PLEASE NOT MOST OF THESE ARTICIAL ARE FROM 2009/10 OR EARLIER AND ARE FOR REFERENCE POINTS ONLY OR TO BE USED IN CASE STUDIES AS BOTH OF THESE VIRUS' MAKE A NEW EMERGENCE ON THE WORLD TODAY





A Single Mutation in the PB1-F2 of H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence



The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N–infected mice compared with wild-type 1918 virus–infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.

http://www.plospathogens.org/article/in ... at.0030141



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Pathogenesis of Avian Influenza A (H5N1) Viruses in Ferrets


Viruses. The avian H5N1 viruses A/Hong Kong/486/97 (HK/486) and A/Hong Kong/483/97 (HK/483) and the human H3N2 viruses A/Sydney/05/97 (Sydney/97) and A/Panama/2007/99 (Panama/99) were used in this study. A U.S. Department of Agriculture permit was obtained for work with avian influenza viruses. H5N1 virus stocks were grown in MDCK cells either once (HK/483) or twice (HK/486) and were then grown in the allantoic cavities of 10-day-old embryonated hens' eggs for 24 to 28 h at 37°C for four passages. H5N1 viruses were handled under biosafety level 3+ (BSL-3+) laboratory conditions (34, 51).
H3N2 viruses were grown in embryonated eggs for 48 h at 34°C; Sydney/97 and Panama/99 were passaged three and five times, respectively, in our laboratory. Virus stocks were aliquoted and stored at -70°C until use. Fifty percent egg infectious dose (EID50) titers were calculated by the method of Reed and Muench (30) after serial titration in eggs.

Infection and monitoring of ferrets. Young adult male or female ferrets (Marshall Farms, North Rose, N.Y.) aged 8 to 10 months and serologically negative by hemagglutination inhibition assay for currently circulating human influenza A or B viruses were moved at least 4 days prior to infection to the BSL-3+ animal holding area, where they were housed in cages contained in bioclean portable laminar flow clean room enclosures (Lab Products, Seaford, Del.). Prior to infection, baseline temperatures were measured twice daily for at least 3 days. Ferrets were anesthetized with ketamine (25 mg/kg), xylazine (2 mg/kg), and atropine (0.05 mg/kg) by the intramuscular route and infected intranasally (i.n.) with a total of 1 ml of 107 EID50 of virus/ml in phosphate-buffered saline (PBS) delivered to the nostrils. For both H5N1 viruses, this dose was approximately equivalent to 105 50% ferret infectious doses (FID50). Control animals were mock infected with an equivalent dilution (1:30) of noninfectious allantoic fluid. Temperatures were measured twice daily using either a rectal thermometer or a subcutaneous implantable temperature transponder (BioMedic Data Systems, Inc., Seaford, Del.). Preinfection values were averaged to obtain a baseline temperature for each ferret. The change in temperature (in degrees Celsius) was calculated at each time point for each animal. Clinical signs of sneezing (before anesthesia), inappetence, dyspnea, and level of activity were assessed daily. A scoring system based on that described by Reuman et al. (32) was used to assess the activity level as follows: 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not playful when stimulated; and 3, neither alert nor playful when stimulated. Based on the daily scores for each animal in a group, a relative inactivity index was calculated as follows: (day 1 to day 7) [score + 1]n/(day 1 to day 7) n, where n equals the total number of observations. A value of 1 was added to each base score so that a score of 0 could be divided by a denominator, resulting in an index value of 1.0. The numbers of animals assessed on different days were as follows: days 0 and 1, n = 9; day 3, n = 7; day 5, n = 5; and days 7 and 9, n = 3.

The FID50 was determined for each virus by i.n. infection of two ferrets each with doses of 104, 103, and 102 EID50 of virus and three ferrets each with 101 EID50 of virus as described above. Nasal wash samples were collected on day 3 p.i. and titrated in eggs to detect the infectious virus. Animals with nasal wash titers of 102 EID50/ml were considered positive for virus. The FID50 was calculated by using the method of Reed and Muench (30).

more at link

http://jvi.asm.org/cgi/content/full/76/9/4420


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H1N1- Ultimate News on influenza A - bird flu, swine flu & avian flu alerts

Ultimate news on H1N1, H5N1, avian, swine & Bird flu including mutation, new sequences from H5N1 to H1N1, with breaking news on H5N1, Influenza , H1N1, H3N2


A 35 year-old Italian tourist is one of three people believed to have been infected with A(H1N1) virus, or swine flu, as well as the H5N1 virus, known as avian flu. The patients are recovering in the Egyptian Red Sea resort of Hurghada, said Mohammed Rifai, director general of preventive medicine.

Rifai also spoke of a 28-year-old man who tested positive for both viruses, after arriving at the port of Safaga after a pilgrimage to Saudi Arabia.

"We are waiting the results of the tests that have already been carried out in some patients suspected of having been infected by the H5N1 and the A(H1N1)," said World Health Organisation spokesman Gregory Hartl in an interview with Adnkronos Salute.

The above comments raise concerns of co-infections of pandemic H1N1 and H5N1, as well as human to human co-transmission. This development would create serious pandemic concerns.


The ability of the pandemic H1N1 to infected multiple species has been demonstrated by the widespread outbreaks in swine. Outbreaks in Canada have been reported in three provinces (Alberta, Quebec, and Manitoba) as well as two locations in Argentina near Buenos Aires, as well as three locations in Australia, These swine infections demonstrate the ease of transmission from humans to swine, as well as swine to swine. Recent results in Chile also demonstrated transmission toturkeys, raising concerns that H1N1 could be silently transmitting to multiple mammalian and avian species.

The jump to avian raised concerns that the H1N1 could interact with H5N1, which is predominantly found in birds, including long range migratory birds. Recent data also demonstrated H5N1 in pikas at Qinghai Lake, which is a mammalian reservoir.

Results from Egypt also raised concerns of co-infections because of the large number of mild H5N1 cases and the concentration in children. The heavy concentration in children raised concerns of silent spread of H5N1 in adults, providing opportunities for co-infection, which could lead to the H1N1 "rescuing" H5N1 leading to co-transmissions.

http://bluefeeling.typepad.com/

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Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets


The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.


http://www.plospathogens.org/article/in ... at.1001022


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What about the possibility that influenza A (H1N1) might recombine with other more virulent viruses?

There is some concern that co-circulation with seasonal influenza A viruses during the winter, or with highly pathogenic H5N1 viruses in countries where those viruses are endemic, might lead to the emergence of more virulent reassortant viruses [8]. But although occasional dual infections with pandemic and seasonal viruses have been detected during the 2009 Southern hemisphere winter, there have been no reports of emergence of such reassortants.


http://jbiol.com/content/8/8/69


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New Vaccination Strategy May Protect Against Both Lethal 1918 And H5N1 Influenza Viruses


ScienceDaily (June 20, 2009) — A new study suggests that vaccination with 1918 H1N1 influenza virus-like particles not only protected mice and ferrets against the lethal 1918 influenza virus, but also displayed cross-reactive immunity against the potentially pandemic H5N1 influenza virus.


In this study researchers generated VLPs from the structural proteins of the 1918 H1N1 virus and compared their ability to protect mice and ferrets against a reconstructed 1918 virus and the highly pathogenic avian H5N1 virus that was isolated from a fatal human case. When immunized twice intranasally with H1N1 VLPs mice were highly protected against a lethal challenge with both the 1918 and H5N1 virus. In contrast, mice receiving two intramuscular immunizations of 1918 VLPs were only protected against the 1918 viral challenge. Mucosal vaccination with 1918 VLPs induced higher levels of cross-reactivity in mice and complete survival in ferrets challenged with a lethal dose of the H5N1 virus. Only a 50% survival rate was noted in intramuscularly immunized animals.

"These results suggest a strategy of VLP vaccination against a pandemic virus and one that stimulates heterotypic immunity against an influenza virus strain with threatening pandemic potential," say the researchers.

http://www.sciencedaily.com/releases/20 ... 112429.htm

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Dangerous Progeny Can Result When Flu Strains 'Hook Up'

Article Date: 14 Jul 2011 - 5:00 PDT


In their current study, the researchers looked at the compatibility of the 2009 pandemic pH1N1 virus - which has some genetic characteristics that may allow it to reassort more easily than other influenza viruses - with an influenza strain known as H9N2.

Published in a recent issue of Proceedings of the National Academy of Sciences (PNAS), this new research builds on earlier findings by Perez and his team of the heightened communicability of the H1N1 virus as well as their work on the airborne communicability of H9N2. And it adds knowledge that may advance modern medicine's longstanding effort to learn how to predict when pandemic flu viruses will arise. An effort that in recent years has focused on study of H5, H7, and H9 subtypes of flu viruses because these all occasionally infect humans and, in the case of H5 viruses, can cause significant disease and death.

For their PNAS study, the researchers created four reassortant viruses with one or two genes from the H9N2 virus and the rest of the genes from pH1N1. They used two different H9N2 viruses to provide the genes. One was a typical H9N2 isolated from a bird in Asia. The other was an avian isolate that had been adapted to infect and transmit in mammals.

Perez and colleagues looked at the growth characteristics of these four viruses and also their infectivity and transmissibility in ferrets. Ferrets are used as a model for human infections as they are susceptible to the same viruses and show similar signs of infection. All four viruses were able to grow to relatively high levels in cell culture. Similarly all four viruses infected ferrets and showed similar signs of disease and levels of replication. Additionally, they were all able to transmit to ferrets housed in the same cage and allowed physical contact. Finally, three of the four viruses were able to transmit to ferrets that were physically separated but shared the same air.

http://www.medicalnewstoday.com/releases/231099.php

Vaccine Selection for the 2011–2012 Influenza Season

The United States Food and Drug Administration (FDA) has chosen the three influenza (flu) viruses for inclusion in the 2011-2012 seasonal flu vaccine based on recommendations from the World Health Organization (WHO). Each year, experts from FDA, WHO, U.S. Centers for Disease Control and Prevention (CDC) and other institutions study virus samples collected from around the world to identify the influenza viruses that are the most likely to cause illness during the upcoming flu season so that people can be protected against them through vaccination.

How are the viruses selected to make flu vaccine?

The influenza (flu) viruses selected for inclusion in the seasonal flu vaccines are updated each year based on information about which influenza viruses are being found, how they are spreading, and how well the previous season's vaccine viruses might protect against any that are being newly identified. Currently, 136 national influenza centers in 106 countries conduct year-round surveillance for influenza viruses and disease activity. These laboratories then send influenza viruses for additional analyses to the five World Health Organization (WHO) Collaborating Centers for Reference and Research on Influenza, which are located in the following places:
•Atlanta, Georgia, USA (Centers for Disease Control and Prevention, CDC);
•London, United Kingdom (National Institute for Medical Research);
•Melbourne, Australia (Victoria Infectious Diseases Reference Laboratory);
•Tokyo, Japan (National Institute for Infectious Diseases); and
•Beijing, China (National Institute for Viral Disease Control and Prevention).

The seasonal flu vaccine is a trivalent vaccine (a three-component vaccine) with each component selected to protect against one of the three main groups of influenza viruses circulating in humans.

Three vaccine viruses are chosen to maximize the likelihood that the influenza vaccine will protect against the viruses most likely to spread and cause illness among people during the upcoming flu season. WHO recommends specific vaccine viruses for influenza vaccine production, but then individual countries make their own decisions for licensing of vaccines in their country. In the United States, the U.S. Food and Drug Administration determines what viruses will be used in U.S.-licensed vaccines.

What flu viruses are included in the Northern Hemisphere seasonal vaccine for 2011-2012?

WHO recommended that the Northern Hemisphere's 2011–2012 seasonal influenza vaccine contain the following three vaccine viruses:
•an A/California/7/2009 (H1N1)-like virus;
•an A/Perth/16/2009 (H3N2)-like virus; and
•a B/Brisbane/60/2008-like virus.

These are the same viruses that were selected for the Northern Hemisphere for the 2010-2011 influenza vaccine.

What flu viruses are included in the Southern Hemisphere seasonal vaccine for 2011?

On September 29, 2010, in Geneva, Switzerland, WHO recommended that the Southern Hemisphere's seasonal influenza vaccine contain the following three vaccine viruses:
•an A/California/7/2009 (H1N1)-like virus;
•an A/Perth/16/2009 (H3N2)-like virus;* and
•a B/Brisbane/60/2008-like virus.

*Note: A/Wisconsin/15/2009 and A/Victoria/210/2009 are A/Perth/16/2009-like viruses.

These are the same virus strains that the Food and Drug Administration (FDA) selected for inclusion in the Northern Hemisphere vaccine being used in the United States during the 2010-2011 season.

http://www.cdc.gov/flu/about/qa/vaccine-selection.htm

Fitness of Pandemic H1N1 and Seasonal influenza A viruses during Co-infection
Evidence of competitive advantage of pandemic H1N1 influenza versus seasonal influenza
On June 11, 2009 the World Health Organization (WHO) declared a new H1N1 influenza pandemic. This pandemic strain is as transmissible as seasonal H1N1 and H3N2 influenza A viruses. Major concerns facing this pandemic are whether the new virus will replace, co-circulate and/or reassort with seasonal H1N1 and/or H3N2 human strains. Using the ferret model, we investigated which of these three possibilities were most likely favored. Our studies showed that the current pandemic virus is more transmissible than, and has a biological advantage over, prototypical seasonal H1 or H3 strains.

Conclusions
Recent reports have speculated on the possibility that the new pandemic H1N1 strain may become more virulent and/or more transmissible if it were to reassort with seasonal influenza strains [8][9][10]. Alternatively, it has been postulated that the new H1N1 virus may not be completely adapted to the human population and that additional changes could lead to more virulent and perhaps more transmissible strains [8][9][10]. Our studies show that the pandemic Ca/04 virus has a clear biological advantage in replication, transmission, tropism and pathogenesis when compared to both seasonal H1 and H3 representative strains. In addition, co-infection of seasonal and pandemic strains did not result in the rapid selection of reassortant viruses that either improved replication or transmission or exacerbated virulence. Although we must be cautious interpreting studies in the ferret model, it is reasonable to speculate that this prototypical pandemic strain, Ca/04, has all the makings of a virus fully adapted to humans. At present, reassortment may not be particularly favored. We must note that more analysis need to be done to rule the possibility that either the inoculated, direct contacts and/or respiratory droplet contacts shed a population of reassortant viruses at any time during the infection. Nevertheless, if any reassortant was present, our data clearly indicates that they did not have an obvious biological advantage over the Ca/04 wild type virus during transmission via respiratory droplets. Alternatively, studies looking at additional combinations of pandemic and seasonal strains may provide a better understanding of the reassortment potential among these viruses. We observed that in the BR/10:Ca/04 co-infection group, respiratory droplet contact ferrets seroconverted to both the Ca/04 H1N1 and the BR/10 H3N2 yet, only high levels of Ca/04 replication were detected. However, since co-infection with the H3 seasonal strain resulted in exacerbation of clinical signs, it is important to determine whether co-infections have been associated in some of the lethal human cases. Surveillance should consider careful examination of clinical cases and determine whether co-infections are related to the morbidity and mortality associated with the pandemic strain. Based on studies in the ferret model, strong emphasis should be placed on determining whether underlying H3 infections are associated with co-infections with the pandemic strain.

http://knol.google.com/k/fitness-of-pan ... infection#