The Avian Influenza Virus - Essay Example

What risks does the avian flu virus (H5N1) present to the human population Introduction: The avian influenza virus is normally an infective strain tothe birds only; however, the world now faces the threat of a human influenza pandemic arising from the recently emerged avian influenza H5N1 virus. Although of avian origin, H5N1 is of particular concern since it has recognised ability to be transmitted directly from birds to human. Historically, the emergence and spread of severe acute respiratory syndrome (SARS) in 2003 caused significant effects on the health, trade, and economies of a number of countries, particularly in Asia. This happened perhaps due to the fact that in the humans, this strain of avian virus may cause severe disease with extremely high mortality. Moreover, it has been known that this H5N1 virus is able to infect other animals other than birds and pigs, such as, cats and larger felines, and transmission of infection from other animals to human may occur unknowingly. An article published in the Journal of Virology in the year 2005, titled "Avian Influenza (H5N1) Viruses Isolated from Humans in Asia in 2004 Exhibit Increased Virulence in Mammals" and researched by Maines and co-workers investigates this in an isolate of the virus of the H5N1 strain, isolated from 2004 Asian epidemic, and in this work, that article will be critiqued. Research Questions: As is apparent from the epidemiologic patterns of the outbreak of these H5N1 infections, the virulence factors responsible for human disease need to be elucidated. The problem remains that the virulence factors of H5N1 viruses is poorly understood from the biologic and molecular perspectives, this being an avian virus transmitted to humans. It points to the need for mammalian models of studies, and since there is evidence that mammalian experiments in nonhuman primates such as ferrets and mice have been studied for influenza virus pathogenesis and other nonhuman mammals may also serve as carriers for human virus, nonhuman primate studies may serve as an acceptable model. In these models, the relative virulence of the H5N1 isolated from 2003-2004 outbreaks in both humans and avian species can be studied, and the authors can have the advantage of comparing them with earlier 1997 H5N1 human isolates to compare the virulence of the virus, so a molecular or biologic correlate is available that may be applied in the management of human epidemics. Relevance of the Question: From the public health point of view, the circulation of H5N1 virus in the poultry may cause more human cases with human-avian reassortant pandemics. Epidemiologic observations have demonstrated that from the time of first recognition and isolation, the virulence of this strain is increasing markedly over the years in terms of mortality and case fatality rates. Although the clinical presentations of the human cases were usual in the form of fever, respiratory symptoms, diarrhea, lymphopenia, and thrombocytopenia, in rare occasions, they indeed presented with just fever and gastrointestinal symptoms. This could have indicated a change in the pattern of virulence, despite the pattern of fatality remaining the same, that is, development of pneumonia with critical compromise in gas exchange, leading to respiratory failure and death despite assisted ventilations. Research in this area is necessary since there is a paucity of research answering this question about how an infection with this virus leads to acute respiratory distress and multiple organ dysfunctions in humans. Although studies in mammalian models do have constraints from the practical, ethical, and economic perspectives, it could be a valid option since ferret is a naturally susceptible host to influenza A virus, has been used in previous studies to evaluate H5N1 virulence and safety and efficacy of vaccine candidate preparations. Moreover, the same authors previously studied H5N1 virus virulence in ferrets and they have valid criteria for assessment, where they demonstrated earlier equivalence in virulence of two strains of virus. They have also evidence that the phenotypic distinctness of strains have been demonstrated in the earlier research in BBALB/c mice with highly pathogenic systemic expression and low pathogenic respiratory viral replication with no systemic disease. Methodology: H5N1 viruses were isolated and propagated in hen egg allantoic cavities. The allantoic fluid was pooled and stored, and tissue culture infectious dose and 50% egg infectious dose were determined through serial titration of viruses. Two strains of viruses, VN1203 and VN1204 were plaque purified, and stocks were produced in the recommended medium. Sequence and phylogenetic analysis of these isolated strains were done following extraction of viral RNA which was subjected to reverse transcription PCR. The PCR products were purified and directly sequenced to construct a phylogenetic tree based on the available databases throughout the world. Differences in their amino acid structures were identified. Six- to eight-week-old female BLAB/c mice were infected intranasally, and 50% mouse infectious dose and 50% lethal dose were determined in separate groups of mice. Three mice from a group of eight in each batch were sacrificed to prepare tissues for examination, and the other five were observed for clinical signs. Replications of the H5N1 virus in different tissues were examined, and in others, blood samples were collected to determine haematologic parameters. In the isolated ferrets, which had been previously tested and found negative for any circulating influenza virus agglutinins, a preexposure baseline serum, weight, and temperature measurement was taken 2 days prior to the experiment. Following adequate anesthesia, these were injected with 107EID50 of the virus, and these were monitored for appearance and persistence of relevant clinical symptoms of viral infection. Apart from other symptoms, the measurement of temperature and lethargy to measure relative inactivity index, demonstrated loss of body weight to the extent of 25%, appearance of neurologic dysfunctions were monitored for selecting candidates who were subjected to postmortem tissue examinations and blood analysis to indicate parameters suggesting pathogenic infection. In these ferrets, virus shedding was measured from nasal washes, and sacrificed ferrets were subjected to histopathologic examination of the tissues from spleen, kidneys, intestines, liver, heart, lungs-trachea, brain, olfactory bulb, and nasal turbinates. Virus titers from tissue homogenates, peripheral blood, and nasal washes were expressed as EID50/g. Lung and brain cortex tissues previously preserved were examined both by conventional H and E stains and immunohistochemistry. Tests were undertaken to detect the attached primary antibody, and the data were subjected to statistical analysis for tests of significance. Validity of the Methods: These methods are appropriate since it replicates the nature of infection of these viruses. It has used appropriate standards of time and medium of culture where indicated and has attempted to replicate the pathogenic process in ferrets from the strains identified by mice experiment. The prior determination of EID50 and its titers to determine the lethal dose have ensured infection, and the authors did not always depend on explicit disease for selecting the specimens. Therefore, infections with strains of different virulence are successfully included in the study, and this experiment has a chance to elucidate the patterns of infections by both the phylogenetically identified strains of the virus and could correlate them to haematologic manifestations, haematologic parameters, antibody titers, asymptomatic disease, mild non lethal disease, and lethal disease with a carrier state estimation by nasal shedding. Moreover, prior labeling of the viral strains by PCR would enable the researchers to correlate virulence pattern with the molecular architecture of the specific virus. Results and Analysis: The authors used a panel of representative viruses from the 2003 and 2004 outbreak, and they were compared to similar panel from 1997 infections. All these viruses had high infectivity titers. The phylogenetic characterisation of these two strains identified based on HA1 gene analysis of nine viruses demonstrated that they fall into two distinct genetic categories and they had separate geographic clads. The lethality of H5N1 virus was compared with lethality of another virus with known patterns of lethality. It was demonstrated that based on MID50 and LD50 titers, all eight human H5N1 isolates replicated in the lungs without prior adaptation, but three of the four avian viruses had minimal lung virus titers with higher MID50 titers. These resulted in two types of infections in these mice, those infected with human isolates demonstrated virulent infection with VN1203 strain, whereas VN1203 strain needed 40 times the previous inoculums to produce the same effect. This could have been due to difference in amino acid sequences of these two, and studies demonstrated that the variation in lethality not due to a mixed strain of viruses. The ability of these strains of these human viruses to replicate in organ systems outside the respiratory tract was determined by tissue studies. It was found that among the human H5N1 isolates, the Thai16 virus was most virulent in the sense that it attained maximum titers in the brain, induced early leucopenia and lymphopenia. In comparison, the virulence and lethality of the avian strains were far less. As highlighted earlier, the ferret experiment indicated that the four human viral strains used to infect ferrets were highly lethal from all parameters, and the results detail the clinical and clinicopathologic parameters well with high onset of progressive abnormalities in clinical parameters and haematologic pictures. With the avian strains, the infection was associated with minimal clinical signs and reversal of pathology in due course. The authors compared their findings from 1997 ferret study and concluded that the strain of 2004 H5N1 is far more virulent causing more severe disease than the 1997H5N1 virus. This could be correlated to the findings from the replication study of H5N1 viruses in the ferrets. The highly virulent 2004H5N1 human isolates would colonize in the nasal mucosa early in the course, would achieve lethal levels in the blood faster, and the animals would not be able to clear them from the upper respiratory tract and would be shed for a longer time. In contrast, the low virulence virus would never achieve the lethal levels in terms of titers and would successfully be cleared by the animals. Viral titers in the major organs were measured to determine the systemic infection potential of a particular strain, and as expected low virulence strain would demonstrate lower titers in other organ systems. The results indicated that the strains with high virulence would replicate to higher titers for a longer duration in the respiratory tract and from there to other organ systems. Both from the macroscopic and microscopic points of views, the high virulence strains would demonstrate discolouration and haemorrhage, tissue changes, and high tissue antigen levels in comparison to low virulence ones which despite infecting other tissues would demonstrate inflammatory tissue reactions without detection of viral antigens. The molecular determinants of virulence could not exactly pinpoint the differences in the amino acid sequences of these two strains as is relevant to their pathogenic qualities, but there is a possibility of existence of many other sequences differences responsible for this. The only identified Lys-to-Glu 627 substitution in PB2 is not enough to explain the differences. Controls: There is no apparent control in this experimental study. However, for the sake of comparison, the authors have utilized their 1997 study and their findings. The influenza SP83 strain has been used to replicate reversible clinically nonfatal infections in the mice. There is no scope to access the details of 1997 study in this work, but from the rigor of the study and methodology, it is very clear that the authors have used appropriate controls for this 2004 H5N1 study. Data Interpretation: From this critique it is evident that the data have been interpreted accurately and appropriately by the researchers. Since the objective of this research was to compare the virulence of viruses of multiple genetic groups in two well-validated animal models, and all these results suggest that 2004H5N1 human isolates were highly virulent and lethal for mice and ferrets, which is not the case with the avian isolates. Where applicable, the authors have used data bases for comparison, control haematologic parameters and clinical parameters for comparison, and statistical analyses to determine the significance of the findings. Therefore, the data have been interpreted appropriately. Conclusion: The mice and ferret mammalian models offer similar results in evaluation of virulence of H5N1 human virus, which had been exemplified in 2004 and 2005 H5N1 human epidemics in terms of the pattern of human disease, case fatality, organ involvement, clinical parameters, haematologic profile, and carrier state. Of the strains studied, the most virulent were four human isolates, namely VN1203, VN1204, Thai16, and Kan 353. The authors have concluded that the uses of animal models are valid options to better understand the virulence of a particular strain of H5N1 virus, and implications in human disease can be predicted. The idea of using both mice and ferrets is also sustainable due to the facts that inbred mice are more sensitive to detection of virulence patterns associated with single amino acid substitution in the viral genome, but the clinical parameters of a virulent disease are more explicit in infected ferrets that almost mimic a human disease of similar fatality. Further in conclusion, the authors suggest that these mammalian systems could be useful to identify molecular correlates of virulence of these viruses that can have predictive value in emergence of new strains of H5N1 that can infect and cause more virulent disease in humans. Support for Conclusions: In this current study, the virulent strains were replicated in high titers in the respiratory tracts and ultimately disseminated to multiple organs. In comparison, the avian isolates only infected the respiratory tract with nominal dissemination with no pathologic significance. Similar pictures have been supported in real human infections in the epidemiologic settings, and these replicate findings in all, clinical, pathologic, haematologic, viral load, organ dissemination, and carrier state parameters. A comparison with Asian 1997 H5N1 virulence pattern indicates that the 2004 strain used in this experiment is far more virulent, and the human lethality in the epidemic was almost simulated in these animal experiments. The authors also present support for their hypothesis of enhanced virulence from other studies, and the multiorgan haemorrhagic lesions and encephalitis in ferrets in their study were identical to the findings in studies conducted in a zoo epidemic. The recent change is virulence thus poses increased risks to human health due to enhanced virulence and expanded host range. Thus it is evident that the results, indeed, support the conclusions. Unanswered Question: This is a study of very high quality with significance and implications in human health in a future time; however, like any other scientific studies, this study has limitations. This study fails to correlate the molecular structure of the viruses to the virulent potential. Although that open the avenue for future research, the authors declare that the amino acid sequences of these two isolates, VN1203 and VN1204 in eight different coding regions are not enough to explain these differences in virulence pattern in the clinical or in the experimental setting. Additional studies are needed to totally delineate the molecular correlates that determine the virulence of H5N1 virus in mice and ferrets, but the authors have successfully established which would be animal models of such experiments. Reference Maines, TR et al. (2005). Avian Influenza (H5N1) Viruses Isolated from Humans in Asia in 2004 Exhibit Increased Virulence in Mammals. Journal of Virology, p. 11788-11800, Vol. 79, No. 18 Read More