Fecal Influenza in Mammals: Selection of Novel Variants

In aquatic birds, influenza A viruses mainly replicate in the intestinal tract without significantly affecting the health of the host, but in mammals, they replicate in the respiratory tract and often cause disease. Occasionally, influenza viruses have been detected in stool samples of hospitalized patients and in rectal swabs of naturally or experimentally infected mammals. In this study, we compared the biological and molecular differences among four wild-type avian H1N1 influenza viruses and their corresponding fecal and lung isolates in DBA/2J and BALB/cJ mice. All fecal and lung isolates were more pathogenic than the original wild-type viruses, when inoculated into mice of both strains. The increased virulence was associated with the acquisition of genetic mutations. Most of the novel genotypes emerged as PB2^E627K, HA^F128V, HA^F454L, or HA^H300P variations, and double mutations frequently occurred in the same isolate. However, influenza virus strain- and host-specific differences were also observed in terms of selected variants. The avian H1N1 virus of shorebird origin appeared to be unique in its ability to rapidly adapt to BALB/cJ mice via the fecal route, compared to the adaptability of the H1N1 virus of mallard origin. Furthermore, a bimodal distribution in fecal shedding was observed in mice infected with the fecal isolates, while a normal distribution was observed after infection with the lung isolates or wild-type virus. Fecal isolates contained HA mutations that increased the activation pH of the HA protein. We conclude that influenza virus variants that emerge in fecal isolates in mammals might influence viral transmission, adaptation to mammals, and viral ecology or evolution.     

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.     

Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments

Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus.     

Lethality to ferrets of H5N1 influenza viruses isolated from humans and poultry in 2004

The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.     

Pathogenesis of avian influenza A (H5N1) viruses in ferrets

Highly pathogenic avian influenza A H5N1 viruses caused outbreaks of disease in domestic poultry and humans in Hong Kong in 1997. Direct transmission of the H5N1 viruses from birds to humans resulted in 18 documented cases of respiratory illness, including six deaths. Here we evaluated two of the avian H5N1 viruses isolated from humans for their ability to replicate and cause disease in outbred ferrets. A/Hong Kong/483/97 virus was isolated from a fatal case and was highly pathogenic in the BALB/c mouse model, whereas A/Hong Kong/486/97 virus was isolated from a case with mild illness and exhibited a low-pathogenicity phenotype in mice. Ferrets infected intranasally with 10(7) 50% egg infectious doses (EID(50)) of either H5N1 virus exhibited severe lethargy, fever, weight loss, transient lymphopenia, and replication in the upper and lower respiratory tract, as well as multiple systemic organs, including the brain. Gastrointestinal symptoms were seen in some animals. In contrast, weight loss and severe lethargy were not noted in ferrets infected with 10(7) EID(50) of two recent human H3N2 viruses, although these viruses were also isolated from the brains, but not other extrapulmonary organs, of infected animals. The results demonstrate that both H5N1 viruses were highly virulent in the outbred ferret model, unlike the differential pathogenicity documented in inbred BALB/c mice. We propose the ferret as an alternative model system for the study of these highly pathogenic avian viruses.     

Characterization of a highly pathogenic avian influenza H5N1 virus isolated from an ostrich

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health.     

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.     

Cross-Reactive Neutralizing Antibodies Directed against Pandemic H1N1 2009 Virus Are Protective in a Highly Sensitive DBA/2 Mouse Influenza Model

Our ability to rapidly respond to an emerging influenza pandemic is hampered somewhat by the lack of a susceptible small-animal model. To develop a more sensitive model, we pathotyped 18 low-pathogenic non-mouse-adapted influenza A viruses of human and avian origin in DBA/2 and C57BL/6 mice. The majority of the isolates (13/18) induced severe morbidity and mortality in DBA/2 mice upon intranasal challenge with 1 million infectious doses. Also, at a 100-fold-lower dose, more than 50% of the viruses induced severe weight loss, and mice succumbed to the infection. In contrast, only two virus strains were pathogenic for C57BL/6 mice upon high-dose inoculation. Therefore, DBA/2 mice are a suitable model to validate influenza A virus vaccines and antiviral therapies without the need for extensive viral adaptation. Correspondingly, we used the DBA/2 model to assess the level of protection afforded by preexisting pandemic H1N1 2009 virus (H1N1pdm) cross-reactive human antibodies detected by a hemagglutination inhibition assay. Passive transfer of these antibodies prior to infection protected mice from H1N1pdm-induced pathogenicity, demonstrating the effectiveness of these cross-reactive neutralizing antibodies in vivo.Respiratory tract infections are the third leading cause of mortality in the world (27). Influenza, a disease of the airways caused by influenza viruses, is responsible for approximately half a million deaths and 3 to 5 million hospitalizations per year (28). In addition to the annual disease burden, influenza A virus is more notoriously known for its ability to cause pandemics. Three pandemics have been reported in the twentieth century: the first that occurred in 1918 (Spanish influenza) killed 20 to 50 million individuals (15); the other two in 1957 and 1968, although less lethal, killed millions due to the lack of preexisting immunity. In April 2009, two cases of febrile illness were confirmed to be caused by swine-origin influenza A virus (H1N1) (4, 8). Continuous spread within North America and other parts of the world has signaled the first influenza pandemic of this century.To study the pathogenicity of influenza A viruses, including the current pandemic A (H1N1) 2009 virus (H1N1pdm), in mammalian hosts and to determine the effectiveness of pharmaceutical interventions, it is essential to have a sensitive animal model. Although influenza has some important differences in mice and humans, a murine model is the only animal model thus far described that allows for relatively high group numbers and any relatively high throughput. Unfortunately, only a few strains of influenza A virus—almost exclusively belonging to the highly pathogenic avian influenza virus isolates of the H5 and H7 subtypes—are pathogenic in most commonly used mouse strains without adaptation through serial passaging. The hemagglutinin (HA) proteins of these H5 and H7 viruses contain a basic amino acid cleavage site, allowing them to spread systemically (12, 19, 26). Most other subtypes of influenza virus, including H1N1 and H3N2, either do not infect or cause very mild disease in mice. The requirement for adaptation of a pandemic virus to commonly used mouse strains can lead to a delay in the gathering of important data to help guide public health control strategies. As such, the lack of a sensitive small-animal model to study the infection dynamics of various subtypes of avian influenza viruses severely hampers the rapid and effective response required during a pandemic or prepandemic situation.This study was designed to demonstrate the utility of DBA/2 mice, previously reported to be susceptible to highly pathogenic influenza viruses (1), to study infections caused by several influenza A virus subtypes isolated from birds or humans without the need for prior adaptation. To assess the utility of the model to respond to emerging strains, we used DBA/2 mice to examine the functional activity of sera from individuals previously shown to have preexisting cross-reactive H1N1pdm antibodies. It is hypothesized that these individuals may be partially protected from infection because of the presence of cross-reactive neutralizing antibodies produced after infection with a different but related H1N1 virus. This hypothesis is supported by in vitro microneutralization and hemagglutination inhibition (HI) assays (2, 10); however, it is not yet known whether these antibodies are also functional in vivo.     

Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.     

Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.     

Replication and pathogenesis of the pandemic (H1N1) 2009 influenza virus in mammalian models

This study aimed to characterize the replication and pathogenic properties of a Korean pandemic (H1N1) 2009 influenza virus isolate in ferrets and mice. Ferrets infected with A/Korea/01/2009 (H1N1) virus showed mild clinical signs. The virus replicated well in lungs and slightly in brains with no replication in any other organs. Severe bronchopneumonia and thickening of alveolar walls were detected in the lungs. Viral antigens were detected in the bronchiolar epithelial cells, in peribronchial glands with severe peribronchitis and in cells present in the alveoli. A/Korea/01/2009 (H1N1) virus-infected mice showed weight loss and pathological lung lesions including perivascular cuffing, interstitial pneumonia and alveolitis. The virus replicated highly in the lungs and slightly in the nasal tissues. Viral antigens were detected in bronchiolar epithelial cells, pneumocytes and interstitial macrophages. However, seasonal H1N1 influenza virus did not replicate in the lungs of ferrets, and viral antigens were not detected. Thus, this Korean pandemic (H1N1) 2009 isolate infected the lungs of ferrets and mice successfully and caused more pathological lesions than did the seasonal influenza virus.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Characterization of a human H5N1 influenza A virus isolated in 2003

In 2003, H5N1 avian influenza virus infections were diagnosed in two Hong Kong residents who had visited the Fujian province in mainland China, affording us the opportunity to characterize one of the viral isolates, A/Hong Kong/213/03 (HK213; H5N1). In contrast to H5N1 viruses isolated from humans during the 1997 outbreak in Hong Kong, HK213 retained several features of aquatic bird viruses, including the lack of a deletion in the neuraminidase stalk and the absence of additional oligosaccharide chains at the globular head of the hemagglutinin molecule. It demonstrated weak pathogenicity in mice and ferrets but caused lethal infection in chickens. The original isolate failed to produce disease in ducks but became more pathogenic after five passages. Taken together, these findings portray the HK213 isolate as an aquatic avian influenza A virus without the molecular changes associated with the replication of H5N1 avian viruses in land-based poultry such as chickens. This case challenges the view that adaptation to land-based poultry is a prerequisite for the replication of aquatic avian influenza A viruses in humans.     

Molecular correlates of influenza A H5N1 virus pathogenesis in mice

Highly pathogenic avian influenza A H5N1 viruses caused an outbreak of human respiratory illness in Hong Kong. Of 15 human H5N1 isolates characterized, nine displayed a high-, five a low-, and one an intermediate-pathogenicity phenotype in the BALB/c mouse model. Sequence analysis determined that five specific amino acids in four proteins correlated with pathogenicity in mice. Alone or in combination, these specific residues are the likely determinants of virulence of human H5N1 influenza viruses in this model.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Clue to the molecular mechanism of virulence of highly pathogenic H5N1 avian influenza viruses isolated in 2004

Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia since 2003. These viruses are highly lethal to birds and humans. Of the 74 confirmed human cases, 49 were fatal (as of Mar 30, 2005), raising concerns of a possible pandemic by these viruses. Despite the well-established pathogenicity of these viruses, the molecular mechanism for expressing such high virulence remains elusive. Thus, we examined the pathogenicity of the H5N1 viruses isolated in Vietnam in 2003-2004 using animal models (mouse, duck, and ferret). Viruses from humans were generally more pathogenic in mice and ferrets than those from birds. Indeed, one human isolate was even lethal to ferrets. The human isolate possessing Lys at amino acid position 627 of PB2 was more virulent than that possessing Glu at this position, underscoring the importance of Lys at this position 627 of PB2 for efficient growth in mammals.     

The Short Stalk Length of Highly Pathogenic Avian Influenza H5N1 Virus Neuraminidase Limits Transmission of Pandemic H1N1 Virus in Ferrets

H5N1 influenza viruses pose a pandemic threat but have not acquired the ability to support sustained transmission between mammals in nature. The restrictions to transmissibility of avian influenza viruses in mammals are multigenic, and overcoming them requires adaptations in hemagglutinin (HA) and PB2 genes. Here we propose that a further restriction to mammalian transmission of the majority of highly pathogenic avian influenza (HPAI) H5N1 viruses may be the short stalk length of the neuraminidase (NA) protein. This genetic feature is selected for when influenza viruses adapt to chickens. In our study, a recombinant virus with seven gene segments from a human isolate of the 2009 H1N1 pandemic combined with the NA gene from a typical chicken-adapted H5N1 virus with a short stalk did not support transmission by respiratory droplet between ferrets. This virus was also compromised in multicycle replication in cultures of human airway epithelial cells at 32°C. These defects correlated with a reduction in the ability of virus with a short-stalk NA to penetrate mucus and deaggregate virions. The deficiency in transmission and in cleavage of tethered substrates was overcome by increasing the stalk length of the NA protein. These observations suggest that H5N1 viruses that acquire a long-stalk NA through reassortment might be more likely to support transmission between humans. Phylogenetic analysis showed that reassortment with long-stalk NA occurred sporadically and as recently as 2011. However, all identified H5N1 viruses with a long-stalk NA lacked other mammalian adapting features and were thus several genetic steps away from becoming transmissible between humans.     

Risk Assessment of H2N2 Influenza Viruses from the Avian Reservoir

H2N2 influenza A viruses were the cause of the 1957-1958 pandemic. Historical evidence demonstrates they arose from avian virus ancestors, and while the H2N2 subtype has disappeared from humans, it persists in wild and domestic birds. Reemergence of H2N2 in humans is a significant threat due to the absence of humoral immunity in individuals under the age of 50. Thus, examination of these viruses, particularly those from the avian reservoir, must be addressed through surveillance, characterization, and antiviral testing. The data presented here are a risk assessment of 22 avian H2N2 viruses isolated from wild and domestic birds over 6 decades. Our data show that they have a low rate of genetic and antigenic evolution and remained similar to isolates circulating near the time of the pandemic. Most isolates replicated in mice and human bronchial epithelial cells, but replication in swine tissues was low or absent. Multiple isolates replicated in ferrets, and 3 viruses were transmitted to direct-contact cage mates. Markers of mammalian adaptation in hemagglutinin (HA) and PB2 proteins were absent from all isolates, and they retained a preference for avian-like α2,3-linked sialic acid receptors. Most isolates remained antigenically similar to pandemic A/Singapore/1/57 (H2N2) virus, suggesting they could be controlled by the pandemic vaccine candidate. All viruses were susceptible to neuraminidase inhibitors and adamantanes. Nonetheless, the sustained pathogenicity of avian H2N2 viruses in multiple mammalian models elevates their risk potential for human infections and stresses the need for continual surveillance as a component of prepandemic planning.     

Distinct pathogenesis of hong kong-origin H5N1 viruses in mice compared to that of other highly pathogenic H5 avian influenza viruses

In 1997, an outbreak of virulent H5N1 avian influenza virus occurred in poultry in Hong Kong (HK) and was linked to a direct transmission to humans. The factors associated with transmission of avian influenza virus to mammals are not fully understood, and the potential risk of other highly virulent avian influenza A viruses infecting and causing disease in mammals is not known. In this study, two avian and one human HK-origin H5N1 virus along with four additional highly pathogenic H5 avian influenza viruses were analyzed for their pathogenicity in 6- to 8-week-old BALB/c mice. Both the avian and human HK H5 influenza virus isolates caused severe disease in mice, characterized by induced hypothermia, clinical signs, rapid weight loss, and 75 to 100% mortality by 6 to 8 days postinfection. Three of the non-HK-origin isolates caused no detectable clinical signs. One isolate, A/tk/England/91 (H5N1), induced measurable disease, and all but one of the animals recovered. Infections resulted in mild to severe lesions in both the upper and lower respiratory tracts. Most consistently, the viruses caused necrosis in respiratory epithelium of the nasal cavity, trachea, bronchi, and bronchioles with accompanying inflammation. The most severe and widespread lesions were observed in the lungs of HK avian influenza virus-infected mice, while no lesions or only mild lesions were evident with A/ck/Scotland/59 (H5N1) and A/ck/Queretaro/95 (H5N2). The A/ck/Italy/97 (H5N2) and the A/tk/England/91 (H5N1) viruses exhibited intermediate pathogenicity, producing mild to moderate respiratory tract lesions. In addition, infection by the different isolates could be further distinguished by the mouse immune response. The non-HK-origin isolates all induced production of increased levels of active transforming growth factor beta following infection, while the HK-origin isolates did not.     

DBA/2 mouse as an animal model for anti-influenza drug efficacy evaluation

Influenza viruses are seasonally recurring human pathogens. Vaccines and antiviral drugs are available for influenza. However, the viruses, which often change themselves via antigenic drift and shift, demand constant efforts to update vaccine antigens every year and develop new agents with broad-spectrum antiviral efficacy. An animal model is critical for such efforts. While most human influenza viruses are unable to kill BALB/c mice, some strains have been shown to kill DBA/2 mice without prior adaptation. Therefore, in this study, we explored the feasibility of employing DBA/2 mice as a model in the development of anti-influenza drugs. Unlike the BALB/c strain, DBA/2 mice were highly susceptible and could be killed with a relatively low titer (50% DBA/2 lethal dose = 10^2.83 plaque-forming units) of the A/Korea/01/2009 virus (2009 pandemic H1N1 virus). When treated with a neuraminidase inhibitor, oseltamivir phosphate, infected DBA/2 mice survived until 14 days post-infection. The reduced morbidity of the infected DBA/2 mice was also consistent with the oseltamivir treatment. Taking these data into consideration, we propose that the DBA/2 mouse is an excellent animal model to evaluate antiviral efficacy against influenza infection and can be further utilized for combination therapies or bioactivity models of existing and newly developed anti-influenza drugs.