The whole thing makes a bit more sense now. Here's hoping it turns out to be small scale, but I suspect those pig farms are not going to change much anytime soon.

Just a few weeks ago, my husband and I decided to eat mainly vegetarian at home, though we still eat meat when out at times. Our reasons were health and ecology. A connection like this reinforces that impulse.

http://bit.ly/vaC5

This site says it well http://www.fairfoodfight.com/blog/el-dragón/grist-cafos-blame-h1n1-swine-flu-really

Flu pandemics existed long before feedlots became the norm.

Pigs probably don't get MRSA infections (Methicillin-resistant Staphylococcus aureus) either, but that originates from pig farms and is spreading in the human population - and it is horrible, no joke.

Erik, Orion Grassroots Network

we're talking about a virus that infects humans that originated from a combination of swine/avian/human strains. no animals are sick - read the news reports. 

this topic is almost off topic on a site like Grist - except its a topic because of some hyper extrapolation from the author of the post. If you have a science background you realize that Grist is mostly long on hyper extrapolations and short on facts.

we don't see the CDC telling us to run far and fast from porkers or birds do we ?

I respectfully suggest that you (a) research how viruses are transmitted and (b) consider the very real impact of poorly informed and erroneous public information regarding a serious public health concern such as this, before publishing further on the subject.

Certainly there are many worthy and valid topics associated with this outbreak / possible pandemic (e.g. commercial hog farming, for example, as an ethically reprehensible and ecologically dangerous practice) and as such this outbreak demands media scrutiny. However to suggest that flies may be the cause of the swine flu outbreak, and to imply a specific farm may be the source, all without any real evidence (and frankly with a demonstrated lack of fundamental understanding about flu virus transmission and epidemiological investigation), can be downright dangerous. As a public health emergency management professional I would ask that you temper your public writing with a healthy dose of proper research and recognition of the known facts before making statements linking a potential pandemic to unlikely if not impossible transmission methods and unconfirmed point sources. You now have people speculating wildly in response to this poorly researched article (although some commenters are on-point with their responses as well) and this does nothing except to mis-inform the public, make it more difficult for real public information officers to do their job, and preempt actual epidemiological investigations with publicly viewed misleading "theories" as to the source and the infection routes. 

Respectfully,

-cw

More as this develops.   @amazingdrx

"...by 2005, MRSA was killing more than 18,000 Americans a year, more than AIDS."

"One of the first clues that pigs could infect people with MRSA came in the Netherlands in 2004, when a young woman tested positive for a new strain of MRSA, called ST398. The family lived on a farm, so public health authorities swept in — and found that three family members, three co-workers and 8 of 10 pigs tested all carried MRSA."

"Now this same strain of MRSA has also been found in the United States. A new study by Tara Smith, a University of Iowa epidemiologist, found that 45 percent of pig farmers she sampled carried MRSA, as did 49 percent of the hogs tested."

These quotes are from a piece in a recent edition of the New York Times. No doubt you have a 'professional' take on this.

Erik, Orion Grassroots Network

Further, one of the nasty things about hog farms is the amount of innoculation of the herds. Due to close quarters, flu of many types is deadly to pig farms. They use a powerful cocktail of influenza innoculations that covers a huge variety of avian, human and swine strains. Frankly, a corporate farm pig may be the last creature on earth to get   the Flu.

http://twitter.com/veratect

They also have a press release on their website that says they were reporting on the event 18 days prior to the World Health Organization posting its first public report

I just found this tid bit of info. It is on the bird flu. It does state that there is a link between flies carrying virus (bird flu in this case) and also mentions the awful conditions in some poultry farms that begin the problem. If you google enough you will find that flies can carry virus. This can happen.  This is just info as I could not verify how legitimate this article is( couldn't find too much info about the scientist), but there are other articles that say the same thing.  Interesting and distressing. Why can't flies spread Swine Flu?

Wednesday, July 11, 2007

Houseflies can spread bird flu virus: Study

INDIA - Recent North American research has made the startling conclusion that several insects, particularly Musca domestica or the common housefly, are capable of carrying and transmitting Newcaslte Disease Virus and the highly pathogenic avian flu strain, H5N1, which is transmittable from animals to humans, writes Asit Jolly, Asian Age.

British scientist, Terry Mabbett, reporting in a recent issue of Poultry International, says the new research findings must come as a big wake up call for the world's poultry industry.

"That the avian influenza (AI) virus can be spread by winged insects as well as wild birds underlines the need for efficient fly control on poultry farms along with other strict biosecurity measures," he says.

According to Mabbett, studies recently carried out at the North Carolina State University, adult houseflies were seen to carry infectious doses of the Newcaslte Disease Virus in their guts for up to three hours after feeding. This he says, "might be important for the spread of the virus when fly populations are high and in contact with highly virulent NDV strains." The scientist has also cited earlier reported instances of houseflies carrying avian influenza virus. A 1985 study based on a serious 1983/84 outbreak of H5N2 in Lancaster County Pennsylvania (USA) where nearly 90 per cent of the affected poultry stocks died.

More than a third of the housefly samples collected from the vicinity of the outbreak contained bird flu virus particles.

Similarly, blow flies caught near a Kyoto poultry farm in Western Japan following an H5N1 outbreak in 2004 also carried doses of the virus. Dr Mabbett says the presence of avian influenza virus in Musca domestica or other flies has opened up a whole new dimension on this virus disease.

Indian poultry experts told Asian Age that "at the very minimum, poultry farm owners need to put their house in order. Our poultry farms could be particularly susceptible to insect-borne transmissions of the virus because of the abysmal sanitation maintained."

I am not saying this swine flu is definitely the product of factory farming: just that it is plausible and worth investigating.

http://www.eluniversal.com.mx/notas/594075.html

In a message broadcast from his office at the Government Palace, made it clear that the origin of the swine influenza began in Asia, so it is not related to agricultural activities in the region of Perote, with companies producing pork .

I am not so sure there is good, verified information coming from Mexico about this. There is a lot of finger pointing though. I am sure the good Govenor has no financial interest in the "companies producing pork", no?

http://www.rollingstone.com/politics/story/12840743/porks_dirty_secret_the_nations_top_hog_producer_is_also_one_of_americas_worst_polluters

Swine flu's ground zero? Residents say nearby farm

"As far back as late March, roughly one-sixth of the residents here in the Gulf Coast state of Veracruz began complaining of respiratory infections that they say can be traced to a farm that lies upwind five miles (8.5 kilometers) to the north, in the town of Xaltepec. But Jose Luis Martinez, a 34-year-old resident of La Gloria, said he knew the minute he learned about the outbreak on the news and heard a description of the symptoms: fever, coughing, joint aches, severe headache and, in some cases, vomiting and diarrhea. "When we saw it on the television, we said to ourselves, 'This is what we had,'" he said Monday. "It all came from here. ... The symptoms they are suffering are the same that we had here." "

http://news.yahoo.com/s/ap/20090428/ap_on_re_la_am_ca/lt_swine_flu_mexico_ground_zero

Everything else I've read (including this 'report' from Tom) is full of conjecture: "The residents believe that.."? Are you kidding me? Surely you mean all of the research scientist residents..

The corporate swine operations should be illegal for many reasons -- health and environmental, but if corporate pig farms could result in new strains of swine flu that can be transmitted human to human,  North Carolina would have been the world's top swine flu breeding ground for going on 20 years.

Sadly, unfounded accusations make it more difficult, not less difficult to shut down these operations. When corrected, the fallacies provide ammunition to the hog industry to say all the criticism of the industry is without basis in fact.

I have a general comment about these factory farms.  The Fort Worth Star and old Knight-Ridder (now McClatchy) news service ran a number of investigative articles about the effect of factory faming introduced after NAFTA into Mexico.  One of the big issues was the destruction of local smaller farming in Mexico.  With that collapse came a marked increase in illegal immigration into the USA due the the lack of opportunity for small farming in Mexico.  In one story there was a report of small hog farmers turning their hogs loose in government offices.

I will agree that there are strong ethical, health and ecological concerns regarding CAFOs; however, the link between them and the flu stated above is nothing but hearsay at best, and malicious posturing at worst. Thus, it is no surprise that the story above is purported as the 'ground-breaking' piece of journalism and is disclosed by someone who has NO training in medicine or public health management. So far, any attempt to point a finger is nothing but an uneducated opinion.

http://www.rollingstone.com/politics/story/12840743/porks_dirty_secret_the_nations_top_hog_producer_is_also_one_of_americas_worst_polluters

http://i.realone.com/assets/rn/img/5/6/8/0/12840865-12840868-slarge.jpg

Remember, that wild migratory waterfowl, rather than farmed ones, are the cause/carriers of Avian flu, should we be killing them to reduce Avian flu?

Influenza will find a path for mutation to more dangerous forms, regardless of how we choose to contain specific hosts. That is just what it does, arguably as a requirement of natural evolutionary change, or a pruning of the species if you prefer.

You write: "Remember, that wild migratory waterfowl, rather than farmed ones, are the cause/carriers of Avian flu..," That has certainly been the industry mantra. Do you have a credible source? Factory farms have been implicated in swine flu. From a paper by an international team of scientists—including Jay Graham and Ellen Silbergeld of Johns Hopkins—published in the May-June 2008 Public Health Reports, entitled “The Animal-Human Interface and Infectious Disease in Industrial Food Animal Production: Rethinking Biosecurity and Biocontainment” (PDF)

An analysis of data from the Thai government investigation in 2004 indicates that the odds of H5N1 outbreaks and
infections were significantly higher in large-scale commercial poultry operations as compared with backyard flocks. These data suggest that successful strategies to prevent or mitigate the emergence of pandemic avian influenza must consider risk factors specific to modern industrialized food animal production.

There are several sources with this conclusion:

http://www.vetmed.ucdavis.edu/vetext/INF-PO_AvianInfluenzaFS.html

A variety of articles are found here. In my examination of them, the poultry industry is a route of transmission, but not considered anything other than incidental. They use birds. The larger the flock of wild or domestic birds, the more likely a major outbreak.

http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=Search&Term=avian flu waterfowl&itool=QuerySuggestion

I am not a proponent of corporate animal farming, quite the opposite. It just appears your focus on same is a bit too intense based on the facts at hand. If you look hard enough for data to support any pre-determined conclusion, you will find some. But that is not investigation, that is supporting (as opposed to testing) your hypothesis.

Have you seen any black helicopters lately?

FDA Announces Ban on Feeding of Poultry Litter

Livestock Update, March 2004

http://www.ext.vt.edu/news/periodicals/livestock/aps-04_03/aps-318.html

I suspect the same is true in Mexico, although I don't have sources to cite nor experience in the Mexican feed industry.

http://www.guardian.co.uk/commentisfree/2009/apr/27/swine-flu-mexico-health

Even Fix It News (FOX) has an piece on how this flu most likely started in a "pig farm" by hogs eating bird and other droppings, and a human with the flu having contact with that hog and the three forms of flu forming this current swine flu.

As far as the piece from FOX news, how much validity do you give that?  I am very conservative and I can't even tolerate Fox News for more than a few minutes. 

For people that can spend time gathering facts on this stuff, how about citing some real, hard to find sources with up to the minute information, like CDC, APHIS, FAO OIE, WHO and the agencies of the Mexican Federal Government?  And please use quotes and give citations with names.

All of this rumor reporting and recylcing of biased blog posts doesn't get us one step closer to the real cause & cure.  It only serves to stir up an already jumpy public.

Here is the actual Lincoln quote and source:

"I see in the near future a crisis approaching that unnerves me and causes me to tremble for the safety of my country. . . corporations have been enthroned and an era of corruption in high places will follow, and the money power of the country will endeavor to prolong its reign by working upon the prejudices of the people until all wealth is aggregated in a few hands and the Republic is destroyed."

-- U.S. President Abraham Lincoln, Nov. 21, 1864
(letter to Col. William F. Elkins)
Ref: The Lincoln Encyclopedia, Archer H. Shaw (Macmillan, 1950, NY)

http://www.ratical.org/corporations/Lincoln.html

As to how this flu can be passed on:

"How does swine flu spread?
Influenza viruses can be directly transmitted from pigs to people and from people to pigs. Human infection with flu viruses from pigs are most likely to occur when people are in close proximity to infected pigs, such as in pig barns and livestock exhibits housing pigs at fairs. Human-to-human transmission of swine flu can also occur. This is thought to occur in the same way as seasonal flu occurs in people, which is mainly person-to-person transmission through coughing or sneezing of people infected with the influenza virus. People may become infected by touching something with flu viruses on it and then touching their mouth or nose." http://www.cdc.gov/swineflu/key_facts.htm

So back to the very strong possibility of these corporate hog farms being the source of this current outbreak, it is more than possible.  For the interference of corporations into our lives for their profits I believe Lincoln was right on - and that is proven every day from these international factory farms to Wall Street "corporate" bankers and their money influence on our government and it's agencies.

For some more detailed research into these corporate farms Pew Research conducted a very lengthy investigation into their effects, both in terms of health issues and actual economics.

 Final Report: Putting Meat on The Table: Industrial Farm Animal Production in America

http://ncifap.org/

Article

Nature Structural & Molecular Biology 16, 265 - 273 (2009)
Published online: 22 February 2009 | doi:10.1038/nsmb.1566

Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses

Jianhua Sui1,4, William C Hwang2,4, Sandra Perez3, Ge Wei2, Daniel Aird1, Li-mei Chen3, Eugenio Santelli2, Boguslaw Stec2, Greg Cadwell2, Maryam Ali1, Hongquan Wan3, Akikazu Murakami1, Anuradha Yammanuru1, Thomas Han1, Nancy J Cox3, Laurie A Bankston2, Ruben O Donis3, Robert C Liddington2 & Wayne A Marasco1

Abstract

Influenza virus remains a serious health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Here we used a human non-immune antibody phage-display library and the H5 hemagglutinin ectodomain to select ten neutralizing antibodies (nAbs) that were effective against all group 1 influenza viruses tested, including H5N1 'bird flu' and the H1N1 'Spanish flu'. The crystal structure of one such nAb bound to H5 shows that it blocks infection by inserting its heavy chain into a conserved pocket in the stem region, thus preventing membrane fusion. Nine of the nAbs employ the germline gene VH1-69, and all seem to use the same neutralizing mechanism. Our data further suggest that this region is recalcitrant to neutralization escape and that nAb-based immunotherapy is a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

Introduction

Seasonal influenza A is a scourge of the young and old, killing more than 250,000 worldwide each year, while creating an economic burden for millions1. Pandemic influenza, which occurs when a new virus emerges and infects people globally that have little or no immunity, represents a grave threat to human health; for example, the 1918 Spanish Flu pandemic caused an estimated 50 million deaths2, 3. Vaccines have historically been the mainstay of infection control. However, owing to rapid antigenic drift, the vaccine antigen must be updated annually based on global influenza surveillance4, 5, and it is not always fully successful. In addition, some recent H5N1 vaccines have shown promising results6, 7, 8, 9, but none has been reported to elicit a broad neutralizing response in humans. Neuraminidase inhibitors, especially oseltamavir (Tamiflu), remain the primary treatment, but they have limited efficacy if administered late in infection, and widespread use is likely to result in the emergence of resistant viral strains10, 11.

Influenza A is subclassified by its two major surface proteins: hemagglutinin, which mediates cell entry, first by recognizing host proteins bearing sialic acid on their surface, and second by triggering the fusion of viral and host membranes following endocytosis, allowing viral RNA to enter the cytoplasm; and neuraminidase, which cleaves sialic acid from host and viral proteins, facilitating cell exit12. There are 16 hemagglutinin subtypes (H1–16) and 9 neuraminidase subtypes (N1–9) that make up all known strains of influenza A viruses by various combinations of hemagglutinin and neuraminidase12 (Supplementary Fig. 1 online).

The recent spread of highly pathogenic avian influenza (HPAI), caused by the H5N1 strain, across Asia, Europe and Africa raises the specter of a new pandemic, should the virus mutate to become readily transmissible from person to person. The evolution of H5N1 into a pandemic threat could occur through a single reassortment of its segmented genome or through the slower process of genetic drift12, 13. Nearly 400 human H5N1 infections have been reported since 1997 from 14 countries, with a case mortality rate in the immunocompetent population above 60%4.

New therapeutic strategies that provide potent and broadly cross-protective host immunity are therefore a global public health priority. Human mAb-based 'passive' immunotherapy is now being used to treat numerous human diseases, including respiratory syncytial virus infection, and we have proposed how immunotherapy could be used strategically in a viral outbreak setting14.

In the present study, we first used a phage-display antibody library and recombinant H5 trimeric ectodomain to isolate a group of high-affinity nAbs that were potent inhibitors of H5N1 viral infection in vitro and in vivo. On the basis of crystallographic and functional studies, we showed that the nAbs bind to a common epitope—a highly conserved pocket in the stem region of hemagglutinin containing the 'fusion peptide'—that rationalizes their ability to block membrane fusion rather than cell attachment. Sequence and structural analysis of all 16 hemagglutinin subtypes point to the existence of just two variants of this epitope, corresponding to the two classic phylogenetic groupings of hemagglutinin (groups 1 and 2). We therefore tested eight more group 1 hemagglutinin subtypes and demonstrated an unprecedented cross-subtype binding and/or neutralization spectrum. Because we had used a group 1 subtype (H5) for our panning, our nAbs, as expected, failed to neutralize group 2 subtypes H3 and H7. These results nevertheless raise the possibility that a cocktail comprising a small subset of nAbs raised against representatives of the two groups could provide broad protection against all seasonal and pandemic influenza A viruses.

Top of page

Results

Identification of nAbs against H5N1

The current H5N1 epidemics involve viruses derived from a single lineage of H5 hemagglutinin. Within this lineage, four distinct clades have been identified as major threats to public health15, 16. We expressed recombinant trimeric ectodomain of H5 hemagglutinin from one of these viruses (strain A/Vietnam/1203/04 (H5N1), 'H5-VN04', clade 1) in insect cells17 (Supplementary Fig. 2 online), immobilized it on a plastic surface and selected antibodies from a 'non-immune' human antibody phage-display library (using single-chain VH-VL fragments ('scFv'))18. Two rounds of panning and the screening of 392 clones identified 10 unique antibodies formed by six distinct VH (variable region of heavy chain) fragments in combination with ten different VL (variable region of light chain) fragments (Supplementary Table 1 online).

We found that all ten nAbs bound trimeric H5-VN04 with similar avidity, but did not bind monomeric HA1 (Fig. 1a). Presented as scFv-Fc constructs, they potently neutralized the clade 1 H5 pseudovirus, A/Thailand/2-SP-33/2004 (H5N1) ('H5-TH04') (Fig. 1b); and, in a stringent plaque-reduction assay, they all showed high levels of neutralization against H5-VN04, as well as the more divergent (clade 2.1) A/Indonesia/5/2005 ('H5-IN05') (Fig. 1c,d). We further found that the nAbs cross-competed with each other in a competition enzyme-linked immunosorbent assay (ELISA; Supplementary Fig. 3 online), suggesting that they share a common epitope. On the basis of this finding, as well as VH sequence diversity and neutralization potency, we converted three of the nAbs (D8, F10 and A66) into full-length human IgG1s for further studies; all three IgG1s bound to recombinant H5-VN04 with high affinity (Kd 100–200 pM) and slow dissociation rates (kd 10-4s-1) (Supplementary Fig. 4 online).

Figure 1: In vitro binding and neutralization of anti-H5 antibodies.

(a) The ten antibodies were converted to soluble scFv-Fcs (scFv linked to the hinge, CH2 and CH3 domains of human IgG1) and evaluated for binding to trimeric H5-TH04 or monomeric HA1 of H5-TH04 coated on an ELISA plate. The H5 scFv-Fcs recognize trimeric H5 but not HA1. An antibody raised against HA1 (2A) recognized both. (b) Neutralization of H5-TH04–pseudotyped viruses (virus-like particles with HIV-1 only cores that display H5 on their surface). Percentage of neutralization at two concentrations is shown with s.d. The mAb 80R18 was used as a negative control (Ctrl.). (c,d) Neutralization of wild-type H5-VN04 and H5-IN05 by the ten scFv-Fcs at three concentrations using a plaque reduction assay. Results are consistent with those obtained from a microneutralization assay (data not shown).

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Prophylactic and therapeutic efficacy in mice

We evaluated the protective efficacy of the three IgG1s against H5N1 virus infection in a BALB/c mouse model (Fig. 2). Mice were treated with IgG1s before (prophylactically) or after (therapeutically) lethal viral challenge. Prophylaxis using 10 mg kg-1 of IgG1s effectively protected (80–100%) mice when challenged with a high lethal dose of H5-VN04 (clade 1) or A/HongKong/483/97 ('H5-HK97') (clade 0) (Fig. 2a,b). Therapeutic treatment with 15 mg kg-1 (an achievable dose in humans) of IgG1 24 h post-inoculation (hpi) also protected 80–100% of the mice challenged with either H5-VN04 or H5-HK97 virus (Fig. 2c,d). Mice treated at later times (48 hpi or 72 hpi) with H5-VN04 showed similar or higher levels of protection (Fig. 2e,f). Furthermore, surviving mice remained healthy and showed minimal body weight loss over the 2-week observation period (data not shown).

Figure 2: Prophylactic and therapeutic efficacy of anti-H5 nAbs in mice.

(a,b) Prophylactic efficacy. Percentage of survival of mice treated with anti-H5 nAbs or control mAb 1 h before lethal challenge by intranasal inoculation with H5-VN04 (a) or H5-HK97 (b) viruses. (c–f). Therapeutic efficacy. Mice were inoculated with H5-VN04 and injected with nAbs at 24 h, 48 h of 72 hpi (c,e,f) or with H5-HK97 at 24 hpi (d).

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Whereas human influenza viruses are typically restricted to the upper respiratory tract, systemic spread is a typical outcome of H5N1 infection in mice, and it has been reported in some humans. We found that the three IgG1s caused potent suppression of viral replication in the lungs (measured 4 d after viral challenge) of mice treated within 48 h of viral challenge; and that two IgG1s, F10 and A66, were effective when given at 72 hpi. The strong impact of antibody therapy on systemic infection was demonstrated by 1,000-fold suppression of virus spread to the spleen, even when given 72 hpi (Supplementary Fig. 5 online). We also observed suppression in the brain, but in this case, systemic spread was too low in control animals for accurate quantitation.

nAbs inhibit cell fusion rather than receptor binding

Two ways in which anti-hemagglutinin antibodies can neutralize infection is by blocking the initial binding of hemagglutinin to its cellular receptor (sialic acid) or by interfering with the subsequent step of hemagglutinin-mediated virus-host membrane fusion, which occurs in acidic endosomes19, 20. We found that none of the nAbs inhibited virus binding to cells (Fig. 3a) or hemagglutination of red blood cells (data not shown). However, we were able to show, using a model system of cell fusion, that the nAbs potently inhibited membrane fusion (Fig. 3b).

Figure 3: Neutralization mechanism.

(a) nAbs do not inhibit cell binding of full-length hemagglutinin from H5-TH04–pseudotyped HIV-1 viruses. None of the three nAb-treated viruses inhibited cell binding. Mouse anti-H5 mAb, 17A2.1.2 and ferret anti-H5N1 serum, which inhibit hemagglutination, were used as positive controls. Anti-SARS spike protein (80R) and anti-HA1 (2A) were used as negative controls. Error bars represent s.d. (b) All three nAbs inhibit cell fusion. HeLa cells were transfected with H5-TH04–expressing plasmid and exposed to a pH 5.0 buffer for 4 min in the presence or absence of nAbs. Syncytia formation induced by the brief exposure to pH 5.0 was completely inhibited by D8, F10 and A66, at 20 g ml-1 ( 0.13 M), whereas controls (80R and anti-HA1 mAb (2A) at the same concentration had no effect.

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Structural characterization of the nAb epitope

To provide a structural basis for neutralization and to explore the prospects for developing even broader-spectrum therapeutics, we determined the crystal structure of F10 (as the scFv fragment) in complex with the H5 (H5-VN04) ectodomain (Fig. 4 and Supplementary Table 2 online). We used H5 activated by cleavage of the single-chain precursor, HA0, into two polypeptides, HA1 and HA2. Cleavage leads to the partial burial of the fusion peptide (the first 21 residues of each HA2) into the stem19, 21, which also contributes to the formation of each of three hydrophobic 'pockets' located below the large trimeric receptor binding head. In the complex, one F10 nAb binds into each pocket, burying 1,500 Å2 of protein surface. Only the heavy chain (VH) participates directly in binding, using all three of its complementarity-determining regions (CDRs). The light chain (VL) points out into solution and makes only nonspecific contacts with the distal end of the oligosaccharide of glycosylated residue Asn331 from a neighboring monomer. The epitope on H5 encompasses the entire pocket, which is formed by the HA2 fusion peptide flanked by elements of HA1 on one side and helix A of HA2 on the other.

Figure 4: Structure of the H5–F10 complex.

(a) Structure of the H5 trimer bound to F10 (scFv). H5 is similar to the uncomplexed structure35 (pairwise r.m.s. deviation (C ) = 1.0 and 0.63 Å for two independent trimers). HA1, HA2, the A helix of HA2, the fusion peptide (FP) and F10 (VH and VL) are color coded. The third F10 molecule is hidden behind the stem. (b) Close-up of the epitope showing H5 as a molecular surface, with selected epitope residues labeled. The fusion peptide is in green. The tip of F10 (red ribbon) and selected CDR side chains are shown. Of 1,500 Å2 buried surface at the interface, 43% involves hydrophobic interactions. (c) Surface of the central stem region, showing two H5 monomers. One monomer has HA1 (yellow) and HA2 (blue) colored differently; the path of the FP through the epitope (red) is outlined, and mutations that do not affect binding are colored cyan (Fig. 4d). The fusion peptides (FP and FP') are labeled in both monomers. Epitope residues are labeled white (HA2) or yellow (HA1), and the position of buried residue H1112 is shown as a black ball labeled 'H'. (d) Binding of the three nAbs to H5 mutants in the A helix, transiently transfected into 293T cells. Note the similar response to all mutants tested. Mutations were made either to alanine or to the corresponding H7 residue; 24 h after transfection, nAbs or ferret anti-H5N1 serum was used to stain the transfected cells. Fluorescent intensity was normalized against ferret anti-serum (100%) to account for different expression levels.

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The key interactions are as follows (Fig. 4b). (i) CDR-H2 adopts the 'type 2' conformation22. Two hydrophobic residues, Met54 and Phe55, from the tip of H2 insert into the pocket. Phe55 lies across a flat hydrophobic surface formed by the main chain of the fusion peptide, residues 182–212; it also makes favorable orthogonal aromatic interactions23 with the side chains of Trp212 at the back of the pocket and His181 at the front (subscripts 1 or 2 refer to HA1 or HA2, and the numbering scheme follows the structure of H3 (PDB 2HMG)17, 24). The Met54 sulfur makes -aromatic interactions25 with the Trp212 ring, hydrophobic interactions with Ile452 from helix A and a hydrogen bond between Met54 C=O and the His381 side chain. (ii) Tyr102 from CDR-H3 extends from the apex of the H3 loop to a location only 3 Å from Phe55, and it complements CDR-H2 by cementing together the fusion peptide (via a main chain hydrogen bond to Asp192) and the A helix of HA2 (by intercalating between Thr412 and Ile452). A large hydrophobic residue at the neighboring position 103 supports the side chain conformation of Tyr102. (iii) The CDR-H1 loop is characterized by small hydrophobic or polar side chains (notably Val27, Thr28 and Ser31) such that CDR-H1 fits snugly beneath the hemagglutinin head while packing against helix A. A somatic mutation of conserved Gly26, G26E, generates a noncanonical conformation for H1, with Thr27 pointing outward and making contact H5.

An N-terminal hairpin (residues Ile292 and Met302) from HA2 of the counterclockwise neighbor packs against the other side of helix A at this point, wrapping around its fusion peptide and further locking it into place (Fig. 4a,c). Thus, the F10 nAb may stabilize the fusion peptide of more than one subunit. One framework (FR3) residue, Gln74, seems to be especially important in stabilizing the CDR-H1 and CDR-H2 loop conformations, by forming hydrogen bonds to the main chain C = O groups of Pro53 and Met54, as well as the side chain of Ser30. The FR3 residue at position 72 is the major determinant of the choice between two distinct conformations of the H2 loop22.

Consistent with the structural data, mutations in three H5 residues on HA2 A, Val522, Asn532 and Ile562, which make important interactions with F10, greatly reduce or ablate nAb binding, whereas the conservative mutation V52L has no effect (Fig. 4c,d). Mutations to other surfaces of the A helix either have no effect (typically exposed residues) or lead to increased nAb binding, perhaps by subtly increasing the flexibility of the epitope (Fig. 4d). Notably, the nine other nAbs show similar mutant binding profiles. Together with the cross-competition noted above, this strongly suggests that the epitopes for all ten nAbs overlap very closely indeed, and that the nAbs bind in a similar location and orientation.

Structural basis of H5 neutralization by the nAb panel

The broad neutralizing behavior against H5 may be attributed in part to the exclusive role of VH in antigen binding and the use of a common germline gene, VH1-69, in five out of the six VHs, although their CDR3 loops are variable in sequence and length (13–17 residues) (Supplementary Fig. 6 and Supplementary Table 1 online). In addition, free-energy calculations26 point to dominant binding contributions ( 70% of the total favorable free energy) of the three conserved residues in the VH segment (Fig. 4b). In CDR-H2 derived from germline VH1-69, position 55 is always phenylalanine, and position 54 is always hydrophobic (methionine, isoleucine, leucine or valine). In our nAbs, CDR-H3 always has a tyrosine predicted to lie at the tip of the CDR3 loop (conserved at the position 6). The conformation and sequence of the CDR1 loop does not seem to be critical, because the other antibodies we isolated do not contain the somatic mutation (G26E) found in F10 and are predicted to have canonical structures. The sixth VH gene we isolated is derived from the germline gene VH1-2; its H2 loop has the same length as VH1-69, but by virtue of a change from alanine to arginine at position 72 (ref. 22) it is predicted to adopt a distinct conformation ('type 3') that presents loop residues 3 and 4 to the antigen (rather than residues 4 and 5 in type 2 loops). The specific somatic mutation at position 4, from asparagine to methionine, presumably promotes H5 binding. It is not possible to predict the structure of the larger H3 loop, but a tyrosine located at the center of the loop may play an analogous role to that in F10.

Thus, the F10-H5 crystal structure suggests a common mechanism of H5 virus neutralization for our nAb panel. They make no contact with the receptor binding sites in the head and so do not inhibit cell attachment. Rather, they lock the fusion peptide and helix A in place, thereby preventing the large structural reorganizations that are required for membrane fusion17, 19, 27, 28, 29, 30. Our data point to this event occurring at an early step in infection, although we cannot rule out the possibility that the nAbs act at a later stage, given the close packing of molecules on the surface of the mature virion, which might restrict early access to the epitope. The only previously published crystal structure of a hemagglutinin-nAb complex that inhibits membrane fusion uses a different mechanism: it prevents conformational changes by cross-linking the upper surfaces of adjacent subunits in the head31.

NAbs bind and neutralize a broad range of group 1 viruses

Next we examined all of the available hemagglutinin sequences (total 6,360) in the public influenza sequence database (Supplementary Table 3 online). Of note, the sequences of the F10 epitope are nearly always conserved within the H5 subtype. Indeed, many epitope residues, especially in HA2, are highly conserved across all 16 hemagglutinin subtypes (Fig. 5). This high sequence conservation provides a rationale for the cross-neutralization of the H5N1 virus clades described above, and this prompted us to test our antibodies against a broader range of hemagglutinin subtypes.

Figure 5: Sequence conservation in hemagglutinin groups, clusters and subtypes at the F10 epitope.

Circles below residue numbers indicate estimated contribution to the binding energy at each position: red, strong; yellow, intermediate; blue, neutral. Residues without a circle are not directly involved in the epitope but are discussed in the text. Colored highlighting on the sequences indicates conservation within clusters and groups, with orange indicating high conservation or invariance. Other colors (for example, yellow, cyan and pink) highlight residues that are cluster or subtype specific. The network of interhelical contacts that stabilize the fusogenic structure61 are indicated below the HA2 sequences. Subtypes that can be recognized/neutralized by F10 are indicated with '+' on the far right. (+) or (-) indicates a predicted positive or negative binding, respectively.

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Group 1 viruses, which contain 10 of the 16 subtypes, are further classified into three 'clusters': H1a, H1b and H9 (refs. 32,33; Fig. 5). We tested nAb binding to eight members of clusters H1a, H1b and H9, which include avian H5 and the most common human influenza subtypes (the major exception is the group 2 subtype, H3). In addition to H5, we found that all three IgG1s bound to cells expressing full-length H1 from three different strains of H1N1, including the 1918 Spanish flu, H2 from H2N2 and H6 from H6N2, the Cluster 1b subtypes H11 from H11N9, H13 from H13N6 and H16 from H16N3, and the Cluster H9 subtypes from three H9N2 strains. However, none of them bound to a group 2 subtype, H7 from H7N1 (Supplementary Fig. 7 online).

The IgG1s also neutralized H5-, H1-, H2-, H6- and H11-pseudotyped virus infections (Fig. 6a). In a microneutralization assay, F10-IgG1 also neutralized H5N1, H1N1, H2N2, H6N1, H6N2, H8N4 and H9N2 influenza viruses (Fig. 6b). However, none of the nAbs neutralized group 2 viruses, for example, H3N2 (Fig. 6b and Supplementary Fig. 8 online). Thus, these nAbs recognize an epitope on hemagglutinin that is conserved among H5 clades and in all members of group 1 viruses. Finally, we demonstrated the in vivo protective efficacy of two of the IgG1s against two lethal H1N1 viral strains in a BALB/c mouse model, using the same protocol as for the H5N1 studies (Fig. 6c,d).

Figure 6: Cross-subtype neutralization by nAbs.

(a) nAbs D8, F10 and A66 all neutralized indicated pseudotyped viruses (strains described below). Error bars indicate s.d. (b) Microneutralization assay. Neutralization titers (0.1 mg ml-1 antibody stock solution) of nAb F10 against wild-type H5N1, H1N1, H2N2, H6N1, H6N2, H8N4, H9N2 and H3N2 virus strains. 80R is the negative control. Vertical bars and whiskers represent the lowest and the highest neutralization titer (2 , values of are shown on the y axis), respectively, of two or three independent experiments. (c,d) Prophylactic efficacy against two H1N1 strains in mice. Percentage of survival of mice treated with anti-H5 nAbs or control mAb are shown before lethal challenge by intranasal inoculation with H1-WSN33 (c) or H1-PR34 (d) viruses. Complete viral strain designations are: H1-OH83 (A/Ohio/83 (H1N1)); H1-PR34 (A/Puerto Rico/8/34 (H1N1)); H1-SC1918 (A/South Carolina/1/1918 (H1N1)); H1-WSN33 (A/WSN/1933 (H1N1)); H2-AA60 (A/Ann Arbor/6/60 (H2N2)); H2-JP57 (A/Japan/305/57(H2N2)); H3-SY97 (A/Sydney/5/97(H3N2)); H6-HK99 (A/quail/Hong Kong/1721-30/99(H6N1)); H6-NY98 (A/chicken/New York/14677-13/1998 (H6N2)); H7-FP34 (A/FPV/Rostock/34 (H7N1)); H8-ON68 (A/turkey/Ontario/6118/68); H9-HK(G9)97 (A/chicken/HongKong/G9/97 (H9N2)); H9-HK99 (A/HongKong/1073/99 (H9N2)); H11-MP74 (A/duck/Memphis/546/74 (H11N9)).

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Basis of the group-specific broad-spectrum virus neutralization

The ability of our nAbs to recognize all group 1 (cluster H1a/b and H9) viruses (H12 was not tested) can be attributed to the key conserved features of the nAbs described above in combination with the highly conserved pocket on hemagglutinin (Figs. 4 and 5). The epitope may be divided into three elements. (i) At its center, the sequence of the N-terminal segment of HA2—fusion peptide residues 182–212—is conserved across all hemagglutinin subtypes (note that the side chain at position 192 does not participate in binding). (ii) A downstream segment of HA2 adopts part of the A helix (residues 392–562), which is nearly invariant; the only significant difference is a threonine to glutamine change at position 492 in the untested H9 cluster subtype, H12. Thr492 lies at the periphery of the epitope and makes one long hydrogen bond (3.5 Å) to Ser31. Simple modeling suggests that there is plenty of space to accommodate the larger glutamine side chain and that it can make comparable hydrogen bonds. (iii) Smaller contributions from segments of the HA1 chain (residues 181 and 381) and a loop at the base of the head (residues 2911 and 2921).

Three-dimensional comparisons of the epitope in the five known crystal structure subtypes (three from group 1 (H1, H5 and H9) and two from group 2 (H3 and H7)21, 32, 34, 35, 36) show that they adopt two distinct structural classes consistent with the phylogenetic groupings32, 33 (Fig. 7). These differences arise from group-specific differences in the location of buried residues, notably histidines (H1112 is unique to group 1; H171 is unique to group 2) that have been proposed to be the 'triggers' for pH-induced conformational changes29. The differences cause the side chain of Trp212 to turn through 90° in group 2 subtypes, eliminating favorable binding to Phe55 from our nAb panel. In addition, four out of six group 2 subtypes are glycosylated at position 381, at the periphery of the F10 epitope; our modeling studies predict steric clashes with the CDR-H1 loop (data not shown). These structural differences rationalize the observed lack of binding and neutralization of group 2 hemagglutinin subtypes and viruses.

Figure 7: Three-dimensional comparison of the F10 epitope in group 1 and group 2 hemagglutinins.

Stereo overlay of crystal structures of the five known hemagglutinin subtypes in the region of the F10 epitope, showing conservation and differences between the two phylogenetic groups. H1, H5 and H9 (group 1) are in shades of red and yellow (PDB 1RU7, 2IBX and 1JSD); H3 and H7 (group 2) are in shades of blue (PDB 1MQL and 1TI8). R.m.s. differences for pairwise overlays are 0.56 0.11 Å (observed range, group 1), 0.75 Å (group 2) and 1.21 0.12 Å between groups. Consistent differences between phylogenetic groups include the orientation of Trp212 and alternative side chain directions at 181 and 381, which are linked to the packing of buried His1112 (the putative pH trigger in group 1; absent in group 2), and the burial of the larger tyrosine (group 1) versus histidine (the putative pH trigger in group 2) at 171. Of particular note, Asn381 is glycosylated in four members of the group 2 clusters. Other epitope residues are indicated by numbered light blue circles.

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Prospects for immune escape

The remarkable transformation to the fusogenic state includes repacking of the central helices of three HA2 protomers to form a new triple-helical bundle, in which residues 34–37 form an N-terminal cap, and the creation of C-terminal arms that extend to the N terminus of the new bundle37. It is straightforward to model the locations of the F10 epitope residues in this model of the fusogenic state (Supplementary Note 1 online). All eight epitope residues, which were fully exposed in the neutral pH structure, become either part of the new hydrophobic bundle core (Thr412, Ile452, Val522 and Ile562) or they make networks of hydrogen bonds with the C-terminal arms and other elements that stabilize the new bundle (Lys 382, Gln422, Thr492 and Asn532). The requirement for adopting two entirely different conformations, each with a distinct hydrophobic core and hydrogen-bonding network may place powerful evolutionary constraints on the sequence of the helix, as evidenced by the almost complete lack of genetic drift within helix A among the 16 hemagglutinin subtypes.

To test this hypothesis, we attempted to select neutralization-escape mutants. We propagated VN/04 (H5N1) virus in MDCK cells for 72 h in the presence of 40 g ml-1 of each of the 3 nAbs as well as a murine antibody, 22F, that targets the receptor binding head. Following three in vitro passages, we readily isolated a mutant VN04 virus (K193E) that was resistant to 22F. In contrast, we failed to identify any viruses resistant to any of our three IgG1s (D8, F10 or A66). Although these experiments cannot prove that escape mutants with unimpaired viral fitness will never arise, they clearly support the notion that the pocket is more refractory than epitopes in the head. Notwithstanding, if such mutants should arise, we can use our in vitro approach to find new reactive nAbs, or further engineer the existing nAbs to have even broader spectrum reactivity38.

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Discussion

Before the present study, the vast majority of nAbs isolated against influenza A virus have targeted the receptor binding head and lacked broad cross-neutralizing activity. However, a murine nAb, termed C179 (ref. 39), was positively selected on the basis of its cross-neutralization properties (of H1 and H2 subtypes) and was subsequently shown to neutralize H5, but not group 2, subtypes39, 40 (Supplementary Note 2 online). Moreover, C179 was shown to block membrane fusion rather than cell attachment and to protect mice against viral challenge41, although a detailed mechanism was not reported. We compared the activities of C179 and F10 and found that both showed similar binding toward H5. We also found that F10 efficiently competed with C179 for binding to H5, but not vice versa (Supplementary Fig. 9 online). Furthermore, the point mutant V522E abrogated binding to both antibodies, whereas T3181K affected only C179 binding. These results suggest that F10 and C179 have partially overlapping epitopes and that their modes of action are similar.

The manner in which hemagglutinin was presented to the antibody phage-display library in this study seems to have been crucial in our success, because similar attempts to isolate broadly nAbs using cell-surface expressed hemagglutinin showed only partial success against H5, and most antibodies recognized linear epitopes42. As noted above, we repeatedly isolated nAbs that use the same VH germline gene (IGHV1-69 or 'VH1-69'). One published study pointed out that this is the only VH gene that consistently encodes two hydrophobic residues at the tip of its CDR-H2 loop43; indeed, it is the only germline gene to encode a phenylalanine at this position, which makes several crucial interactions with H5. Moreover, the 'type 2' H2 loop, which is long and compact, is predicted to occur in only 4 out of the 50 human germline genes. These factors may explain at least in part the ability of nAbs derived from this germline gene to cross-react with viral epitopes through their unusual ability to bind to conserved hydrophobic pockets. Such pockets are likely to have an important function and for this reason they are often cryptic in the unactivated state of the antigen. For example, VH1-69 is the predominant gene used by a group of CD4-induced nAbs raised against the HIV-1 surface glycoprotein, gp120, where the pocket is part of a conserved co-receptor binding site that is exposed only transiently upon binding to its primary receptor, CD4 (ref. 43). Similarly, an antibody raised against the HIV gp41 trimeric 'inner-core' fusion protein intermediate uses the hydrophobic tip of its VH1-69 CDR-H2 loop to insert into a conserved hydrophobic pocket that blocks further assembly to the fusion-competent six-helix structure44. In vivo, B cells carrying the VH1-69 gene are the primary mediators of innate defense against HCV infection, generating antibodies against its membrane-fusion glycoprotein, E2 (ref. 45), although the epitope and mode of action have not been determined. Notably, as we found here, VH1-69 is not the only germline gene that is suitable for achieving neutralization in a similar manner. Another recent example is a nAb against the Ebola virus surface glycoprotein, KZ52, which uses the VH3-21 germline gene46. However, their common ability to lock viral envelope proteins into a nonfusogenic conformation suggests a general strategy for broad-spectrum and/or potent viral neutralization.

Recent work using immune-based phage-display libraries generated from B cell populations of patients who survived H5N1 infection resulted in the isolation of three human nAbs that neutralized both H1 and H5 viral strains. The authors postulated that the reason for survival was an effective humoral im

But you still aren't convincing in your case that the Grandjas Carroll farms are the source of this outbreak.    As Enviroperk pointed out, the facts still point to negative where the Grandjas farms are concerned.  Facts, give me facts.  Not might possibly could be's.  You can say that these farms might be the source of the hybrid H1N1 virus becasue pigs are there.  Well, you have all the parts necesary to be a prostitutue.  But that doesn't mean you are.  In both cases, something keeps the accusations from being true.

The Pew report is mostly opinionm not science.  The commission was very one sided.  Most of the few progressive agriculture representatives quit in disgust early on.  Conclusions were drawn and the report was written before much fo the research (which was mostly just review of existing literature) was even completed. I'll have to get back to you with references.  It is supper time right now.

Re-reading my post, especially after reading FormerAgTeachers comments does necessitate a further  comment. My comments were too abrupt to express my true  feelings about the matter.

Yes, I too  have been correctly challenged on far too many well-meaning opinions of cause and effect and who is to blame. I did take many to heart for a while.

Then I started reading a lot of back and forth theories among scientists about areas of crystalline structures. Not applicable here is the science (though I am not a scientist ), but applicable here is how that communication worked. It was fully respected as part of the process to voice a wild idea to hear what the storms of critique would come from your peers.

Highly valued was that critique and it was never taken personally. Much more highly valued among the group was the individual willing to express the wild idea. For they knew that only through one of the wild ideas was a breakthrough discovered.

Keep posting and I too appreciate the effort and the references. Take the criticism for what it is and learn from it. It is not personal.

Thanks!

Banning Poultry Litter was just so much Bush Co chatter to appease the people annoyed by the Mad Cows showing up here and there. As soon as they were replaced by some other issue it was put aside:

Published on Saturday, June 18, 2005 by the Associated Press
Critics: US Doing Too Little to Prevent 'Mad Cow'
by Libby Quaid
http://www.commondreams.org/headlines05/0618-02.htm

WASHINGTON -- American cattle are eating chicken litter, cattle blood and restaurant leftovers that could help transmit mad cow disease -- a gap in the U.S. defense that the Bush administration promised to close nearly 18 months ago.

"Once the cameras were turned off and the media coverage dissipated, then it's been business as usual, no real reform, just keep feeding slaughterhouse waste," said John Stauber, an activist and co-author of Mad Cow USA: Could the Nightmare Happen Here?

He contended, "The entire U.S. policy is designed to protect the livestock industry's access to slaughterhouse waste as cheap feed."

Loopholes in Ban on Cattle Remains in Feed

The Food and Drug Administration promised in January 2004 to close loopholes in a ban on putting cattle remains in cattle feed, but it has failed to act. The government calls the ban a "firewall" against the spread of mad cow disease. Eating the mad cow disease protein is the only way cows are known to get the disease.

The Food and Drug Administration promised to tighten feed rules shortly after the first case of mad cow disease was confirmed in the U.S., in a Washington state cow in December 2003.
"Today we are bolstering our BSE firewalls to protect the public," Mark McClellan, then-FDA commissioner, said on Jan. 26, 2004. The FDA said it would ban blood, poultry litter and restaurant plate waste from cattle feed and require feed mills to use separate equipment to make cattle feed.

However, last July, the FDA scrapped those restrictions. McClellan's replacement, Lester Crawford, said an international team of experts assembled by the Agriculture Department was calling for even stronger rules and that the FDA would produce new restrictions in line with those recommendations.

Today, the FDA still has not done what it promised to do. The agency declined interviews, saying in a statement only that there is no timeline for new restrictions.

"It's just a lot of talk," said Rep. Rosa DeLauro, D-Conn., a senior House Democrat on food and farm issues. "It's a lot of talk, a lot of press releases, and no action."

No reason to think that Mexico has done what the United States hasn't done either.

2.  Earth Justice = lawyers. 

3.  Lawyers = promoters of unbiased, science based information.

Which of the statements above is not true?

Best to get two confirming references and consider the movitves of all sources cited before relying on or propogating information. 

No, I don’t take these postings personal, although on most the news source blogs/comments I don’t usually do more than make a comment and if I have a reference item I’ll link to it.

Many of the pro-factory farm arguments I read remind me the folks in the 60s and 70s that argued against the relationship between cancer, breathing problems and cigarettes.

As for the Pew research folks, I put more confidence on their research, opinions than an unknown blogger, including myself.

As to the relationship between Grandjas farm and this outbreak I would only say the relative distance to the first breakout is more than just suspicious.

What I noticed in this Wall Street item was this paragraph:

“When news surfaced last week that a virus called swine flu was killing people in Mexico, C. Larry Pope, president and chief executive of Smithfield, dispatched two of his lieutenants, Chief Financial Officer Robert "Bo" Manly and Gregg Schmidt, president of international operations for the company's hog-production business, to Perote.”

http://online.wsj.com/article/SB124105320874371313.html

Note the CFO, money man, and the president of international operations were sent.  Having worked in Mexico over the last 15 years in the telecomm business one thing I noticed was the obvious way many folks there have their hands out ready for gratuities.  One would think if Smithfield was interested in a real determination on the effects of their hog farm they would have asked for perhaps WHO and Mexican, American health officials in for an inspection immediately.  The old nothing to hide thing.  Maybe they have and I have missed it.

From a strictly personal taste point of view my wife and I have started buying what meat we eat from two sources.  One is a local chain that sells good grass feed beef, beef that both of us compare the taste to beef we had as kids growing up in north Texas.  The second source we use is a near-local market, with their own small slaughter house for both local beef and local pork, in Munenster, TX.  Just has a much better, fresher taste, to us.

Thanks and cheers to all.

British press suffers from a case of swine fever hysteria

http://www.thefirstpost.co.uk/47411,news,british-press-gets-swine-flu-hysteria-over-mexico-factory-farming

Unsubstantiated and sensationalised coverage of pig flu and its possible origins at a Mexican farm has made the British media a laughing stock