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PostPosted: Sat Jul 20, 2013 6:52 am 
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Harbin Science paper describes H7N9 transmission in ferrets by respiratory droplets.

http://www.sciencemag.org/content/early ... ce.1240532

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PostPosted: Sat Jul 20, 2013 6:52 am 
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Published Online July 18 2013
< Science Express Index
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Science DOI: 10.1126/science.1240532 •Report


H7N9 Influenza Viruses Are Transmissible in Ferrets by Respiratory Droplet

Qianyi Zhang1,2,*,
Jianzhong Shi1,*,
Guohua Deng1,*,
Jing Guo1,*,
Xianying Zeng1,*,
Xijun He1,
Huihui Kong1,
Chunyang Gu1,2,
Xuyong Li1,
Jinxiong Liu1,
Guojun Wang1,2,
Yan Chen1,
Liling Liu1,
Libin Liang1,
Yuanyuan Li1,
Jun Fan1,
Jinliang Wang1,
Wenhui Li1,
Lizheng Guan1,
Qimeng Li1,2,
Huanliang Yang1,
Pucheng Chen1,
Li Jiang1,
Yuntao Guan1,
Xiaoguang Xin1,
Yongping Jiang1,
Guobin Tian1,
Xiurong Wang1,
Chuanling Qiao1,
Chengjun Li1,
Zhigao Bu1,
Hualan Chen1,2,†

+ Author Affiliations

1State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, People’s Republic of China.


2College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730030, People’s Republic of China.

↵†Corresponding author. E-mail: chenhualan@caas.cn

↵* These authors contributed equally to this work.

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PostPosted: Sat Jul 20, 2013 6:53 am 
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Abstract



A newly emerged H7N9 virus has caused 132 human infections with 37 deaths in China since 18 February 2013. Control measures in H7N9 virus–positive live poultry markets have reduced the number of infections; however, the character of the virus, including its pandemic potential, remains largely unknown. We systematically analyzed H7N9 viruses isolated from birds and humans. The viruses were genetically closely related and bound to human airway receptors; some also maintained the ability to bind to avian airway receptors. The viruses isolated from birds were nonpathogenic in chickens, ducks, and mice; however, the viruses isolated from humans caused up to 30% body weight loss in mice. Most importantly, one virus isolated from humans was highly transmissible in ferrets by respiratory droplets. Our findings indicate nothing to reduce the concern that these viruses can transmit between humans.

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PostPosted: Sat Jul 20, 2013 6:58 am 
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Originally published in Science Express on July 18 2013



H7N9 Influenza Viruses Are Transmissible in Ferrets by Respiratory Droplet

Qianyi Zhang, Jianzhong Shi, Guohua Deng, Jing Guo, Xianying Zeng, Xijun He, Huihui Kong, Chunyang Gu, Xuyong Li, Jinxiong Liu, Guojun Wang, Yan Chen, Liling Liu, Libin Liang, Yuanyuan Li, Jun Fan, Jinliang Wang, Wenhui Li, Lizheng Guan, Qimeng Li, Huanliang Yang, Pucheng Chen, Li Jiang, Yuntao Guan, Xiaoguang Xin, Yongping Jiang, Guobin Tian, Xiurong Wang, Chuanling Qiao, Chengjun Li, Zhigao Bu, Hualan Chen


Materials/Methods, Supplementary Text, Tables, Figures, and/or References
Download SupplementMaterials and Methods
Supplementary Text
Figs. S1 to S4
Tables S1 to S5
References


http://www.sciencemag.org/content/early ... /suppl/DC1

http://www.sciencemag.org/content/suppl ... ang.SM.pdf

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PostPosted: Sat Jul 20, 2013 7:10 am 
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niman wrote:
Originally published in Science Express on July 18 2013



H7N9 Influenza Viruses Are Transmissible in Ferrets by Respiratory Droplet

Qianyi Zhang, Jianzhong Shi, Guohua Deng, Jing Guo, Xianying Zeng, Xijun He, Huihui Kong, Chunyang Gu, Xuyong Li, Jinxiong Liu, Guojun Wang, Yan Chen, Liling Liu, Libin Liang, Yuanyuan Li, Jun Fan, Jinliang Wang, Wenhui Li, Lizheng Guan, Qimeng Li, Huanliang Yang, Pucheng Chen, Li Jiang, Yuntao Guan, Xiaoguang Xin, Yongping Jiang, Guobin Tian, Xiurong Wang, Chuanling Qiao, Chengjun Li, Zhigao Bu, Hualan Chen


Materials/Methods, Supplementary Text, Tables, Figures, and/or References
Download SupplementMaterials and Methods
Supplementary Text
Figs. S1 to S4
Tables S1 to S5
References


http://www.sciencemag.org/content/early ... /suppl/DC1

http://www.sciencemag.org/content/suppl ... ang.SM.pdf

Above supplement shows that EVERY avian PB2 sequence had K627E.

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PostPosted: Sat Jul 20, 2013 7:42 am 
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Solid-phase binding assay
The HA genes of the 37 viruses isolated from the birds and the environment of the live poultry markets are quite similar; 18 of them are identical at the amino acid level, and the others differ
5
by only one or two amino acids. The amino acid changes at positions 226 and 243 presented consistently in 6 of the viruses. We therefore selected four viruses isolated from birds (two from chickens and two from pigeons) for the receptor binding analysis: two viruses (chicken virus CK/S1053 and pigeon virus PG/S1421) from the 18 viruses that have identical HA genes, one that has an amino acid change at position 226 in HA (chicken virus CK/S1410), and one that has an amino acid change at position 243 in HA (pigeon virus PG/S1069). The direct receptor-binding capability of the viruses was examined by using a solid-phase binding assay as described previously (
17) with four different biotinylated glycans [two α-2,6 glycans (6'SLN: Neu5Acα2-6Galβ1-4GlcNAcβ-SpNH-LC-LC-Biotin; 6′S-Di-LN: Neu5Acα2-6[Galβ1-4GlcNAcβ1-3]2β-SpNH-LC-LC-Biotin) and two α-2,3 glycans (3′SLN: Neu5Acα2-3Galβ1-4GlcNAcβ-SpNH-LC-LC-Biotin; 3′S-Di-LN: Neu5Acα2-3[Galβ1-4GlcNAcβ1-3]2β-SpNH-LC-LC-Biotin)] that were kindly provided by the Consortium for Functional Glycomics (Scripps Research Institute, Department of Molecular Biology, La Jolla, CA). Each test was performed with three repeats. Briefly, a streptavidin-coated high-binding capacity 96-well plate (Pierce) was incubated at 4°C overnight with 100 μl of PBS containing different concentrations (starting from 2.4 μM) of biotinylated glycans. After the glycan solution was removed, the plates were washed four times with ice-cold PBS and then incubated at 4°C overnight with 100 μl of PBS containing 64 HA units of purified influenza virus. After being washed with ice-cold PBS containing 0.05% Tween-20 and PBS, the plate was incubated for 4 hours at 4°C with chicken antiserum against H7N9 virus CK/S1053. After four successive washes with ice-cold PBS containing 0.05% Tween-20, the plate was incubated with horseradish peroxidase (HRP)-conjugated goat-anti-chicken antibody (Sigma-Aldrich) for 2 hours at 4°C. After four washes with PBS containing 0.05% Tween-20, the plate was incubated with O-phenylenediamine (Invitrogen) in substrate
6
solution containing 0.01% H2O2 for 10 minutes at room temperature, and the reaction was stopped with 50 μl of 2M H2SO4. Absorbance was determined at 490 nm.

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PostPosted: Sat Jul 20, 2013 7:50 am 
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Chinese researchers who did extensive work on H7N9 viruses from birds and humans found that one of the human strains was highly transmissible by aerosol droplets in ferrets, fueling more concerns that the new virus could spread between people.

The potential for aerosol spread is one of the key factors health officials use in gauging a new virus's pandemic potential, and the new study follows closely on the heels of two others that also found evidence of respiratory droplet transmission in ferrets.

The newest findings were reported today in an early online edition of Science by a team based at the Harbin Veterinary Research Institute, a World Health Organization (WHO) collaborating center.

Genetic comparisons produce new clues

Scientists ran multiple tests on H7N9 viruses obtained during poultry surveillance and isolated from human cases to get a clearer picture of its pathogenicity, virulence, replication, and transmissibility.

They sequenced the genomes of 37 representative H7N9 samples, most of which came from live-poultry markets, and compared them with five human isolates. The hemagglutinin (HA) and neuraminidase (NA) genes were highly similar, but they found more diversity in the six internal genes. They also found that the viruses are still capable of frequent reassortment and rapid evolution.

When they examined the basic polymerase 2 (PB2) gene for amino acids associated with flu virulence and transmission in mammals, they found that all of the bird and environment samples had the amino acid combination 627E/701D. The human isolates, in contrast, had either the 627K or 701N mutation, both of which are important for virulence and transmission.

The group wrote that the findings suggest the mutations may have occurred during replication in humans.

Experiments to explore receptor binding, another factor that plays a role in flu virus replication and transmission, identified a 1243V mutation that—similar to the Q226L mutation—may play a key role in exclusive binding to humanlike receptors for two of the avian isolates and two of the human samples.

Tests track aerosol transmission, other factors

Virulence and infection tests in birds confirmed that the H7N9 virus was low pathogenic in poultry and that infected chickens shed the virus for up to 7 days.The researchers said this finding suggests that chickens "may be one of the major carriers and spreaders of H7N9 viruses in the live poultry markets."

In tests on mice that were given lethal doses of H7N9, no signs of disease or death were seen in the ones that received viruses isolated from birds, but the animals infected with human isolates lost weight, got very sick, or died. Similarly, the group's replication tests on mice infected with human strains found higher viral titers in the nasal passages and lungs when compared with animals infected by the bird strains.

Replication experiments in ferrets also showed differences between the bird and human H7N9 strains. The group's pathology tests on ferret lung samples found severe bronchopneumonia and prominent viral antigen expression in the animals infected with three human strains and one of the bird strains. Ferret lungs, though, appeared normal after infection with a poultry H7N9 strain.

Aerosol transmission studies involved placing uninfected ferrets in cages adjacent to those housing infected ferrets. The investigators found H7N9 in one ferret exposed to those infected with one of the bird strains and two human strains isolated from some of the first patients in Shanghai. However, the virus was detected in all three ferrets exposed to animals infected with a human H7N9 strain isolated form a patient in Anhui province (AH/1).

To assess reproducibility, they repeated the aerosol transmission test with the AH/1 isolate and got the same result.

Senior author Hualan Chen, PhD, told CIDRAP News that there was no significant difference in transmission among four of the five viruses they tested in aerosol transmission testing. "The transmission of AH/1 to all three ferrets suggests that the H7N9 virus has great pandemic potential," she said.

The team noted that it's difficult to pinpoint which amino acid substitution alone makes the virus highly transmissible, but the amino acid differences between the avian viruses and the Anhui virus range from 1 to 27, suggests that only a few changes are needed to make the virus highly transmissible in mammals.

"Moreover, these changes can occur easily during replication in humans," they added.

Overall, the team said their tests found that the H7N9 viruses from poultry and humans can bind to human airway receptors and can replicate efficiently in ferrets, and that one human isolate can transmit efficiently among ferrets by aerosol droplets.

Chen said she was surprised that all of the viruses tested are able to bind to humanlike receptors and that the PB2 gene of the virus so easily gains mutations during replication in humans that boost its virulence and transmissibility.

Experts weigh pandemic potential

She said the group's findings are useful for weighing the threat from the virus. "This study suggests that the H7N9 virus is likely to transmit in humans, and immediate action, not only in China, is needed to prevent a possible pandemic caused by such a virus," Chen said.

The ability of the virus to transmit easily in poultry across a large part of China over a brief period points to the importance of control measures in poultry markets, the group said. But stamping out H7N9 will be a big, long-term challenge, because the virus spreads silently in chickens and also spreads to humans.

Ian Mackay, PhD, a virologist at the Australian Infectious Diseases Research Centre at the University of Queensland, told CIDRAP News that the study's molecular epidemiology component is the largest of its kind to date and adds many more complete H7N9 genomes to the publicly accessible GenBank database. Mackay also authors the Virology Down Under Web site.

He noted that the group's comparison between the human and avian strains found that they differed by less than 4% at the nucleotide level, "Sometimes there are no differences," he said, noting that the most divergent strains came from the Shanghai region, where 20% of the samples originated.

The team's infection experiments on chickens confirm that H7N9 is a silent spreader and that the birds shed the virus for about a week before their illness resolves, Mackay said.

Findings revealed that major differences between H7N9 viruses are at the amino acid level, with the most divergent segment at the PB1 gene. "But it is the PB2 and HA segment that harbors mutations of particular interest to document the journey from infrequent spillover events to sustained human-to-human pandemic-level transmission," he said.

Mackay added: "We know that pandemic potential does not rest solely on one or other amino acid change, but rather a collection of changes. We also don't know what we don't know yet."

The Harbin group showed the potential of H7N9 viruses to bind to both avian and human receptors and that human isolates replicated well in the upper airways, a site for efficient transmission, he noted.

"The study reinforces that even 'lowly' or inefficient transmission—only 33% of ferrets, for example—is still transmission," Mackay said. "That proportion would lead to a lot of human cases in densely populated or frequented areas."

Those factors might help explain the wide clinical spectrum that has been seen, as well as difficulties in tracking the source and the proportion of patients who get severely ill and die, he said.

Zhang Q, Shi J, Deng G, et al. H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science 2013 Jun 18 [Abstract]

See also:

Jul 10 CIDRAP News story "New studies on H7N9 raise pandemic concerns"

Virology Down Under Web site

http://www.cidrap.umn.edu/news-perspect ... orne-route

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PostPosted: Sat Jul 20, 2013 12:42 pm 
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Map updated

https://maps.google.com/maps/ms?ie=UTF8 ... =6&vpsrc=6

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PostPosted: Sun Jul 21, 2013 11:06 am 
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do they say which virus is transmissible in ferrets and why
(what mutations) ?


> one virus isolated from humans was highly transmissible in ferrets
> by respiratory droplets


--------edit-------------------------------

CIDRAP:
" Aerosol transmission studies involved placing uninfected ferrets in cages adjacent to those housing infected ferrets. The investigators found H7N9 in one ferret exposed to those infected with one of the bird strains and two human strains isolated from some of the first patients in Shanghai. However, the virus was detected in all three ferrets exposed to animals infected with a human H7N9 strain isolated form a patient in Anhui province (AH/1). "

that one is almost identical to the index
so apparently the ability of ferret transmission decreased during
subsequent evolution

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PostPosted: Sun Jul 21, 2013 12:38 pm 
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gsgs wrote:
do they say which virus is transmissible in ferrets and why
(what mutations) ?


> one virus isolated from humans was highly transmissible in ferrets
> by respiratory droplets


--------edit-------------------------------

CIDRAP:
" Aerosol transmission studies involved placing uninfected ferrets in cages adjacent to those housing infected ferrets. The investigators found H7N9 in one ferret exposed to those infected with one of the bird strains and two human strains isolated from some of the first patients in Shanghai. However, the virus was detected in all three ferrets exposed to animals infected with a human H7N9 strain isolated form a patient in Anhui province (AH/1). "

that one is almost identical to the index
so apparently the ability of ferret transmission decreased during
subsequent evolution

Please. READ the paper (you can access the SUPPLEMENT without a subscription).

http://www.sciencemag.org/content/suppl ... ang.SM.pdf

ALL of the polymorphisms are listed for all 8 gene segments for all 37 avian sequences as well as 5 human. Anhui/1 transmitted to ALL THREE ferrets - NO PASSAGE required.

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