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Win-Shift and Win-Stay Protocol on the Radial Arm Maze
By John Agnew February 28, 2018 July 29th, 2019 No Comments
Home » Win-Shift and Win-Stay Protocol on the Radial Arm Maze
The Radial Arm Maze is an experimental protocol that is used to test working memory [1] as well as reference memory or spatial memory [2] in rats and mice. It has been used in studies of aging, dementia, and pharmacology [3][4]. Working memory is a cognitive process in which an individual uses and manipulates information. Reference memory refers to information that is constant over time, which is related to spatial memory. Spatial memory refers to information about how objects are arranged in space. All three of these processes rely on the hippocampus [5-7].
Physiological Basis of Working and Spatial Memory
The hippocampus is part of the medial temporal lobe of the brain and has been associated with many aspects of memory. The hippocampus proper includes three major regions, known as CA1, CA2, and CA3 [8]. The larger hippocampal formation includes the hippocampus proper and the adjacent dentate gyrus, subicular complex, and entorhinal cortex [8]. Connections between the entorhinal cortex and the hippocampus proper allow sensory information to enter the hippocampus. A large variety of pathways within the hippocampus processes the sensory information and generate output from the hippocampus back to the cortex, which is processed through the subicular cortex [9]. Research in human subjects has suggested that different parts of the hippocampal formation are specialized for spatial memory. Functional imaging studies in taxi drivers suggested that the posterior hippocampus plays a role in spatial memory [10][11]. A review of different studies of hippocampal function has also suggested that the right hippocampus plays a role in spatial memory while the left hippocampus is more involved in autobiographical memory [12].
The Radial Arm Maze
The Radial Arm Maze protocol assesses hippocampal function, both by using spatial cues to remember which arms have been visited previously and by using working memory to process and manipulate information. There are several variants on this procedure, but this post will focus on the win-shift and the win-stay procedures.
Ethanol and towels or wipes to clean the maze after each use.
Experimental animals, typically rats or mice.
a. Animals are housed under normal conditions, but should be food-deprived to about 80% of normal caloric intake prior to the start of the experiment.
b. The animals are typically separated into at least two groups, an experimental group and a control group.
Standard food for the experimental animals.
Radial Arm Maze apparatus.
a. The standard version has eight arms radiating off a central chamber.
b. The length of the arms and size of the central chamber varies depending on whether the experimental subjects are rats or mice.
c. At the end of each arm proximal to the central chamber is a door controlling access to the arm.
d. At the distal end of each arm is a well in which food pellets will be placed.
Visual cues to place outside of the Radial Arm Maze to enable the subjects to navigate the environment based on the external cues.
The use of a video camera is recommended to record the movements of the subjects for later analysis.
Experimental Procedure
Familiarization with the Maze
Over the course of two days, the animals are placed into a central chamber of the maze.
Multiple food pellets are placed along each arm to encourage the animal to explore the arm thoroughly and food is placed in the food well at the end of each arm.
The animal is allowed to explore the maze for 10 minutes.
Once an arm has been explored, it is not re-baited to encourage the animal to explore the other arms of the maze.
The animal is removed from the maze after 10 minutes and returned to the home cage.
The apparatus is thoroughly cleaned with ethanol and the protocol is repeated with another animal.
Typically, animals participate in two familiarization sessions per day, separated by four hours. These sessions occur on two sequential days to completely familiarize the animal with the apparatus and the procedure.
Win-Shift Procedure
Training Phase
One day after the Familiarization procedure, the animals are placed back into the central chamber of the maze and encouraged to explore for five minutes [1].
Salient visual cues outside the maze allow the animals to establish a mental map of the maze.
Only four of the arms are baited with a food reward at the end of the arm and the other four arms are closed off by the doors and unavailable for exploration.
The blocked arms are chosen in a pseudo-random fashion, such that no more than two adjacent arms are blocked at a time.
After the five minutes, the animals are removed from the maze for a period of time (the Inter-Trial Interval; ITI), typically about five minutes.
While the rodents are out of the maze, the maze is cleaned with ethanol. The blocks are removed and the arms that were previously unbaited and blocked are now baited and opened/unblocked. The arms that were previously baited no longer contain a reward.
Experimental Phase
After the ITI, the animals are returned to the central compartment of the maze and allowed to explore all eight arms for five minutes. The entire experimental phase can be recorded for later scoring or scored in real time.
After each animal, the entire apparatus is again washed with ethanol.
The experiment is run twice per day per animal, with a break of about five hours between sessions, until the experimental criteria have been met. One standard criterion is that the control group does not make more than one error per session [1].
Common Difficulties
The animals will sometimes fail to explore the maze entirely during the Training phase. This is addressed by providing a longer Training time for all experimental subjects or increasing the food reward for exploring the maze.
Animals are sometimes observed to jump from one arm of the maze to another. To discourage this behavior, the animal should be picked up and returned to the central chamber, if such jumping occurs.
Data Analysis and Scoring
The rodent is considered to have entered the arm if all four limbs leave the central compartment.
A correct response in this experiment would be the animal visiting the arms that were initially not rewarded.
A Working Memory Error occurs when the animal enters an arm of the maze that it previously entered in the same trial.
A Spatial Memory or Reference Memory Error occurs when the animal enters an arm of the maze that was baited during the training phase.
Data from Training and Experimental Phases are analyzed separately, as are data from Working Memory Errors and Reference Memory Errors.
In the standard analysis, the errors made in four sequential trials (spread over two days) are averaged to form a data point. Data from all animals and all data points are then analyzed using an ANOVA.
Win-Stay Procedure
While the rodents are out of the maze, the doors are opened and the arms that were previously baited are baited again. The arms that were previously unbaited remain unrewarded [13].
Correct responses in this experiment would be the animal visiting the arms that were initially rewarded.
A Spatial Memory or Reference Memory Error occurs when the animal enters an arm of the maze that was unbaited during the training phase.
Applications of the Radial Arm Maze
This test has been applied to study cognitive functions under many different conditions. Drugs that affect the hippocampus and memory can be administered to affect performance (Experimental Group). Similarly, the length of the ITI can be altered to make working memory more or less effective [14]. A version of this win-shift protocol has been used in a rat model of depression, to assess how learned helplessness can adversely impact cognitive functions such as memory and attention [15]. A version of this test has even been adapted for human participants in a virtual-reality environment to assess human spatial learning [16].
The Radial Arm Maze is a traditional test of spatial and working memory, two functions that depend on the complex circuitry of the hippocampus and the interaction of this structure with the rest of the cerebral cortex. The win-shift and win-stay protocols enable researchers to manipulate the complexity of the memory task and apply various experimental manipulations that will affect learning, memory, and attention. It provides them with a means to study and understand memory and learning in rats and mice and newer, virtual version of this protocol have been applied to human subjects as well.
DeLuca, S.N., L. Sominsky, and S.J. Spencer, Delayed Spatial Win-Shift Test on Radial Arm Maze. Bio-Protocol, 2016. 6(23): p. e2053.
Schmidt, A.T., et al., Dissociating the long-term effects of fetal/neonatal iron deficiency on three types of learning in the rat. Behavioral Neuroscience, 2007. 121(3): p. 475-82.
Ward, M.T., C.R. Stoelzel, and E.J. Markus, Hippocampal dysfunction during aging II: deficits on the radial-arm maze. Neurobiol Aging, 1999. 20(4): p. 373-80.
Yuede, C.M., H. Dong, and J.G. Csernansky, Anti-dementia drugs and hippocampal-dependent memory in rodents. Behavioural pharmacology, 2007. 18(5-6): p. 347-363.
Broadbent, N.J., L.R. Squire, and R.E. Clark, Spatial memory, recognition memory, and the hippocampus. Proceeding of the National Academy of Sciences, USA, 2004. 101(40): p. 14515-20.
Leszczynski, M., How does hippocampus contribute to working memory processing? Frontiers of Human Neuroscience, 2011. 5: p. 168.
Ramos, J.M., Remote spatial memory and the hippocampus: effect of early and extensive training in the radial maze. Learning and Memory, 2009. 16(9): p. 554-63.
Schultz, C. and M. Engelhardt, Anatomy of the hippocampal formation. Frontiers of neurology and neuroscience, 2014. 34: p. 6-17.
Cappaert, N.L.M., N.M. Van Strien, and M.P. Witter, Chapter 20 – Hippocampal Formation, in The Rat Nervous System (Fourth Edition). 2015, Academic Press: San Diego. p. 511-573.
Maguire, E.A., et al., Navigation-related structural change in the hippocampi of taxi drivers. Proceeding of the National Academy of Sciences, USA, 2000. 97(8): p. 4398-403.
Maguire, E.A., K. Woollett, and H.J. Spiers, London taxi drivers and bus drivers: a structural MRI and neuropsychological analysis. Hippocampus, 2006. 16(12): p. 1091-101.
Burgess, N., E.A. Maguire, and J. O'Keefe, The Human Hippocampus and Spatial and Episodic Memory. Neuron, 2002. 35(4): p. 625-641.
UCLA Behavioral Testng Core. Radial Arms Maze. No Date [cited 2017; Available from: https://btc.psych.ucla.edu/behavioral-testing-core-facilitys-available-tasks/mouse-and-rat-services/radial-arms-maze/.
Stafstrom, C.E., CHAPTER 49 – Behavioral and Cognitive Testing Procedures in Animal Models of Epilepsy, in Models of Seizures and Epilepsy, P.A. Schwartzkroin and S.L. Moshé, Editors. 2006, Academic Press: Burlington. p. 613-628.
Richter, S.H., et al., Where Have I Been? Where Should I Go? Spatial Working Memory on a Radial Arm Maze in a Rat Model of Depression. PLOS ONE, 2013. 8(4): p. e62458.
Shelton, A.L., S.A. Marchette, and A.J. Furman, Chapter Six – A Mechanistic Approach to Individual Differences in Spatial Learning, Memory, and Navigation, in Psychology of Learning and Motivation, B.H. Ross, Editor. 2013, Academic Press. p. 223-259.
John Agnew is a cognitive neuroscientist living in Colorado. He has a Ph.D. in Neuroscience from Georgetown University and a B.A. in Chemistry and Biochemistry from Haverford College. For the past 15 years, he has taught biology, psychology, and neuroscience courses at the community college and university level and is currently a Contributing Faculty Member in the School of Psychology at Walden University and Adjunct Faculty Member in the Biology Department at Front Range Community College. John is also involved in neuroscience research, attempting to understand some of the behavioral aspects of autism spectrum disorders and evaluating the effectiveness of interventions for individuals with autism. As a freelance editor, John has worked with textbook publishers to update and develop new content for their psychology and neuroscience texts, including James Kalat's Biological Psychology, Michael Gazzaniga's Cognitive Neuroscience, Bob Garrett's Brain & Behavior, and for David Eagleman's Cognitive Neuroscience texts.
About John Agnew
Effect of Fish Oil on Mouse Behavior
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Allow me to take you back in time a little bit, to a young kid wandering the streets of a neighborhood that you'd probably roll up your windows and lock your doors as you drove through. This was the area I called home, and though there's not that many good memories of that place, it's where I came up, where I learned the lessons that shaped me today, and where I made most of the worst decisions in my life. You see, in this area, we didn't spend our time thinking of where we were going to go to college, or what we were going to do as careers, it was a constant thought of how we were going to get through the day, and how we were going to take care of ourselves in the moment.
School was a place for everyone to gather, group up and try to intimidate the other groups who were doing the same to us. What block you lived on typically represented how many people were in your crew, and those on a block with more people their relative age had the fortune of having bigger crews. There was a spot two streets over from mine where there was seven kids on the strip, my street had four. So you either grouped with another street of kids, or you stuck to low numbers and tried to be harder than everyone else.
Fights on the school yard weren't over trivial things like someone said something, it was usually retaliatory for someone in your group getting jumped by another group. We didn't just circle each other taunting and then throwing a punch or two, let's just say that the ambulances knew pretty direct routed to our school yard. Across the halls you would see group after group with little mingling in between, and you formed a close bond with those within your group, even if you didn't like them that much. They were part of the set you represented, and therefore were people you needed to worry about, if you wanted them to return the favor.
Being stuck on a street with a bunch of scrawny and timid kids was usually the worst situation to be in, the others wouldn't represent you, they would hide out in the gym or cafeteria at all times, and try to stay out of everyone's way. These stragglers of people left on their own would usually jump to joining a bigger street, yet this wasn't always met warmly. Some kids needed to be jumped into their groups, others had to jump someone else to gain cred. The teachers and other members of the school knew about all this of course, but they also knew that trying to get into the middle of it would just turn everyone's negative attention towards them, and that was something above their pay grade, can't say I blame them, I would have given up on us to. Just try to educate, and then get the hell out when the bell rang.


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What we know about 'Gabby' Petito's fiance Brian Laundrie
Sarasota Herald-Tribune
A North Port woman has been missing since Saturday, and agencies from several states and the FBI are searching for Gabrielle 'Gabby' Petito.
Petito set out on a cross-country trip with her fiancé, Brian Laundrie, 23, who travelled back to North Port in their van 10 days before her family reported her missing and who refuses to cooperate with law enforcement, according to authorities.
North Port Police Department officials said during a news conference on Wednesday that Laundrie is now a person of interest in the case.
What we know about Petito:What we know about missing Florida woman Gabby Petito
Background:Gabby Petito's fiancé now a person of interest in her disappearance, Florida officials say
More:Missing North Port woman Gabby Petito's vehicle recovered, woman still not found
Here is what we know about Laundrie:
Who is 'Gabby' Petito's fiancé Brian Laundrie?
Laundrie, 23, is a YouTuber from North Port, who has documented his travels with his fiance, Gabrielle 'Gabby' Petito, whose disappearance has captured global attention. Petito documented her cross-country trips on her YouTube account called 'Nomadic Statik.' The last video was posted three weeks ago and includes clips of Laundrie.
Authorities have identified him as a "person of interest" in the case.
Why is Brian Laundrie a person of interest?
Petito was reported missing to the Suffolk County Police Department by her family in New York just before 7 p.m. Saturday.
According to her family, they were last in contact with her during the final week of August. Prior to that, they believed she was in Grand Teton National Park in Wyoming -- possibly with Laundrie.
The van was recovered at Petito's residence in North Port. Laundrie was at the home, but he refused to speak with authorities, according to a recent story by the Sarasota Herald-Tribune.
Police in Utah released new bodycam footage this week showing an emotional Petito during an apparent altercation with Laundrie weeks before her disappearance.
What car were Gabrielle Petitot and Brian Laundrie traveling in?
Petito and Laundrie were traveling in a white 2012 Ford Transit van with Florida license plate QFTG03.
Law enforcement believes Laundrie drove Petito's van from an unknown location back to North Port on Sept. 1 — 10 days before her family reported her missing.
What has the Laundrie family said?
The family has said nothing regarding Petito's whereabouts and only communicated through the attorney.
Steve Bertolino, Laundrie's attorney, released the following statement Tuesday:
"This is understandably an extremely difficult time for both the Petito family and the Laundrie family. It is our understanding that a search has been organized for Miss Petito in or near Grand Teton National Park in Wyoming. On behalf of the Laundrie family, it is our hope that the search for Miss Petito is successful and that Miss Petito is reunited with her family. On the advice of counsel, the Laundrie family is remaining in the background at this juncture and will have no further comment."
What does Gabrielle Petito look like?
Petito is described as a white female, approximately five feet, 5 inches tall, and 110 pounds. She has blonde hair, blue eyes, and several tattoos, including one on her finger and one on her forearm that reads "Let it be."
Do you have information about the case?
The FBI has set up a national hotline to receive tips at 1-800-CALLFBI (225-5324). It is the primary line for all information on Petito. They are asking for anyone with information to reach out.

redpajama_set_name: RedPajamaCommonCrawl


We had the honor of leading the parade on Remembrance Sunday and what a parade it was! It was lovely to see so many people on parade and remembering the fallen followed by the Church Service in St Mary's Church.
Thank you to Steve who played the Last Post outside and in the Church.
Denstone Christmas lights switch on!
We played our first Christmas pieces on Sunday 18th November for the annual Denstone Christmas Light Switch on! What a night it was. The main theme of the night was the Greatest Showman to which the fireworks and lighting was choreographed too.
We played as the audience lined up to see Santa whilst enjoying the festive refreshments.
A brilliant evening with lots of more Christmas tunes to come!!
We attended the WW1 Commemoration Closing Ceremony and Dedication of the New Memorial in the Coltman V C Peace Wood in Winshill on Saturday 17th November. It was a very moving ceremony where we provided hymns and the last post.


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Check out our products mastered by some of the best tech engineers in the world with all the most distinctive features! Choose from the wide range of design including top technology of Bi-Symmetrical and Tri-Symmetrical Slot Design developed to increase number of out-gassing exit points for brake pad friction gasses to escape through.
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Come and check out our new materials including New European Application guide on stand #7630!


text:
Human Infection with Highly Pathogenic Avian Influenza A(H7N9) Virus, China - Volume 23, Number 8—August 2017 - Emerging Infectious Disease journal - CDC
Volume 23, Number 8—August 2017
Human Infection with Highly Pathogenic Avian Influenza A(H7N9) Virus, China
Technical Appendices
PDF[2.04 MB - 9 pgs]
RIS[TXT - 2 KB]
Changwen Ke1, Chris Ka Pun Mok1, Wenfei Zhu1, Haibo Zhou1, Jianfeng He, Wenda Guan, Jie Wu, Wenjun Song, Dayan Wang, Jiexiong Liu, Qinhan Lin, Daniel Ka Wing Chu, Lei Yang, Nanshan Zhong, Zifeng Yang2 , Yuelong Shu2 , and Joseph Sriyal Malik Peiris2
Author affiliations: Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China (C. Ke, J. He, J. Wu); First Affiliated Hospital of Guangzhou Medical University, State Key Laboratory of Respiratory Disease, Guangzhou (C.K.P. Mok, W. Guan, D.K.W. Chu, N. Zhong, Z. Yang, J.S.M. Peiris); The University of Hong Kong, Hong Kong, China (C.K.P. Mok, J.S.M. Peiris); National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China (W. Zhu, D. Wang, L. Yang, Y. Shu); The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China (H. Zhou, J. Liu, Q. Lin); Jinan University, Guangzhou (W. Song); Macau University of Science and Technology, Macau, China (N. Zhong, Z. Yang)
The recent increase in zoonotic avian influenza A(H7N9) disease in China is a cause of public health concern. Most of the A(H7N9) viruses previously reported have been of low pathogenicity. We report the fatal case of a patient in China who was infected with an A(H7N9) virus having a polybasic amino acid sequence at its hemagglutinin cleavage site (PEVPKRKRTAR/GL), a sequence suggestive of high pathogenicity in birds. Its neuraminidase also had R292K, an amino acid change known to be associated with neuraminidase inhibitor resistance. Both of these molecular features might have contributed to the patient's adverse clinical outcome. The patient had a history of exposure to sick and dying poultry, and his close contacts had no evidence of A(H7N9) disease, suggesting human-to-human transmission did not occur. Enhanced surveillance is needed to determine whether this highly pathogenic avian influenza A(H7N9) virus will continue to spread.
Avian influenza A(H7N9) viruses with zoonotic potential emerged in East China in early 2013. From February 2013 through February 20, 2017, a total of 1,222 patients with A(H7N9) disease had been reported to the World Health Organization, with 304 of these patients being reported January 19–February 14, 2017 (1,2). The overall case fatality ratio was ≈37%. Most patients with A(H7N9) disease primarily had viral pneumonia, with some progressing to acute respiratory distress syndrome (ARDS). Unlike patients with influenza A(H5N1) disease, patients with A(H7N9) disease were more likely to be older and have underlying comorbidities (3). In healthy young persons with A(H7N9) infections, symptoms were often mild, and potentially a substantial proportion of these infections was asymptomatic and not recognized (4–6).
The A(H7N9) virus is a low pathogenicity avian influenza (LPAI) virus that typically causes no signs of disease in birds and has become enzootic in poultry. A(H7N9) infections have been reported in poultry and humans from 22 provinces and municipalities in mainland China, with cases acquired in mainland China sometimes being reported in Hong Kong, China; Macao, China; Taiwan; Malaysia; and Canada (2).
Phylogenetic analyses of the A(H7N9) hemagglutinin (HA) genes showed that the viruses that emerged in the Yangtze River Delta region in 2013 rapidly spread to other parts of South and South East China and that distinct virus clades became established in these different geographic regions (7). Genetic analysis of A(H7N9) viruses has revealed some adaptations in the virus HA that allow binding to the sialic acid receptors on the epithelium of the mammalian upper respiratory tract. Upon infection in humans, these viruses rapidly acquire mutations in the viral polymerase basic 2 (PB2) gene (8). Unlike A(H5N1) viruses, A(H7N9) viruses efficiently infect and replicate in ex vivo cultures of the human bronchus (9) and are transmitted between ferrets, albeit inefficiently, by the airborne route (10), indicating the substantial potential for efficient human-to-human transmission. Although there have been occasional clusters of cases, some likely caused by limited human-to-human transmission, no evidence of sustained human-to-human transmission has been reported (2).
When transmitting among domestic, terrestrial poultry, LPAI type A viruses of the H5 and H7 subtypes can undergo mutations that lead to multiple basic amino acids being present at the HA cleavage site, a signature associated with increased pathogenicity in chickens. Such viruses can disseminate beyond the respiratory and intestinal tracts to affect the brain, liver, spleen, and pancreas and are lethal in chicken; these viruses are then designated highly pathogenic avian influenza (HPAI) (11,12). Since their emergence in 2013, the A(H7N9) viruses have remained LPAI viruses in poultry, which has made detection and containment of these viruses more challenging than these activities were for A(H5N1), an HPAI virus that usually caused severe disease in poultry flocks.
We report the investigation of illness in a patient in China infected with an A(H7N9) virus with a novel polybasic amino acid sequence at its HA cleavage site, a change that corresponds with increased pathogenicity in poultry and potentially (although not invariably) increased pathogenicity in humans. Relevant epidemiologic information, such as history of contact with poultry and evidence of transmission of infection to close contacts, is provided.
The patient was a 56-year-old man with diabetes and hypertension who lived in Lianzhou, China, in Guangdong Province. He raised chickens in his backyard and noticed some of them were sick and dying weeks before his illness. Some of these chickens were slaughtered, cooked, and consumed by the patient and his family members.
Figure 1. Clinical course of 56-year-old man with diabetes and hypertension infected with highly pathogenic avian influenza A(H7N9) virus, China, 2017. CT, computed tomography; ECMO, extracorporeal membrane oxygenation; ICU, intensive care unit; MDR,...
On January 7, 2017, day 1 of illness, he had a fever (39.8°C) and cough; shortly thereafter, shortness of breath developed, and on January 10, day 4 of illness, he was admitted to a local hospital (Figure 1). Bilateral pneumonia was diagnosed based on his clinical signs and computed tomography (CT) results. The patient's throat swab from a point-of-care test was negative for influenza A/B. Because he had a history of contact with poultry, oseltamivir treatment (75 mg 2×/d) was commenced. On day 6, his clinical condition deteriorated markedly, with progression to severe respiratory failure, hypoxia (oxygen saturation 78.6%), and coma. He was transferred to the intensive care unit for invasive mechanical ventilation.
Figure 2. Chest and brain imaging of 56-year-old man infected with highly pathogenic avian influenza A(H7N9) virus, China, 2017: radiograph imaging of chest at day 7 (A) and day 40 (D); computed tomographic...
RNA extracted from an endotracheal aspirate acquired on day 6 was positive for influenza A(H7N9) virus. On day 7, he had mild gastrointestinal bleeding and a cardiac arrest. No endoscopy was performed, and he was rescued by cardiopulmonary resuscitation and intravenous injection of norepinephrine. The size and shape of his heart were within reference range. Chest radiography showed he had extensive bilateral infiltration of the lungs with prominent hilar shadowing (Figure 2, panel A). The patient had progressed to severe ARDS. Antiviral therapy was changed from oseltamivir to peramivir (0.6 g intravenous 4×/d), and he was given antibiotics (cefoperazone sodium/sulbactam sodium [2:1], 3 g/8 h). Extracorporeal membrane oxygenation (ECMO) was commenced.
Figure 3. Kinetics of viral load, oxygenation index, and HI antibody titers in 56-year-old man infected with highly pathogenic avian influenza A(H7N9) virus, China, 2017. Arrows indicate the days HI titers and viral...
Laboratory tests showed high total leukocyte and neutrophil counts and high levels of procalcitonin, aspartate aminotransferase, serum creatinine, lactate dehydrogenase, blood urea nitrogen, and d-dimers throughout the hospitalization (Technical Appendix[PDF - 605 KB - 7 pages] Table 1). His hemoglobin level remained low. Continuous renal replacement therapy was applied because of increasing renal dysfunction (days 8–14). His oxygenation index was maintained at around 200 mm Hg by the use of ECMO (Figure 3). On day 10, treatment with peramivir was stopped and oseltamivir recommenced. Viral load in the endotracheal aspirate started to decline on day 16 and was undetectable on day 20 (Figure 3). However, his clinical condition continued to deteriorate gradually after removal from ECMO. Secondary bacterial infections developed with multidrug-resistant Pseudomonas aeruginosa (detected in sputum days 9 and 19) and Acinetobacter baumannii (detected in blood cultures day 25). Combination antibiotic therapy was used to treat these infections (Technical Appendix[PDF - 605 KB - 7 pages] Table 2).
Cytomegalovirus (CMV) DNA was detected in patient serum on day 11 of illness, suggesting CMV reactivation. Because of the lack of clinical response to oseltamivir and antibiotics and the detection of CMV DNA in serum, ganciclovir therapy was initiated on day 32 to suppress possible systemic CMV disease. A CT scan of the lungs on day 30 showed multiple bilateral patchy hyperdense lesions and bilateral pleural effusions (Figure 2, panel B). Repeated electrocardiography tracings did not reveal evidence of myocarditis, and Doppler echocardiography examinations showed effective myocardial contraction. CT scan of the brain on the same day showed patchy hypodense lesions with unclear borders in the left corona radiate and right centrum semiovale (Figure 2, panel C), the largest measuring 8 × 10 mm with a CT value (8 H) indicative of lacunar infarction. The CT scan also showed symmetric, bilateral cerebral hemispheres; preservation of gray-white differentiation; no obvious lesions in the brain parenchyma; no deepening or widening of sulci, fissures, and cisterns; no dilatation or deformity in the ventricular system; and no mid-line shift. However, intracerebral edema was evident (Figure 2, panel C).
On day 40, extensive bilateral shadowing of the lung fields with blurring of the costophrenic angles continued to be observed on chest radiography (Figure 2, panel D). On day 43, the patient died.
Clinical and Epidemiologic Data Collection
The clinical history and epidemiologic information were obtained from the patient at hospital admission and from relatives during interviews. Progression of clinical symptoms and laboratory and radiologic findings were obtained by retrospective chart review.
Viral Diagnosis
Serially acquired throat swabs, endotracheal aspirates, serum samples, and fecal samples were collected from the patient and stored in viral transport medium. We extracted viral RNA by using QIAamp MinElute Virus Spin kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The extracted RNA was subjected to reverse transcription and amplification with SuperScript III One-Step RT-PCR System (ThermoFisher, Waltham, MA, USA). We synthesized complementary DNA by using uni-12 primers (5′-AGCAAAAGCAGG-3′), and conducted real-time PCR with an A(H7N9) detection kit (Shanghai Zj Bio-Tech Co., Ltd, Shanghai, China) to detect avian influenza A(H7N9) virus.
Genome Sequencing and Phylogenetic Analysis
Staff of the Guangdong Provincial Center for Disease Control and Prevention isolated virus from the endotracheal aspirate specimen obtained on day 6 of illness onset. Whole-genome sequencing was implemented on the Ion Torrent S5 platform (ThermoFisher) with a mean read length of ≈200 bp. We analyzed data predominantly with CLC Genomics Workbench 7.5.1 software (https://www.qiagenbioinformatics.com/products/clc-genomics-workbench/). Low-quality reads were trimmed by using CLC trimmer with a quality limit set at 0.05. We assembled filtered reads de novo in CLC under default parameters. Contigs with coverage >10 bp were extracted and blasted against the GISAID (Global Initiative on Sharing All Influenza Data) databases (http://platform.gisaid.org). Sequences with the highest similarity were selected as references for read mapping (parameters: length fraction = 0.8, similarity fraction = 0.8). We obtained influenza A genome sequences by extracting consensus sequences from the mapping results with a coverage depth of >30× at each nucleotide site of the 8 gene segments. The viral sequences generated in this study (Technical Appendix[PDF - 605 KB - 7 pages] Table 3) were submitted to the GISAID database.
We used the MEGA software version 5.05 (http://www.megasoftware.net) to construct phylogenetic trees, and maximum likelihood trees were constructed with PhyML (http://www.atgc-montpellier.fr/phyml/) by using the general time reversible plus gamma distribution plus proportion of invariable sites model. We estimated node support by the SH-like aLRT method and report values >0.8. Bootstrap values from 1,000 replicates were calculated to assess the reliability of the phylogenetic trees.
A(H7N9)-specific antibody titers were quantified in serially acquired serum samples by hemagglutination inhibition (HI) assay by using horse erythrocytes according to the World Health Organization recommended protocol (http://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_hai_a_h7n9_20131220.pdf). A recombinant A/PR/8/34 virus with the HA and neuraminidase (NA) genes of A/Zhejiang/DTID-ZJU01/2013 (H7N9) was used for serologic tests. All bioassays were conducted in a Biosafety Level 3 laboratory. Seropositivity was defined as an HI titer >1:40.
Guangdong Center for Disease Control and Prevention is legally tasked with data collection on patients in the course of a public health investigation during an emerging infectious disease outbreak. Thus, informed consent was waived.
This patient initially tested negative for influenza A/B, but on day 6, real-time reverse transcription PCR performed using an endotracheal aspirate showed positive results for A(H7N9) virus RNA. Viral RNA was detectable in the patient's endotracheal aspirate at high levels (>4 log RNA copies/mL) until day 17 (Figure 3), was undetectable in serum throughout the course of illness, and was transiently detected in the feces on day 11. Antibody to A(H7N9) virus was first detected in serum collected on day 8 (HI titer 1:80); serum titers increased until day 29 (1:640) (Figure 3).
Figure 4. Phylogenetic analysis of the hemagglutinin gene of highly pathogenic avian influenza A(H7N9) viruses in Guangdong Province, China, and reference viruses. Maximum likelihood trees were constructed with PhyML by using the general...
We performed phylogenetic analyses on the virus (designated A/Guangdong/17SF006/2017 [17SF006/17]) isolated from the patient on day 6. The HA segment of 17SF006/17 was related to A(H7N9) clade W2-C (7) (Figure 4). An insertion that led to the addition of multiple basic amino acid residues (PEVPKRKRTAR/GL) was found at the HA cleavage site, suggesting that this virus might be highly pathogenic in birds (Table) (13). A similar mutation was found for 2 other cases of A(H7N9) infection identified in Taiwan (A/Taiwan/1/2017) (14) and Guangdong Province (A/Guangdong/17SF003/2016, reported as A/Guangdong/Th008/2017) (15). The HA and matrix sequences of these 3 viruses clustered together in our phylogenetic analysis.
We also sequenced and studied the HA and NA genes of 7 A(H7N9) viruses isolated from environmental samples (from birds at live poultry markets) collected previously in the Guangdong Province. Six of the 7 were mixed with A(H9N2) viruses, and thus, the genes for the internal proteins could not be analyzed. All of the A(H7N9) viruses isolated from the environmental samples had the same HA cleavage site sequence motif as the A(H7N9) human viruses (except for an amino acid change [G] at position −6 from the cleavage site) and clustered with them in the phylogenetic tree, suggesting the emergence of a single subclade of A(H7N9) virus with an HA progressively acquiring HPAI mutations. Similar to other A(H7N9) viruses, the amino acid substitution G180V in the HA protein, which is known to enhance the binding to mammalian α-2,6–linked sialic acid receptors, was found.
The NA genes of 17SF006/17, A/Guangdong/17SF003/2016, and the environmental isolates grouped together, but A/Taiwan/1/2017 did not cluster, suggesting that the origin of this NA gene was distinct (online Technical Appendix Figure, panel A). A mutation associated with oseltamivir resistance (one leading to R292K substitution in NA) was identified in the NA gene of 17SF006/17 virus. The patient had been on oseltamivir for 2 days before the mutation was detected. Both A/Guangdong/17SF003/2016 and A/Taiwan/1/2017 also had the mutation associated with oseltamivir resistance, but the viruses from the Guangdong environmental samples did not. Similar to other A(H7N9) viruses, 17SF006/17 had a mutation leading to the S31N amino acid substitution in the matrix protein, which is associated with amantadine resistance. A mutation in PB2 that causes substitution E627K, which is a key signature of mammalian adaptation, was also found in this virus. The other viral gene segments of the 3 human viruses are not monophyletic and appear to be derived from reassortment with genetically diverse A(H7N9) viruses (Technical Appendix[PDF - 605 KB - 7 pages] Figure, panels A–G).
To investigate the potential for human-to-human transmission of HPAI A(H7N9), an epidemiologic investigation was conducted immediately after virus detection in the patient. Seventy close contacts (5 family members and 65 healthcare workers) were placed under medical observation for 2 weeks. None of them showed signs of illness.
We report the illness and death of a patient in Guangdong Province who was infected with a putative HPAI A(H7N9) virus carrying an HA cleavage site with multiple basic amino acids. Previous A(H7N9) viruses were low pathogenicity, containing only a single arginine residue at the HA cleavage site. Although the HA cleavage site we describe in this report was not a typical HPAI motif for H7 viruses, similar motifs have been reported in H7 viruses in Chile and Canada that were shown to be highly pathogenic in chickens by intravenous pathogenicity tests (13). More studies are needed to determine the pathogenicity of these putative HPAI A(H7N9) viruses (e.g., direct isolation from sick and dying poultry and intravenous pathogenicity index testing in chickens). Viruses isolated from live poultry markets had a sequence motif similar to that of the human isolates (identical except for a glycine at the position −6 from the cleavage site). An additional patient identified in Guangdong Province was infected with an A(H7N9) virus (A/Guangdong/Th005/2017) with HA cleavage site KGKRIAR/GL (15). This evidence suggests that the polybasic HA cleavage site was acquired progressively through multiple mutations within the avian species in a subclade of the A(H7N9) viruses.
The patient had reported that chickens in his backyard had started dying in the weeks before his disease onset, indicating the possibility that these chickens were infected with an HPAI and that the patient acquired his infection from them. However, we do not have direct evidence of A(H7N9) infection in these birds, and alternative explanations for their deaths exist.
A(H7N9) viruses having HA cleavage sites with multiple basic amino acids were reported in 2 human cases in the Guangdong Province and Taiwan (Table) (14,15). The HA sequences of these 2 viruses clustered with that of A/Guangdong/17SF006/2017 in our phylogenetic tree. These viruses also clustered with viruses from the environmental samples from Guangdong, which had a similar sequence motif (1 amino acid difference) at the cleavage site as the human isolates, suggesting the progressive emergence of this HPAI motif in a subclade of A(H7N9) viruses in this region. Although the HA gene segments of these viruses are monophyletic, the other gene segments are not and have diverse origins, suggesting that these HPAI viruses continue to reassort their gene segments. Introduction of the polybasic cleavage site into A(H5N1) was associated with increased viral titers in the respiratory tract, increased virus dissemination to distant organs, increased death in mice and ferrets (but not in macaques), and increased virus replication in endothelial cells (16,17).
A question of public health interest is whether acquisition of the polybasic cleavage site in A(H7N9) HA enhances pathogenicity of A(H7N9) viruses in humans. Similar to most patients with A(H7N9) disease (18), the patient in this report had underlying comorbidities (diabetes and hypertension), which probably contributed to the increased disease severity. The clinical progression of the illness to fulminant viral pneumonia and ARDS was relatively rapid, in spite of commencing oseltamivir on day 4 of illness. The patient required mechanical ventilation and became comatose on day 6 of illness and had a sudden cardiac arrest on day 7, necessitating ECMO. Myocarditis and encephalitic illness were not evident by either clinical observation or imaging investigations, and the most likely cause of the coma was respiratory failure. One of the other patients in Guangdong who had a similar HPAI A(H7N9) virus infection had less severe illness, indicating that the HA cleavage sequence did not invariably increase pathogenicity in humans (15).
By day 6 of patient disease onset, 2 days after commencement of oseltamivir, the virus had acquired a mutation leading to an R292K change in the NA protein known to be associated with oseltamivir resistance (19,20). The other 2 viruses with the same HA cleavage site mutation (A/Guangdong/17SF003/2016 and A/Taiwan/1/2017) also had this oseltamivir resistance mutation. It is likely that the viruses from all 3 patients acquired this resistance mutation after commencement of oseltamivir therapy. Although the emergence of R292K has been reported before in a minority of patients following oseltamivir treatment and treatment failure (19), mutations causing this amino acid substitution have not been found in poultry as of May 30, 2017 (7), and accordingly, the resistance mutation was not observed in the related environmental samples collected from live poultry markets. The frequency with which R292K has been detected in patients with the polybasic HA cleavage site is a cause for concern. Whether acquisition of the polybasic HA cleavage site in the A(H7N9) virus contributes to the enhanced probability of the virus acquiring the NA R292K during oseltamivir treatment is unknown. Studies of HPAI A(H5N1) viruses in vitro and in vivo have demonstrated that viruses with polybasic residues at the HA cleavage site or the PB2 E627K amino acid change enhances replication efficiency, which might increase the likelihood of resistance emerging under the selective pressure of oseltamivir therapy (21–23).
NA R292K confers resistance to both oseltamivir and peramivir, the antiviral drugs used in this patient's therapy (20). Viral load in the endotracheal aspirates remained high for 10 days following commencement of antiviral therapy, probably reflecting lack of efficacy of the antiviral regimens. Viral load only began to decline on day 18 of illness, probably because of increasing antibody titers. Thus, the uncontrolled viral replication and emergence of antiviral resistance in this patient possibly contributed to the adverse clinical outcome. In spite of the prolonged infection and high virus titer within the respiratory tract, virus RNA was not detectable in the serum (Figure 3). Thus, systemic dissemination of the virus was unlikely. This patient was not treated with corticosteroids until day 41, shortly before his demise; thus, corticosteroid therapy did not increase viral load or facilitate the emergence of antiviral resistance, a phenomenon speculated to have occurred in other patients in whom oseltamivir resistance mutations arose (19).
The emergence of antibiotic-resistant Acinetobacter and other bacteria might also have contributed to the adverse clinical outcome. Detection of CMV DNA in serum (rather than in leukocytes) is suggestive of clinically significant CMV viremia following CMV reactivation in a seriously ill patient. CMV viremia is an indication for ganciclovir therapy in immunocompromised patients, which was the rationale for initiating ganciclovir therapy. However, the benefit of using ganciclovir in this context remains unknown.
The patient had increasing levels of serum creatinine and blood urea nitrogen, indicating moderate renal dysfunction, and moderately elevated aspartate aminotransferase, indicating moderate liver dysfunction. Similar features have been previously reported in A(H7N9) patients and are not necessarily indicative of systemic virus dissemination (8).
No evidence of human-to-human transmissibility was apparent; family members and healthcare workers who were in contact with the patient did not have evidence of clinical disease. The HA cleavage site mutation that makes avian influenza viruses highly pathogenic in birds does not necessarily affect the transmissibility of the virus between humans. However, unlike LPAI viruses, which are restricted to the chicken respiratory and intestinal tracts, HPAI viruses spread systemically within chickens and are likely to be found at high titer in multiple organs, including muscle. Thus, the risk for zoonotic transmission through handling or butchering infected poultry and consuming undercooked poultry is likely to increase with HPAI viruses.
In summary, we report the clinical disease progression of a patient infected with a mutant A(H7N9) virus that acquired sequence motifs similar to those found in HPAI viruses. The clinical features of human disease with this isolate did not appear to differ from previous infections with low pathogenicity A(H7N9) viruses, and the clinical and virologic evidence suggested that systemic dissemination of the virus did not occur. The emergence of R292K in NA, which is associated with NA inhibitor resistance, probably contributed to the adverse clinical outcome. In China, heightened surveillance of A(H7N9) in humans with severe respiratory disease and poultry is needed to determine how widespread the polybasic HA cleavage sequence has become and to monitor for evidence of oseltamivir resistance.
Mr. Ke works at the Guangdong Provincial Center for Disease Control and Prevention. His research interests include the emerging outbreak of influenza virus.
We acknowledge the contributions of Professor Honglin Chen and his colleagues from the Department of Microbiology at the University of Hong Kong and the colleagues from Qingyuan Center for Disease Control and Prevention. We also thank the Global Initiative for Sharing Influenza Data for providing sequences.
This study was financially supported by National Key Research and Development Program of China (2016YFC1200200 to Y.S. and 2016YFD0500208 to D.W.), National Natural Science Foundation of China (grant nos. 81761128014 and 81490534 to N.Z.), Science Research Project of the Guangdong Province (grant no. 2016A050503047), Municipal Science and Technology Bureau Foundation of Guangzhou (grant no. 2014Y2-00031), the US National Institute of Allergy and Infectious Diseases under Centers of Excellence for Influenza Research and Surveillance (contract no. HHSN272201400006C), and Research Grants Council of Hong Kong, China, through the Theme-based Research Scheme (ref. T11-705/14N).
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Figure 1. Clinical course of 56-year-old man with diabetes and hypertension infected with highly pathogenic avian influenza A(H7N9) virus, China, 2017. CT, computed tomography; ECMO, extracorporeal membrane oxygenation; ICU, intensive care...
Figure 2. Chest and brain imaging of 56-year-old man infected with highly pathogenic avian influenza A(H7N9) virus, China, 2017: radiograph imaging of chest at day 7 (A) and day 40 (D);...
Figure 3. Kinetics of viral load, oxygenation index, and HI antibody titers in 56-year-old man infected with highly pathogenic avian influenza A(H7N9) virus, China, 2017. Arrows indicate the days HI titers...
Figure 4. Phylogenetic analysis of the hemagglutinin gene of highly pathogenic avian influenza A(H7N9) viruses in Guangdong Province, China, and reference viruses. Maximum likelihood trees were constructed with PhyML by using...
Table. Key molecular signatures of avian influenza A(H7N9) virus from patient in Guangdong Province, China, compared with other closely related viruses
Technical Appendix. Virus isolate information submitted for inclusion into the Global Initiative on Sharing All Influenza Data database, laboratory test results of and clinical treatments for the 56-year-old man infected with... 605 KB
DOI: 10.3201/eid2308.170600
1These authors contributed equally to this article.
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