw. gut konservierten Virusproben — als Maßstab für die Virusinfektiosität, und der Hemmungstiter des Immunserums für die Wertbemessung virusneutralisierender Antikörper verwendet werden. Schließlich lieferten Ultrazentrifugierungsversuche den stringenten Nachweis, daß das Haemagglutinin in der Sedimentierungsfraktion der Viruspartikel von 80–100 m μ angereichert wird und von diesen Viruselementen nicht abzutrennen ist. Dessen ungeachtet ist die haemagglutinierende Eigenschaft nicht unbedingt an das infektiöse Vermögen gebunden, da schonend inaktivierte Virusproben einen kaum verminderten Haemagglutinationstiter aufweisen können, und-umgekehrt-liegen Beobachtungen vor, wonach Influenzavirus in bestimmten Gewebesuspensionen trotz hoher Infektiosität kein haemagglutinierendes Vermögen besitzt. Eine selche „Dissociation“ von infektiöser und haemagglutinierender Qualität ist jedoch im einen Fall lediglich auf die unterschiedliche Stabilität dieser Virusfunktionen gegenüber äußeren Einflüssen zurückzuführen und im anderen dadurch verursacht, daß das Haemagglutinin durch gewebliche Hemmstoffe reversibel inaktiviert ist, bzw. maskiert wird.
Article
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In this study the adaptation of a influenza virus strain A/Kasauli/97/77 (H3N2) to mouse lung has been described. After four passages in the mouse lung the strain got adapted to mouse. Adaptation of the strain to the mouse lung resulted in production of typical clinical symptoms of influenza in mice. As the mouse adapted strain of influenza virus is further passaged in embryonated chicken eggs, the virulence for mouse decreased. The course of infection also changed with the serial egg passages of the mouse adapted influenza virus strain (A/HongKong/I/68).
Article
Some of the peculiarities of strains of influenza A and B virus from two epidemics have been described. The influenza B virus of 1945-46, when compared with influenza A virus, proved to be much more difficult to isolate from human sources by any known means. Its adaptation to the chick embryo (by any route) or to mice was much slower than that of A virus. It did not keep nearly as well on storage at -72 degrees C. either in throat garglings or as passage material. Its adaptation to amniotic growth was usually much better than to allantoic growth even after repeated allantoic passages. It failed to show primary evidence of occurring in the O form, although many of the secondary O characteristics were present and persisted. Its titer in throat washings was not demonstrably high as compared with certain strains of A virus, which were demonstrated in garglings at dilutions of 10(-5) and 10(-6). The antigenic patterns of influenza A strains from two epidemics were compared. No antigenic differences of significant degree were found among the strains of either epidemic and the difference between the strains of the two epidemics was very slight. A similar study was made of the influenza B strains of the epidemic of 1945-46. This also showed complete lack of significant strain differences. The implications of these findings for influenza prophylaxis are discussed.
Article
A study of cross inhibition tests among strains of influenza A virus and their antisera showed that the results obtained were subject to a certain amount of variation due to the red cells, the virus suspensions, and the ferret antisera employed. Methods have been demonstrated for handling the data obtained from such tests, so that these variables were corrected or avoided, making it possible to use the agglutination technique for antigenic comparisons. The antigenic pattern of eighteen strains of influenza A virus, obtained from the 1940-41 epidemic in the United States, has been compared by means of agglutination inhibition tests with ferret antisera. No significant antigenic differences were found among sixteen of these strains (all isolated from throat washings by the inoculation of chick embryos) although they were obtained from individuals in widely separated regions of the country. Two strains, from cases occurring early in the epidemic and isolated from throat washings by ferret and mouse passage, showed a slight but significant strain difference from the other strains and from each other. One of the 1940-41 strains on cross test resembled the PR8 strain more closely than any other stock strain tested.
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