Pakistan - BF Suspected Human Cases December 18, 2007 to Feb 2, 2008

[cartski]

Re: Pakistan: December 18+, WHO Begins Investigations

Since we're into the realm of speculation, why not delve into what might have happened while caring for an ill patient in a Pakistan hospital.

I do not know the norm in Pakistan hospitals, but I do know that in China hospitals the family members are the nurses. Is it similar? What role did the brothers play in the care of the patient?

The two brothers could have been charged with cleaning up vomit or incontenance. A simple, messy clean up could have exposed them to an overwhelming viral load, even if they weren't genetically related. Frankly, we need much more detailed information about the care of the index case.

I admit being frustrated with the rampant use of "H2H" in these situations.

"H2H" is such an ill-defined term in all these news reports and commentaries. If it is the WHO's phase 3 definition that we're concerned about: [ "phase 3: a new influenza virus subtype is causing disease in humans, but is not yet spreading efficiently and sustainably among humans" ] surely the contact with large quantities of shedding material is insufficient to meet this definition.

"Casual contact" H2H, as mentioned yesterday in a report, would be very alarming. How can being immersed large amounts of viral material, which may plausible in this hospital envrionment, qualify?

J.

[HenryN]

Re: Pakistan: December 18+, WHO Begins Investigations

Since we're into the realm of speculation, why not delve into what might have happened while caring for an ill patient in a Pakistan hospital.

I do not know the norm in Pakistan hospitals, but I do know that in China hospitals the family members are the nurses. Is it similar? What role did the brothers play in the care of the patient?

The two brothers could have been charged with cleaning up vomit or incontenance. A simple, messy clean up could have exposed them to an overwhelming viral load, even if they weren't genetically related. Frankly, we need much more detailed information about the care of the index case.

I admit being frustrated with the rampant use of "H2H" in these situations.

"H2H" is such an ill-defined term in all these news reports and commentaries. If it is the WHO's phase 3 definition that we're concerned about: [ "phase 3: a new influenza virus subtype is causing disease in humans, but is not yet spreading efficiently and sustainably among humans" ] surely the contact with large quantities of shedding material is insufficient to meet this definition.

"Casual contact" H2H, as mentioned yesterday in a report, would be very alarming. How can being immersed large amounts of viral material, which may plausible in this hospital envrionment, qualify?

J.

Spreading "efficiently and sustainably" used to be PHASE 6.

[HenryN]

Re: Pakistan: December 18+, WHO Begins Investigations

WHO team begins investigating bird flu outbreak
ISLAMABAD: A WHO team began investigating Pakistan?s first human bird flu cases on Tuesday to try to determine whether human-to-human transmission may have occurred. The health experts visited a hospital in Peshawar that had treated most of the eight patients suspected of being infected with the H5N1 virus, said Gregory Hartl, a WHO spokesman. One of the survivors said he had exhibited flu symptoms after slaughtering chickens without wearing protective clothing. His deceased siblings had visited him, reported AP. Health Secretary Khushnood Lashari said the man who had been culling poultry might have inadvertently brought the virus to his home, where the brothers fell sick, through his equipment. Separately, WHO?s global influenza programme coordinator Keiji Fukuda said the suspected human bird flu cases in Pakistan are likely a combination of infections from poultry and limited human-to-human transmission. He said there was no need for alarm, adding that WHO was not raising its level of pandemic alert for the time being, reported Reuters. agencies

http://www.pakistanlink.com/Headlines/Dec07/19/10.htm

[Theresa42]

Re: Pakistan: December 18+, WHO Begins Investigations

Yes, I would agree in regards to the source of infection in these cases. But don't you think that suspectibility among individuals varies and may possibly be related to genetics?

No. Not at this level. This is an AVIAN virus and the genetic composition of infected humans is VERY diverse (as is the dogs, cats, tigers, leopards, pigs, minks, foxes, stone martins, civet cats infected).

Perhaps the different genetics of different people (e.g. families) affects not the susceptibility to being infected, but the severity of the infection? How one's system reacts to the infection? Then we see a bunch of brothers in Pakistan who's systems don't handle the infection (or handle the infection too well maybe!) whereas some other people around them don't get such severe infections? I dunno -- just speculating.

Obviously antibody studies of all the contacts of this family would help clarify.

[HenryN]

Re: Pakistan: December 18+, WHO Begins Investigations

Commentary at

http://www.recombinomics.com/News/12190703/H5N1_Pakistan_H2H_Specifics.html

[HenryN]

Re: H2H suspected in Pakistan?

<TABLE cellSpacing=1 cellPadding=1 width="100%" border=0><TBODY><TR><TD>WHO says Pakistan bird flu cluster no cause for alarm as yet

</TD></TR><TR><TD class=article-author>Disease/Infection News</TD></TR><TR><TD class=article-date>Published: Wednesday, 19-Dec-2007 </TD></TR><TR><TD><!-- Printer Friendly / Email to a Friend Links --><TABLE cellSpacing=0 cellPadding=0 align=right border=0><TBODY><TR><TD width=15></TD><TD> Printer Friendly</TD><TD> </TD><TD width=18></TD><TD> Email to a Friend
</TD></TR></TBODY></TABLE></TD></TR><TR><TD><TABLE cellSpacing=1 cellPadding=2 width="100%" border=0><TBODY><TR><TD style="BACKGROUND: url(aspvirtualnews/template_images/smalldot.gif)" height=25> </TD></TR><TR><TD>According to an expert at the World Health Organization, the cluster of suspected bird flu cases in Pakistan may be a combination of infections from poultry with limited person-to-person transmission.

In the cluster, eight people in North West Frontier Province, Pakistan's "poultry belt", have tested positive for the H5N1 bird flu virus since late October; of the confirmed cases one man has died along with his brother who was never tested and is not included in the tally.
Six have recovered and one remains under medical supervision in the cities of Abbotabad and Mansehra.
The WHO says though this is Pakistan's first outbreak of bird flu among people, several outbreaks of H5N1 influenza have occurred among poultry and spread to the country's wild birds earlier this year.
Keiji Fukuda, the coordinator of WHO's global influenza program, says though still unconfirmed, it appears that any human-to-human spread was the result of close contact by family caring for sick loved ones as in previous outbreaks in Thailand and Indonesia.
The cases initially caused alarm as there has always been the very real fear that the H5N1 bird flu virus will ultimately mutate and acquire the ability to pass from person to person causing a pandemic.
Almost all human cases of bird flu to date have been the result of close contact with infected birds.
The WHO however says there is no immediate cause for alarm and they are not raising the level of pandemic alert as the cases do not appear to be the result of just human to human transmission.
It seems a veterinarian, who was at the centre of the outbreaks and a number of other suspect cases were exposed to poultry.
The veterinarian assisted with culling operations, became infected but did recover whereas his two brothers who cared for him also became infected and died.
Fukuda says it is quite possible that there was a mixed scenario where poultry to human infection occurred and then possibly human to human transmission within a family.
While this has not as yet been verified experts say human to human transmission would not be particularly surprising or unprecedented.
The H5N1 strain of bird flu remains a disease of animals, but any mutation enabling the deadly virus to pass between humans could trigger a pandemic which could kill millions of people.
So far the largest known cluster of human bird flu cases worldwide was in May 2006 in Indonesia's North Sumatra province, where as many as seven people in an extended family died.
The WHO has dispatched three experts, led by Hassan El-Bushra from it's Cairo office, to help Pakistan investigate the outbreak.
The WHO has a set of six phases of pandemic alert which gauges the level of threat and the world is currently in phase 3, which indicates a new influenza virus subtype which is causing disease in humans, but is not yet spreading efficiently or sustainable among humans.
In terms of public health implications, the WHO are watching for human to human transmission where casual contact leads to infections and fosters large outbreaks in communities.
The U.S. Naval Medical Research Unit NAMRU-3 laboratory from Cairo is now in Pakistan and will conduct further tests on the samples from the suspected cases.
According to the WHO since 2003 there have been 341 cases among people in 14 countries, and 210 of them were fatal.

http://www.news-medical.net/print_article.asp?id=33693

</TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>

[Laidback Al]

Re: Pakistan: December 18+, WHO Begins Investigations

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<TABLE cellSpacing=0 cellPadding=6 width="100%" border=0><TBODY><TR><TD class=alt2 style="BORDER-RIGHT: 1px inset; BORDER-TOP: 1px inset; BORDER-LEFT: 1px inset; BORDER-BOTTOM: 1px inset">Originally Posted by niman
Quote:
<TABLE cellSpacing=0 cellPadding=6 width="100%" border=0><TBODY><TR><TD class=alt2 style="BORDER-RIGHT: 1px inset; BORDER-TOP: 1px inset; BORDER-LEFT: 1px inset; BORDER-BOTTOM: 1px inset">Originally Posted by LaidbackAl
Yes, I would agree in regards to the source of infection in these cases. But don't you think that suspectibility among individuals varies and may possibly be related to genetics?


</TD></TR></TBODY></TABLE>
No. Not at this level. This is an AVIAN virus and the genetic composition of infected humans is VERY diverse (as is the dogs, cats, tigers, leopards, pigs, minks, foxes, stone martins, civet cats infected).</I>

</TD></TR></TBODY></TABLE>
Perhaps the different genetics of different people (e.g. families) affects not the susceptibility to being infected, but the severity of the infection? How one's system reacts to the infection? Then we see a bunch of brothers in Pakistan who's systems don't handle the infection (or handle the infection too well maybe!) whereas some other people around them don't get such severe infections? I dunno -- just speculating.

Obviously antibody studies of all the contacts of this family would help clarify.

I see Niman?s point. H5N1 is a novel avian virus that has made the jump to numerous species, including humans. Humans are not special to this virus. Therefore it would be unlikely that many human individuals have innate defense mechanisms to protect from this particular virus and by extension there is no reason to believe that some individuals might be more susceptible to this new virus.

Perhaps a real doctor can weigh in on this, but maybe the severity of the infection results from a combination of local environmental conditions, the overall health and biology of the individual, and possibly, the susceptibility of the person to influenza viruses in general. The body can not recognize or respond to a virus (H5N1) it has never encountered, it only responds as best it can to the invasion of a novel viral insult.
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Perhaps we could have a discussion about why some people do not get infected during a pandemic. It would seem that the attack rate should be 100% once it becomes airborne and easily transmissible. Yet, that does not seem to happen and didn?t happen in 1918.
<o:p></o:p>
Certainly non-pharmaceutical interventions and sheltering in place may allow people to avoid exposure to the virus. But why is it that there are many people (at least in past pandemics) who escape immediate infection even though they are exposed to a novel virus. What is the mechanism that keeps these individuals from becoming infected immediately or at least stay asymptomatic and recover? Or is it strictly a function of the viral load in the initial exposure?

[AlaskaDenise]

Re: Pakistan: December 18+, WHO Begins Investigations

Commentary at

http://www.recombinomics.com/News/12...Specifics.html

Commentary

H5N1 Human to Human Tranmission in Pakistan Specifics?

Recombinomics Commentary
December 19, 2007

The eight individuals in Pakistan who are suspected to have bird flu probably have a combination of infections from poultry and limited person-to-person transmission from close contact, a top World Health Organization expert said on Tuesday. Keiji Fukuda, coordinator of WHO's global influenza program, said while unconfirmed, any human-to-human spread seemed similar to previous outbreaks in Thailand and Indonesia -- affecting close family members caring for sick loved ones.

The above comments confirm the data represented by the consensus media reports. However, these reports lack hard dates, including disease onset dates, which suggest the cluster may be the longest in term of links in the chain, as well as persistance of the chain. The metrics could raise concerns that the H5N1 in circulation will generate similar chains in Pakisitan, as well as downstream locations in Europe, the Middle East and Africa, which could generate unprescedented numbers of confirmed H5N1 cases.

Although Pakistan has not released any H5N1 sequence data from their poultry outbreaks in 2006 and 2007 (see satellite map), public 2006 sequences from adjacent Afghanistan and India have been published. These sequences have some similarities, and it is likely that earlier sequences from Pakistan are similar to those from neighboring countries. However, neither India nor Afghanistan have released sequences from reported outbreaks this year.

Sequences have been released by Krasnodar and Germany, and these sequences are related to 2006 sequences from Uva Lake. These Uva Lake sequences are also related to the earlier sequences from India and Afghanistan, so it is likely that the current sequences in Pakistan are also related to these Uva Lake sequences.

These sequences are widespread. The first appeared in Kuwait at the beginning of this year. Then they appear in multiple countries in Europe of the summer (Czech Republic, Germany, France). Similar sequences were the published from Krasnodar, and subsequent infections in England and Germany are also said to be similar, suggesting that recent outbreaks in Romania, Poland, Rostov, and Saudi Arabia are also similar.

Therefore the ability of these sequences to transmit human-to-human (H2H) is of broad interest, so prompt reporting of the specifics of the H2H transmission is important. The larger cluster includes the index case, four brothers, a cousin, and one or two health care workers. Media reports indicate this cluster began with exposure(s) during a cull on October 21-23. The index case developed symptoms on October 25, and two brothers died November 19 and 25. Moreover, a health care worker may still be hospitalized, suggesting the cluster may be H2H2H2H or longer and may have persisted for almost 2 months. In addition, media reports suggest there are three additional confirmed patients representing another familial cluster.

Local confirmation of the initial lab positives was projected to be completed yesterday, and disease onset dates for the cluster members should have been known for some time. This information is usually released in WHO situation updates, which give the age and gender of confirmed cases, as well as disease onset dates, hospitalization dates, and dates of death as well as relationships between cluster members.

Virtually all of the data has not been released even though the outbreak began almost 2 months ago, and WHO has been aware of this cluster for at least a week.

Yesterday, media reports described a new suspect case in Kuwait, which when coupled with the massive outbreaks in poultry in Saudi Arabia and the high concentration of pilgrims at the Hajj, create cause for concern.


.

[HenryN]

Re: Pakistan: December 18+, WHO Begins Investigations

<TABLE cellSpacing=0 cellPadding=0 width="96%" align=center border=0><TBODY><TR bgColor=#f4faff><TD class=small_txt height=20>Bird flu: KMU lists precautionary steps</TD></TR><TR><TD></TD></TR><TR><TD bgColor=#efefef></TD></TR><TR><TD></TD></TR><TR><TD class=small_txt>Thursday, December 20, 2007
PESHAWAR: The Khyber Medical University's (KMU), Directorate of Research and Development, has issued a precautionary message to create awareness among the people of the NWFP about the precautions to be taken to contain bird flu influenza.

The directorate said that transmission of the H5NI virus was from poultry to human beings. Direct contact with infected poultry, their nasal secretions, or surfaces and objects contaminated by their faces, is presently considered the main route of human infection.

If this mode of transmission is not contained, there is a risk of the virus mutating to a form that is highly infectious for humans and it spreads easily from person to person, which will lead to a worldwide epidemic.

Health professionals around the world are working to avoid this situation.

The Directorate of Research and Development clarified that poultry and poultry products from areas experiencing outbreaks could be safely consumed provided these items were properly handled during food preparation and thoroughly cooked (no "pink" parts or bleeding near bones as in barbecued chicken) and that eggs too were properly cooked (no runny yolks).

Persons handling the poultry either in the poultry farm or in the kitchen need to wash their hands thoroughly with soap and hot water and also clean surfaces (cutting boards, knives, etc) used for poultry preparation with soap and hot water.

The directorate said that in the event of any person suffering from flu symptoms like (runny nose, nasal congestion, chest congestion, fever, etc), especially poultry farm workers, should consult a physician immediately.

If it is Avian Influenza, early detection and management through treatment with Oseltamivir and Zanamivir may reduce the severity of the illness and improve the prospects of survival, if administered early.

Healthcare workers need to practice "barrier-nursing" that is precautionary measures with the use of facial mask, hand gloves and sterile gowns.

It is better to be safe than sorry, let us be informed and protected, concludes the press release.
</TD></TR></TBODY></TABLE>

http://www.thenews.com.pk/daily_detail.asp?id=87283

[AlaskaDenise]

Re: Pakistan: December 18+, WHO Begins Investigations

I see Niman?s point. .......unlikely that many human individuals have innate defense mechanisms to protect from this particular virus ......... The body can not recognize or respond to a virus (H5N1) it has never encountered, it only responds as best it can to the invasion of a novel viral insult.
<?xml:namespace prefix = o /><o:p></o:p>
Perhaps we could have a discussion about why some people do not get infected during a pandemic. It would seem that the attack rate should be 100% once it becomes airborne and easily transmissible. Yet, that does not seem to happen and didn?t happen in 1918.
<o:p></o:p>
........ But why is it that there are many people (at least in past pandemics) who escape immediate infection even though they are exposed to a novel virus. What is the mechanism that keeps these individuals from becoming infected immediately or at least stay asymptomatic and recover? Or is it strictly a function of the viral load in the initial exposure?

Al, I believe you meant humoral for the bolded/underlined word.

Since both 1918 and current H5N1 have the NS1 mutation allowing it to bypass the impact of most of the innate immune system, we could look at the health of a person's humoral system....that might contribute to different responses.

But since this influenza has the polybasic cleavage site, which 1918 did not (however apparently some strains had a deletion in the NA stalk which allowed it to be cleaved by plasminogen), we should expect a much higher CFR.

Hopefully, there will be some mechanism that will allow at least a small percentage of people to survive an infection. A very small number of people cannot get HIV due to "fake" receptors for the virus, thereby causing an ineffetive "docking", & that person cannot get HIV.

Survivors or the seemingly-immune may have had a recent influenza infection, thereby utilizing some aspects of cross-reactive immunity. Remember the HK H9Nx ducks that didn't get H5N1?

.

[Shannon Bennett]

Re: Pakistan: December 18+, WHO Begins Investigations

There appears to be evidence supporting low viral loading may allow some people to fend off the virus. However, a large viral lode results in severe illness. The difference is how much of a dose you get when you are first infected.

[AlaskaDenise]

Re: Pakistan: December 18+, WHO Begins Investigations

There appears to be evidence supporting low viral loading may allow some people to fend off the virus. However, a large viral lode results in severe illness. The difference is how much of a dose you get when you are first infected.

In that case, which part of the immune system is eliminating the virus? Before the humoral can create appropriate responders, any virus could replicate considerably.

I believe it's a good theory, but wouldn't those folks have antibodies? Haven't most contact testing projects found no antibodies in uninfected contacts?

I sure hope the theory holds, as it will allow "social distancing" to be a major deterrent.

.

[HenryN]

Re: Pakistan: December 18+, WHO Begins Investigations

ISLAMABAD, Pakistan (AP) – A second team of health experts were on their way to Pakistan on Wednesday to analyze samples from suspected birdflu cases to determine how the virus spread and whether human–to–human transmission may have occurred.
The experts from the U.S. Naval Medical Research Unit No. 3 in Cairo, scheduled to arrive Thursday, were expected to retest samples already gathered from a number of patients who were positive for the H5N1 birdflu virus in initial government analysis. Once the cases are confirmed, work will begin to piece together how the victims became infected.
Orya Maqbool Jan Abbasi, spokesman for Pakistan’s Health Ministry, said earlier the team had arrived Wednesday, but WHO spokesman Gregory Hartl in Geneva said they were due a day later.
Five brothers were sickened last month in Abbotabad, north of Islamabad. Two died, one of whom was buried before tests were conducted. The other four tested positive for the virus. Up to six more people were suspected of being infected, including several who were in contact with poultry. WHO and the Health Ministry had initially said only four brothers were suspected of being infected, but one patient identified as a cousin was actually another brother.
Outbreaks were reported among birds in the area before the human cases. However, Abassi stressed that there have been no new reports of birdflu in poultry or people.
A separate WHO team visited a hospital Tuesday in the northwestern city Peshawar that treated some of the patients. They were working with doctors and nurses on how to handle suspected cases and improve infection control measures.
’’They want to go through the records in the hospital for the last month or two to see if there’s been any upsurge in respiratory cases that weren’t identified as H5N1 but which could actually be,’’ said Gregory Hartl, a WHO spokesman in Geneva.
The team will look to see which patients could have been exposed to the virus by infected birds and also whether human–to–human transmission could have occurred.
One of the brothers who survived, Mohammed Ishtiaq, said he was hospitalized with flu symptoms after slaughtering chickens suspected of carrying birdflu without wearing protective clothing last month.
His brothers who died visited him in a hospital, he said.
Hartl said no new cases have been discovered, but increased awareness has led to more people with flu–like symptoms being checked.
’’What this is showing is that they’re taking everything very, very seriously,’’ Hartl said. ’’Surveillance has been enhanced, more people are reporting cases and more people have been sensitized on the heath care worker side of the need to notice.’’
At least 209 people have died worldwide from the virus, which began plaguing Asian poultry stocks in late 2003, according to the WHO. It remains hard for people to catch, but scientists worry it could mutate into a form that spreads easily among people, potentially sparking a pandemic.

http://www.caycompass.com/cgi-bin/CFPnews.cgi?ID=1027348

[Shannon Bennett]

Re: Pakistan: December 18+, WHO Begins Investigations

This looks intersting. Was looking for supporting studies of low viral loading.

http://www.jimmunol.org/cgi/content/abstract/179/8/5220

The Journal of Immunology, 2007, 179: 5220-5227.
Copyright ? 2007 by The American Association of Immunologists, Inc.
<TABLE class=content_box_outer_table align=right><TBODY><TR><TD><!-- beginning of inner table --><TABLE class=content_box_inner_table><!-- citation --><TBODY><TR><TD class=content_box_title_highlight colSpan=2>This Article</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Full Text </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Full Text (PDF) </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me when this article is cited </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me if a correction is posted </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Citation Map </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>Services</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Similar articles in this journal </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Similar articles in PubMed </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Alert me to new issues of the journal </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Download to citation manager </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>Google Scholar</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Thitithanyanont, A. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Pichyangkul, S. </TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_title colSpan=2>PubMed</TD></TR><TR><TD class=content_box_space_between_sections colSpan=2></TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>PubMed Citation </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Thitithanyanont, A. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Articles by Pichyangkul, S. </TD></TR><TR><TD class=content_box_arrow vAlign=top width=4></TD><TD class=content_box_item>Pubmed/NCBI databases <TABLE class=content_box_inner_table style="FONT-SIZE: 100%" summary="Entrez Links for this Article"><TBODY style="FONT-SIZE: 100%"><TR style="FONT-SIZE: 100%"><TD style="FONT-SIZE: 100%; WHITE-SPACE: nowrap">Gene</TD><TD style="FONT-SIZE: 100%; WHITE-SPACE: nowrap">GEO Profiles</TD></TR><TR style="FONT-SIZE: 100%"><TD style="FONT-SIZE: 100%; WHITE-SPACE: nowrap">Nucleotide</TD><TD style="FONT-SIZE: 100%; WHITE-SPACE: nowrap">Protein</TD></TR><TR style="FONT-SIZE: 100%"><TD style="FONT-SIZE: 100%; WHITE-SPACE: nowrap" colSpan=2>UniGene</TD></TR><TR style="FONT-SIZE: 100%"><TD style="FONT-SIZE: 100%; WHITE-SPACE: nowrap" colSpan=2>Substance via MeSH</TD></TR></TBODY></TABLE></TD></TR></TD></TR></TBODY></TABLE></TD></TR></TBODY></TABLE>
High Susceptibility of Human Dendritic Cells to Avian Influenza H5N1 Virus Infection and Protection by IFN- and TLR Ligands<SUP>1</SUP>

</NOBR><NOBR>Arunee Thitithanyanont<SUP>*</SUP></NOBR>, <NOBR>Anneke Engering<SUP></SUP></NOBR>, <NOBR>Peeraya Ekchariyawat<SUP>*</SUP></NOBR>, <NOBR>Suwimon Wiboon-ut<SUP>*</SUP></NOBR>, <NOBR>Amporn Limsalakpetch<SUP></SUP></NOBR>, <NOBR>Kosol Yongvanitchit<SUP></SUP></NOBR>, <NOBR>Utaiwan Kum-Arb<SUP></SUP></NOBR>, <NOBR>Watcharoot Kanchongkittiphon<SUP>*</SUP><SUP>,</SUP></NOBR>, <NOBR>Pongsak Utaisincharoen<SUP>*</SUP></NOBR>, <NOBR>Stitaya Sirisinha<SUP>*</SUP></NOBR>, <NOBR>Pilaipan Puthavathana<SUP></SUP></NOBR>, <NOBR>Mark M. Fukuda<SUP></SUP></NOBR> and <NOBR>Sathit Pichyangkul<SUP>2</SUP><SUP>,</SUP></NOBR>

<SUP>*</SUP> Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand; <SUP></SUP> Department of Immunology and Medicine and <SUP></SUP> Department of Enteric Diseases, U.S. Army Medical Component of the Armed Forces Research Institute of the Medical Sciences, Bangkok, Thailand; and <SUP></SUP> Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand
<!-- ABS -->There is worldwide concern that the avian influenza H5N1 virus,<SUP> </SUP>with a mortality rate of >50%, might cause the next influenza<SUP> </SUP>pandemic. Unlike most other influenza infections, H5N1 infection<SUP> </SUP>causes a systemic disease. The underlying mechanisms for this<SUP> </SUP>effect are still unclear. In this study, we investigate the<SUP> </SUP>interplay between avian influenza H5N1 and human dendritic cells<SUP> </SUP>(DC). We showed that H5N1 virus can infect and replicate in<SUP> </SUP>monocyte-derived and blood myeloid DC, leading to cell death.<SUP> </SUP>These results suggest that H5N1 escapes viral-specific immunity,<SUP> </SUP>and could disseminate via DC. In contrast, blood pDC were resistant<SUP> </SUP>to infection and produced high amounts of IFN-. Addition of<SUP> </SUP>this cytokine to monocyte-derived DC or pretreatment with TLR<SUP> </SUP>ligands protected against infection and the cytopathic effects<SUP> </SUP>of H5N1 virus.<SUP> </SUP>
<!-- FN --><!-- null -->The costs of publication of this article were defrayed in part<SUP> </SUP>by the payment of page charges. This article must therefore<SUP> </SUP>be hereby marked advertisement in accordance with 18 U.S.C.<SUP> </SUP>Section 1734 solely to indicate this fact.<SUP> </SUP>
<!-- null --><SUP>1</SUP> This work was supported by the National Center for Genetic Engineering<SUP> </SUP>and Biotechnology (BIOTEC) Thailand, the Ellison Medical Foundation<SUP> </SUP>prime grant, by Thailand Research Fund for Advanced Research<SUP> </SUP>Scholar, and by Grant Y1-AI-5026-01 from the National Institutes<SUP> </SUP>of Health, National Institute of Allergy and Infectious Diseases<SUP> </SUP>International Research in Infectious Disease.<SUP> </SUP>
<!-- null --><SUP>2</SUP> Address correspondence and reprint requests to Dr. Sathit Pichyangkul,<SUP> </SUP>Department of Immunology and Medicine, U.S. Army Medical Component<SUP> </SUP>of the Armed Forces Research Institute of the Medical Sciences,<SUP> </SUP>315/6 Rajvithi Road, Bangkok 10400, Thailand. E-mail address:<SUP> </SUP>sathitp@afrims.org<SCRIPT type=text/javascript><!-- var u = "sathitp", d = "afrims.org"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></SCRIPT> <SUP></SUP><!-- null --><SUP>3</SUP> Abbreviations used in this paper: DC, dendritic cell; mDC, myeloid<SUP> </SUP>DC; pDC, plasmacytoid DC; MOI, multiplicity of infection.<SUP> </SUP>

[Shannon Bennett]

Re: Pakistan: December 18+, WHO Begins Investigations

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<!-- end : Open Access Block --><!-- start title area -->A Single Mutation in the PB1-F2 of H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence

<!-- end title area --><!-- start authors -->Gina M. Conenello<SUP>1</SUP>, Dmitriy Zamarin<SUP>1</SUP>, Lucy A. Perrone<SUP>2</SUP>, Terrence Tumpey<SUP>2</SUP>, Peter Palese<SUP>1,</SUP><SUP>3</SUP><SUP>*</SUP>
<!-- end authors --><!-- start affiliations -->1 Department of Microbiology, Mount Sinai School of Medicine, New York, New York, United States of America, 2 Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 3 Department of Medicine, Mount Sinai School of Medicine, New York, New York, United States of America
<!-- end affiliations --><!-- start: abstract -->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.

<!-- end abstract --><!-- start footnote section -->Funding. GC and DZ were partially supported by NIH/NIAID 1 T32 AI07647-Training Program: Mechanisms of Virus-Host Interactions. This work was also partially supported by National Institutes of Health grants RO1-AI8998, 1 PO1 AI058113, and UO1AI070469, and the Center for Research on Influenza Pathogenesis HHSN2662000700010C.
Competing interests. The authors have declared that no competing interests exist.
Editor: Yoshihiro Kawaoka, University of Wisconsin-Madison, United States of America
Citation: Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P (2007) A Single Mutation in the PB1-F2 of H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence. PLoS Pathog 3(10): e141 doi:10.1371/journal.ppat.0030141

Received: June 7, 2007; Accepted: August 10, 2007; Published: October 5, 2007 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Abbreviations: aa, amino acid; LD<SUB>50</SUB>, 50% lethal dose; MDCK, Madin Darby canine kidney; MOI, multiplicity of infection; PBS, phosphate-buffered saline; PFU, plaque-forming unit
* To whom correspondence should be addressed. E-mail: peter.palese@mssm.edu
<!-- end footnote section --><!-- start special linking abstract - (editorial commentary/summary, author summary) -->Author Summary

PB1-F2 is the most recently discovered protein produced by the influenza A virus. It has been previously shown that PB1-F2 is present in the mitochondria, where it induces cell death; our laboratory has demonstrated that PB1-F2 is a contributor to pathogenesis in the mouse model of infection. To study PB1-F2 further, we examined highly pathogenic strains of avian influenza virus and located an amino acid change that seemed to be associated with increased death in mice. We studied this amino acid change in PB1-F2 at position 66 in two different viruses. A recombinant virus that has a PB1 gene from an H5N1 virus was used as well as a fully reconstructed 1918 pandemic virus. In this study, we show that a mutation in PB1-F2 found in highly pathogenic influenza A virus isolates causes nonpathogenic viruses to induce disease in mice. In addition, we show that the increased pathogenicity is associated with higher levels of virus and cytokines in the lungs. We conclude that PB1-F2 does affect pathogenicity, and that position 66 seems to play an important role in contributing to the effects of PB1-F2 in the mouse model.

<!-- end special linking abstract - editorial commentary --><!-- start: body -->Introduction

Influenza A virus causes 300,000–500,000 deaths worldwide each year, and in pandemic years, this number can increase to 1 million (in 1957–1958 ) or as high as 50 million, as was seen in 1918–1919 [1–3]. More recently, H5N1 highly pathogenic avian influenza viruses have generated great concern regarding their potential to cause a pandemic. H5N1 infections in humans were seen in Hong Kong in a small outbreak in 1997 that resulted in 18 human infections and six fatalities, and since 2003, 309 human cases of H5N1 have been confirmed with a 61% fatality rate (6/1/07) [4–7]. Recent work on these viruses has aimed to elucidate the virulence factors that account for the severe illness observed in humans and mice [4,8–12].
The viral PB1 segment is of particular interest, since, in addition to the glycoprotein genes, the PB1 gene was the only other segment that was exchanged in the pandemic viruses of 1957 and 1968 [13]. Introduction of a novel PB1 gene into the 1998 swine reassortant viruses further implicates the role of this gene in the pathogenesis of (animal) influenza [14]. Moreover, while changes in the surface glycoproteins allow the viruses to overcome the preexisting humoral immune response, they may not be solely responsible for the high virulence of the pandemic influenza viruses. In particular, the 1918 pandemic was associated with significantly higher morbidity and mortality than the subsequent pandemics [15]. Recent reconstruction of the 1918 virus has confirmed that the viral polymerase from the 1918 influenza virus is required for full pathogenicity of the recombinant 1918 virus in mice [16]. In fact, substitution of the viral polymerase genomic segments with those of the modern H1N1 strain severely attenuated the virus in mice [16]. Recent identification and characterization of a novel influenza virus protein called PB1-F2 encoded by the PB1 gene introduced a potential virulence factor that could play a role in pathogenesis of infection with pandemic influenza viruses and explain the selection of the PB1 gene in these viruses [17].
The influenza virus PB1-F2 is a 90–amino acid (aa) protein that is associated with the induction of cell death. The protein directly permeabilizes mitochondria, resulting in the dissipation of the mitochondrial membrane potential and the release of cytochrome c [17–19]. We have previously shown that PB1-F2 contributes to viral pathogenesis in the mouse model and wanted to further investigate whether the PB1-F2 proteins encoded by highly pathogenic viruses have conserved mutations in their aa sequence that are associated with pathogenicity [20]. We chose to study the PB1-F2 proteins of the Hong Kong 1997 H5N1 viruses that caused an outbreak in humans. Characterization of the isolated viruses in mice revealed that the viruses could be subdivided into three different groups based on the pathogenicity phenotype: high-virulence, intermediate-virulence, and low-virulence [21]. Further studies provided molecular correlates of pathogenicity in the high-virulence group, though such studies were not conducted for the PB1-F2 protein [22].
Herein, we assess the contribution of the PB1-F2 protein to the pathogenicity of a highly pathogenic H5N1 virus and the 1918 pandemic strain virus. An alignment of the aa sequences of isolates from the Hong Kong 1997 H5N1 outbreak revealed that a mutation, N66S, was associated with high pathogenicity phenotype in mice. Using a recombinant A/WSN/33 virus with the PB1 segment of A/HK/156/97, we observed increased morbidity and mortality of mice infected with a virus that contained the N66S mutation. In addition, infection with the reconstructed A/Brevig Mission/18 virus, which has an S at position 66, resulted in increased pathogenicity when compared with a reconstructed A/Brevig Mission/18 virus in which position 66 was changed to N [16]. We thereby show that PB1-F2 proteins from highly virulent viruses can contribute to pathogenicity, and identify a single aa change that confers a virulent phenotype in mice.
Materials and Methods

Cell Lines

Madin Darby canine kidney (MDCK), 293T, and A549 cells were obtained from ATCC (http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.atcc.org<WBR style="content: attr(alt)" alt="">/) and were maintained in MEM and DMEM culture media (Gibco, http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.invitrogen.com<WBR style="content: attr(alt)" alt="">/), respectively, supplemented with 10% fetal calf serum (Hyclone, http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.hyclone.com<WBR style="content: attr(alt)" alt="">/) and penicillin/streptomycin (Gibco).
Constructs and Cloning

The pPolI vectors encoding viral genomic RNA of the WSN strain have been described previously [23]. The PB1 gene of the A/HK/156/97 virus was reverse transcribed from purified genomic RNA, amplified by PCR with PB1 segment-specific primers, and cloned into the pPolI vector. The cloning of genes for A/Brevig Mission/18 has been described previously [16]. To generate pPolI vectors encoding the N66S PB1-F2 mutants, the pPolI vectors encoding the A/HK/483/97 PB1 or A/Brevig Mission/18 PB1 were subjected to site-directed mutagenesis using the Stratagene Quick-Change mutagenesis kit (Stratagene, http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.stratagene.com<WBR style="content: attr(alt)" alt="">/). Sequences of each construct were confirmed by automated sequencing performed at the Mount Sinai sequencing core facility.
Reverse Genetics for Recombinant Viruses

The reverse genetics technique for the generation of recombinant influenza viruses has been described previously [23]. Briefly, 293T cells were transfected with eight pPolI vectors encoding the viral genomic RNA segments and four pCAGGS protein expression vectors encoding the subunits of viral polymerase and the nucleocapsid protein. The transfected 293T cells were cocultured with MDCK cells, and virus released into the supernatant was isolated by plaque purification on MDCK cells. The presence of the introduced mutations was confirmed by reverse transcription and sequencing of the PB1 genes of the newly generated viruses. Viruses possessing 1918 genes were generated under biosafety level 3 (BSL-3 with enhancements) containment [24] to ensure the safety of laboratory workers, the environment, and the public. All subsequent laboratory and animal work with live virus containing A/Brevig Mission/18 genes also was performed under these high-containment conditions.
Mouse Experiments

Female C57/BL6 mice 6 to 7 wk old (Jackson Laboratories, http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.jax.org<WBR style="content: attr(alt)" alt="">/) were anesthetized with intraperitoneal injection of 0.07 ml of ketamine/xylazine (0.15 mg ketamine and 0.03 mg xylazine), and infectious virus was diluted in PBS/BSA/PS (phosphate-buffered saline/bovine serum albumin/penicillin and streptomycin) and inoculated intranasally in a volume of 30 μl. To assess virus pathogenicity, groups of four mice were inoculated with appropriate dose and were monitored daily for weight loss over 8 d. Mice that lost more than 25% of their initial body weight were killed according to institutional guidelines and scored as dead. To determine viral replication in the lungs, lungs were collected on days 1, 2, 3, 5, 7, and 8 after infection from 2 (days 1 and 2) or 4 (days 3, 5, 7, and 8) mice from each group and two mice in the PBS group. The lungs were homogenized in PBS using a Dounce homogenizer and processed for virus titering. Virus titers in the supernatant of lung homogenates were determined by plaque assay in MDCK cells.
For 1918 recombinant virus infections, female BALB/c mice, 6 to 7 wk old (Jackson Laboratories) were anesthetized with an intraperitoneal injection of 0.2 ml of 2,2,2-tribromoethanal in tert-amylalcohol (Avertin; Aldrich Chemical Co., http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.sigmaaldrich.com<WBR style="content: attr(alt)" alt="">/), and 50 ul of infectious virus diluted in PBS was inoculated intranasally. The 50% lethal dose (LD<SUB>50</SUB>) titers were determined by inoculating groups of three mice intranasally with serial 10-fold dilutions of virus. LD<SUB>50</SUB> titers were calculated by the method of Reed and Muench [25]. Individual body weights from eight mice were recorded for each group daily and monitored daily for disease signs and death for 14 d after infection. For determination of lung virus titers, 18 additional mice were infected intranasally with the intermediate inoculating dose (10<SUP>4</SUP> plaque-forming unit [PFU]) of virus. On days 1–3 and 5–8 after infection, three mice from each group were killed, and whole lungs were removed aseptically and homogenized in 1 ml of sterile PBS. Homogenates were titrated for virus infectivity using a standard plaque assay. The statistical significance of virus titer data was determined by using analysis of variance.
Cytokine Quantitation

To determine the in vivo levels of cytokine supernatants from the lung, homogenates of the lungs of WH-infected mice were assayed for IFN-γ and TNF-α (assay sensitivity, 2 pg/ml) by use of enzyme-linked immunosorbent assay kits purchased from R&D Systems (http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.rndsystems.com<WBR style="content: attr(alt)" alt="">/).
For high-containment laboratory work with 1918 recombinant viruses, the in vivo levels of cytokine proteins were determined from three individual mice per group. On day 4 after infection, mice were exsanguinated from the axilla and killed, and lung tissues were removed from naive and infected mice. Individual whole-lung samples were immediately frozen at −70 &#176;C. On the day of analysis, tissues were thawed, homogenized in 1 ml of cold PBS, and centrifuged at 150g for 5 min. Cytokine protein levels were measured from clarified lung homogenates by the Bioplex Protein Array system [26] (Bio-Rad, http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.bio-rad.com<WBR style="content: attr(alt)" alt="">/) using beads specific for mouse IL-1β, IFN-γ, and TNFα. Cytokine protein levels were measured according to the manufacturer's instructions by fluorescently conjugated monoclonal antibodies in duplicate against a standard curve.
Results

Conserved Mutations in Highly Pathogenic Influenza A Viruses

It has been previously shown that H5N1 viruses from the Hong Kong 1997 outbreak fall into three separate pathogenicity phenotypes: low, intermediate, and high [22]. The intermediate phenotype had aa sequence identity with the high-pathogenicity phenotype and all of the previously identified molecular correlates of a high pathogenicity phenotype, but caused a less severe disease in mice. However, PB1-F2 was not examined in the study by Katz et al. because it was not known at the time [22]. Alignment of the 1997 human H5N1 PB1-F2 sequences available in the National Center for Biotechnology Information database (http:<WBR style="content: attr(alt)" alt="">/<WBR style="content: attr(alt)" alt="">/www.ncbi.nlm.nih.gov<WBR style="content: attr(alt)" alt="">/) revealed several aa changes that separated the high-virulence from the low-virulence groups. These were (low-virulence versus high-virulence) E6D, R53K, N66S, and R75H. These changes were silent in the open reading frame of the PB1 gene. Alignment of the proteins with other PB1-F2 sequences available in the database revealed that with the exception of the N66S substitution, all of the described mutations were previously present in other influenza viral strains. The N66S mutation was of a particular interest, since it was found only to be present in the highly virulent 1997 H5N1 group, in the PB1-F2 proteins of some avian isolates, and in the 1918 A/Brevig Mission/18 PB1-F2 (Figure 1). Interestingly, the A/HK/156/97 virus (from the intermediate-virulence group), which was previously shown to possess all of the molecular signatures of the high-virulence group, possesses N at position 66 of the PB1-F2 protein.
Figure 1. Alignment and Location of N66S Mutation in the PB1-F2 Protein

Alignment of human isolates of influenza A viruses from a H5N1 Hong Kong outbreak and from the 1918 H1N1 pandemic. Viruses in red are of the high-pathogenic phenotype, those in purple are of intermediate pathogenicity, and those in blue are of low pathogenicity in mice. Yellow indicates C-terminal region with α-helical structure, and green indicates the minimal mitochondrial targeting sequence.

aa residue 66 resides in the C-terminal α-helical region of PB1-F2. This region is the interacting domain for ANT3 and VDAC1 and contains the mitochondrial targeting sequence, making the C-terminal region essential for the function of PB1-F2 [19,27]. The location of the N66S mutation in the structure of PB1-F2 and its presence in the C-terminal region supports the hypothesis that this aa change could impact PB1-F2′s effects in vivo. We hypothesized that this aa substitution may be responsible for the decreased pathogenicity phenotype observed for the A/HK/156/97 virus. Given these findings, we proceeded to determine whether the PB1-F2 mutation in position 66 (N66S) in the 1997 H5N1 viruses contributed to viral pathogenicity.
Impact of PB1-F2 aa 66 on Viral Growth and Virulence In Vitro and In Vivo

In vitro. To examine the effect of the mutation in position 66 on the pathogenicity of influenza A viruses, we created recombinant viruses containing either an asparagine (N) or serine (S) at that site of the PB1-F2 protein. A chimeric virus was created in the A/WSN/33 background that contained the A/HK/156/97 PB1 gene (WH). These viruses were rescued in a BSL-2 environment, making them easier to study. In addition, the WH virus has been characterized in a previous paper [20]. Site-directed mutagenesis was used to introduce the N66S mutation in PB1-F2 without changing the amino acid sequence of the PB1 protein (WH N66S). These viruses were then grown in MDCK cells to determine their growth kinetics in vitro. Cells were inoculated at two different multiplicities of infection (MOIs), 0.1 and 0.001. The two viruses have similar replication kinetics in MDCK cells (Figure 2A). The r1918 and r1918 S66N viruses also have similar growth kinetics in MDCK cells when inoculated at MOIs of .01 and .001 (Figure 2B).
Figure 2. In Vitro Growth Curve of Recombinant Viruses

(A) MDCK cells were inoculated at an MOI of 0.1 and 0.001, and virus growth of WH and WH N66S was assessed at the time points indicated. The figure is representative of three similar experiments.
(B) MDCK cells were inoculated at an MOI of .01 and .001, and virus growth of r1918 and r1918 S66N was assessed at the time points indicated.

In vivo. Next, 1 &#215; 10<SUP>4</SUP> PFU of the viruses were inoculated into mice to determine pathogenicity and viral growth in vivo. Body weights were monitored for up to 8 d after infection. The WH N66S virus caused the mice to start losing weight at day 3, and weight loss continued in all WH N66S–infected mice until day 8, resulting in 50% of the mice succumbing to infection. The WH virus, while causing a slight decrease in weight at day 7, did not cause significant weight loss, and all of the inoculated mice survived the infection (Figure 3A). This difference in pathogenicity is mirrored by the viral replication in the lungs. The WH N66S virus was found to replicate to higher titers in the lungs and exhibited peak virus titers 2 d earlier than the WH virus. WH N66S replication in the lungs was significantly higher than WH on days 2, 5, and 8 after infection, with WH N66S replicating to almost 100 times higher titers on each day (Figure 3B). However, virus levels in the lung were equal on day 7, suggesting that the continued weight loss of WH N66S–infected mice is partially the result of increased cytokine production in the lung. In addition, the high virus titer on day 7 in WH-infected mice corresponds to the mild weight loss seen in Figure 2B. WH N66S–infected mice exhibited slower viral clearance with persisting high viral titers, whereas the WH virus was cleared more effectively from the lung, with 3 out of 4 mice completely clearing the virus by day 8 (Figure 3B). Increased viral load and slowed viral clearance during WH N66S infection could suggest an impaired cellular immune response.
Figure 3. Contribution of PB1-F2 N66S Mutation to Pathogenicity of Recombinant Virus

(A) Mice were inoculated with 1 &#215; 10<SUP>4</SUP> PFU of virus or PBS, and their weights were recorded every day after infection.
(B) Virus titers from lung homogenates were measured from mice infected with WH or WH N66S virus at days 1, 2, 3, 5, 7, and 8 after inoculation. Error bars represent 1 standard deviation.

The 1918 pandemic virus contains an S at position 66 in PB1-F2 corresponding to increased virulence as seen in the A/Hong Kong/483/97 virus. To examine this aa in the context of a fully reconstructed 1918 virus, a single aa change was made (S66N) in PB1-F2 without changing the aa sequence of PB1. To evaluate the virulence and pathogenicity of the 1918 S66N mutant virus, the morbidity (measured by weight loss), virus replication, and LD<SUB>50</SUB> titers were determined in BALB/c mice and compared with a group of animals infected with wild-type 1918 virus, previously shown to be highly lethal in mice [28].
As shown in Figure 4A, mice infected with doses of 10<SUP>3</SUP>, 10<SUP>4</SUP>, 10<SUP>5</SUP>, and 10<SUP>6</SUP> PFU of the wild-type 1918 virus began to lose weight within 3 d. The mice showed progressive signs of illness, such as ruffled fur and listlessness during the first week of infection before succumbing to infection (LD<SUB>50</SUB> = 10<SUP>2.5</SUP>) by day 10 after inoculation. In contrast to the highly virulent wild-type 1918 virus infection, higher amounts of inoculating virus (10<SUP>5</SUP> and 10<SUP>6</SUP> PFU) were required to cause severe disease and weight loss among the mice infected with 1918 S66N mutant virus (Figure 4B). Furthermore, the lethality was substantially lower (LD<SUB>50</SUB> = 10<SUP>5.25</SUP>), requiring 500 times more virus than wild-type 1918 virus to kill mice. Infection of mice with the 10<SUP>4</SUP> PFU of 1918 S66N mutant virus resulted in lung virus titers, on days 2 and 3 after infection, that were at least 12-fold lower than those of mice infected with the same dose of wild-type 1918 virus (Figure 4C).
Figure 4. The Effect of the S66N Mutation on Pathogenicity in Mice

(A) Eight mice were inoculated with recombinant wt 1918 virus at 10<SUP>2</SUP>, 10<SUP>3</SUP>, 10<SUP>4</SUP>, 10<SUP>5</SUP>, or 10<SUP>6</SUP> PFU, and their weights were measured every day. Average weights are represented in the graph.
(B) Eight mice were inoculated with recombinant 1918 S66N virus at 10<SUP>2</SUP>,10<SUP>3</SUP>, 10<SUP>4</SUP>, 10<SUP>5</SUP>, or 10<SUP>6</SUP> PFU, and their weights were measured every other day. Average weights are represented in the graph.
(C) Effect of S66N mutation on mouse lung titers in r1918 virus. Virus titers from lung homogenates were measured from 3 mice infected per time point with r1918 or r1918 S66N virus at days 1, 2, 3, 6, 7, and 8 after inoculation. Error bars represent 1 standard deviation.

Cytokine Dysregulation in the Lungs of Infected Mice

To better understand the increased pathogenicity in the infected mice, we examined the levels of TNF-α and IFN-γ in the lungs. IFN-γ levels were observed to be higher in mice infected with WH N66S virus, especially at days 7 and 8 after infection, when the levels were approximately two times higher than the levels in the WH virus-infected mice (Figure 5A). Levels of TNF-α in the lung also showed significant differences late in infection. At days 7 and 8 after infection, TNF-α levels in mice infected with WH N66S virus had a two times higher increase over levels in WH-infected mice (Figure 5B).
Figure 5. Cytokine Levels in Lungs of Infected Mice

(A) IFN-γ enzyme-linked immunosorbent assay was performed on lung homogenates with four mice per time point for each virus and is represented here in ng/ml. *p < .05.
(B) TNF-α enzyme-linked immunosorbent assay was performed on lung homogenates with four mice per time point for each virus represented here in ng/ml. *p < .05.
(C) Lung homogenates collected on day 4 after inoculation were measured for the levels of IL-1β, IFN-γ, and TNF-α using the Bioplex Protein Array system. All error bars represent 1 standard deviation.

Individual lung tissues were also collected on day 4 after infection from 1918 virus–infected mice. A single timepoint (day 4 after infection) was chosen because it was previously determined that maximal lung cytokine/chemokine levels occurred at this time among mice infected with highly virulent influenza strains [27,28]. Tissues were homogenized and lysates were assayed for cytokines by the Bioplex Protein Array system. Determination of IL-1α, IFN-γ, and TNF-α levels demonstrated that these cytokines were produced above their constitutive levels 4 d after infection with both 1918 S66N mutant and wild-type virus (Figure 5C). All three cytokines were detected at significantly higher levels (p 0.5, analysis of variance) in 1918 wild-type–infected than in 1918 S66N mutant–infected mice. Together, these data indicate that PB1-F2 may play a role in immunomodulation, especially later in infection during viral clearance.
Discussion

Previous studies by our lab have shown that PB1-F2 contributes to the pathogenesis of the influenza A virus [20]. When expression of PB1-F2 was knocked out of a moderately virulent virus in mice, there was a significant loss in pathogenicity, indicating that PB1-F2 plays an important role in virulence [20]. In the present study, we show that a single aa change in PB1-F2 from highly virulent viruses increases pathogenicity in mice and modulates the immune response. It has been proposed that PB1-F2 causes apoptosis of immune cells, which may lead to decreased antigen presentation and a decrease in the adaptive immune response [17]. Humans infected with highly pathogenic viruses consistently have decreased lymphocytes and impaired immune response to influenza virus infection [4,8,10,21,29,30]. We wondered if these effects could be caused in part by PB1-F2. In this study, we provide evidence that PB1-F2 does contribute to the high pathogenicity phenotype and that the N66S mutation, also found in the 1918 H1N1 virus, contributes to virulence in highly pathogenic viruses.
After aligning the PB1-F2 sequences from H5N1 viruses that exhibited high- and low-pathogenicity phenotypes, a single aa change was found to correlate with high pathogenicity. The location of the N66S mutation also made it an excellent candidate for affecting the proapoptotic function of PB1-F2. Position 66 is in the α-helical structure of PB1-F2, in the mitochondrial targeting sequence. The location of aa 66 in the C-terminal mitochondrial targeting sequence of the protein could affect PB1-F2 interactions with ANT3 and VDAC1, potentially increasing the induction of apoptosis by PB1-F2 [19].
Recombinant A/WSN/33 viruses were created to specifically examine the effects of the N66S mutation during viral infection. The recombinant virus WH has decreased pathogenesis in mice compared with that of A/WSN/33 (unpublished data), likely due to the mismatched polymerase genes, resulting in less efficient replication in the host. The N66S mutation within the PB1-F2 protein partially reversed this attenuating effect.
Within a natural setting, the presence of a “virulent” PB1-F2 may be important when influenza viruses cross species barriers or when new pandemic strains are generated by reassortment. In fact, the PB1 gene has been one of the segments found to reassort to create the pandemic strains of 1957 and 1968, potentially giving these viruses a more pathogenic PB1-F2 and thus a higher virulence [13]. It is possible that the PB1-F2 protein could allow a newly reassorted virus to replicate in a new host efficiently enough to spread, and develop mutations to create a more efficient polymerase complex. In addition, influenza surveillance data shows that in recent history (1970 onward), H3N2 infections cause almost 14 times the number of influenza related deaths than H1N1 infections and are associated with a higher epidemic severity index (as measured by the rate of increase in pneumonia and influenza mortality) [31–33]. Interestingly, recent H1N1 isolates contain a truncated PB1-F2, which possibly plays a role in their decreased virulence [19,34]. The mutation we investigate here is not currently found in recent H5N1 isolates; however, it is possible for those viruses to acquire the mutation either through the error-prone RNA polymerase or through reassortment with a virus that contains the N66S mutation.
The observation that the WH N66S virus grew to higher titers in the lung and persisted at high titers for a longer time than the WH virus supports the role of PB1-F2 in allowing for increased replication. This may also explain the impairment of viral clearance in the mice infected with WH N66S. In addition, the 1918 wt virus showed higher lung titers and slower viral clearance when compared with the 1918 S66N virus. We suspect that the delay in viral clearance due to expression of PB1-F2 protein may allow for prolonged viral replication and development of irreversible pulmonary immunopathology, the findings observed with highly pathogenic influenza strains. CD8<SUP>+</SUP> T cells are mainly responsible for viral clearance in the host, and it is possible that their function could be impaired by PB1-F2 [35,36]. In support of this, we observed that the WH N66S and wt 1918 viruses caused a significant increase in IFN-γ and TNF-α cytokine production over the WH and 1918 S66N viruses, respectively. Whether this change in cytokine levels is through the direct action of PB1-F2 or through its impact on viral replication in the lung is difficult to determine. However, the cytokine dysregulation is of special interest because it has been associated with both H5N1 and 1918 H1N1 virus infections. In previous studies, cytokine dysregulation was associated with high virulence and death in animal models [29,37]. Our study supports these findings and suggests that PB1-F2 could be one of the factors contributing to the cytokine dysregulation seen in H5N1 virus–infected patients and 1918 H1N1 virus–infected animals [4,37].
Acknowledgments

LAP is supported by a postdoctoral fellowship sponsored by the American Society for Microbiology and the Coordinating Centers for Infectious Diseases, Centers for Disease Control and Prevention. PP is a Senior Scholar of the Ellison Medical Foundation.
<!-- start after acknowledgements footnote section -->Author contributions. All authors conceived and designed the experiments and analyzed the data. GMC, DZ, LAP, and TT performed the experiments. GMC, DZ, TT, and PP wrote the paper.
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