Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013
We describe a novel spike pseudoparticle neutralisation
assay (ppNT) for seroepidemiological studies on
Middle East respiratory syndrome coronavirus (MERSCoV)
and apply this assay together with conventional
microneutralisation (MN) tests to investigate 1,343
human and 625 animal sera. The sera were collected
in Egypt as a region adjacent to areas where MERS has
been described, and in Hong Kong, China as a control
region. Sera from dromedary camels had a high prevalence
of antibody reactive to MERS-CoV by MERS NT
(93.6%) and MERS ppNT (98.2%) assay. The antibody
titres ranged up to 1,280 and higher in MN assays
and 10,240 and higher in ppNT assays. No other
investigated species had any antibody reactivity to
MERS-CoV. While seropositivity does not exclude the
possibility of infection with a closely related virus, our
data highlight the need to attempt detection of MERSCoV
or related coronaviruses in dromedary camels. The
data show excellent correlation between the conventional
MN assay and the novel ppNT assay. The newly
developed ppNT assay does not require Biosafety Level
3 containment and is thus a relatively high-throughput
assay, well suited for large-scale seroepidemiology
studies which are needed to better understand the
ecology and epidemiology of MERS-CoV.
Spatio temporal dynamics of global H5N1 outbreaks match bird migration patternsHarm Kiezebrink
The global spread of highly pathogenic avian influenza H5N1 in poultry, wild birds and humans, poses a significant pandemic threat and a serious public health risk.
An efficient surveillance and disease control system relies on the understanding of the dispersion patterns and spreading mechanisms of the virus. A space-time cluster analysis of H5N1 outbreaks was used to identify spatio-temporal patterns at a global scale and over an extended period of time.
Potential mechanisms explaining the spread of the H5N1 virus, and the role of wild birds, were analyzed. Between December 2003 and December 2006, three global epidemic phases of H5N1 influenza were identified.
These H5N1 outbreaks showed a clear seasonal pattern, with a high density of outbreaks in winter and early spring (i.e., October to March). In phase I and II only the East Asia Australian flyway was affected. During phase III, the H5N1 viruses started to appear in four other flyways: the Central Asian flyway, the Black Sea Mediterranean flyway, the East Atlantic flyway and the East Africa West Asian flyway.
Six disease cluster patterns along these flyways were found to be associated with the seasonal migration of wild birds. The spread of the H5N1 virus, as demonstrated by the space-time clusters, was associated with the patterns of migration of wild birds. Wild birds may therefore play an important role in the spread of H5N1 over long distances.
Disease clusters were also detected at sites where wild birds are known to overwinter and at times when migratory birds were present. This leads to the suggestion that wild birds may also be involved in spreading the H5N1 virus over short distances.
Dossier transmission: Transmission of Avian Influenza Virus to DogsHarm Kiezebrink
Avian influenza was found in a dog on a farm in South Gyeongsang Province amid growing concerns that the disease could spread to other animals, officials the Ministry of Agriculture, Food and Rural Affairs said. The dog ― one of three at a duck farm in Goseong-gun, South Gyeongsang Province ― had antigens for the highly pathogenic H5N8 strain of bird flu, the Ministry of Agriculture, Food and Rural Affairs said. The disease affected the farm on Jan. 23.
Since the first case of a dog being infected with the poultry virus in March 2014, there have been 55 dogs found with antibodies to the bird flu virus. The antibody means the immune system of the dogs eliminated the virus. This is the first time bird flu has been found in a dog in Korea through the detection of antigens.
“None of these dogs had shown symptoms. No antigens or antibodies for the virus were found in the two other dogs, which means that dog-to-dog transmission is unlikely to have happened,” quarantine officials said.
The ministry suspected that the dog may have eaten infected animals at the farm. All poultry and dogs at the concerned farm were slaughtered as part of the preventive measures right after the farm was reported to have been infected with the disease, officials said.
Meanwhile, quarantine officials rejected the possibility of viral transmission to humans. According to the ministry’s report, about 450 workers at infected farms across the country had been given an antigen test, with none showing signs of infection. None of Korea’s 20,000 farm workers have reported any symptoms so far, officials added.
“It is thought that infected dogs do not show symptoms of the disease as they are naturally resistant to bird flu,” the ministry said. Meanwhile, the Agriculture Ministry has toughened the quarantine measures in Goseong-gun. The region is a frequented by migratory birds, which are suspected to have spread the viral disease.
Reseach on H9N2: evidence that link outbreaks in Eurasia, China, South Korea,...Harm Kiezebrink
In this study, scientists from the U.S. Geological Survey and U.S. Fish and Wildlife Service harnessed a new type of DNA technology to investigate avian influenza viruses in Alaska. Using a “next generation” sequencing approach, which identifies gene sequences of interest more rapidly and more completely than by traditional techniques, scientists identified low pathogenic avian influenza viruses in Alaska that are nearly identical to viruses found in China and South Korea.
The viruses were found in an area of western Alaska that is known to be a hot spot for both American and Eurasian forms of avian influenza.
“Our past research in western Alaska has shown that 70 percent of avian influenza viruses isolated in this area were found to contain genetic material from Eurasia, providing evidence for high levels of intercontinental viral exchange,” said Andy Ramey, a scientist with the USGS Alaska Science Center and lead author of the study. “This is because Asian and North American migratory flyways overlap in western Alaska.”
The new study, led by the USGS, found low pathogenic H9N2 viruses in an Emperor Goose and a Northern Pintail. Both of the H9N2 viruses were nearly identical genetically to viruses found in wild bird samples from Lake Dongting, China and Cheon-su Bay, South Korea.
“These H9N2 viruses are low pathogenic and not known to infect humans, but similar viruses have been implicated in disease outbreaks in domestic poultry in Asia,” said Ramey.
There is no commercial poultry production in western Alaska and highly similar H9N2 virus strains have not been reported in poultry in East Asia or North America, so it is unlikely that agricultural imports influenced this result.
The finding provides evidence for intercontinental movement of intact avian influenza viruses by migratory birds. The USGS recently released a publication about the detection of a novel highly pathogenic H5N8 virus in the U.S. that is highly similar to the Eurasian H5N8 viruses. This suggests that the novel re-assortment may be adapted to certain waterfowl species, enabling it to survive long migrations. That virus, and associated strains, have now spread from early detections in wild and domestic birds in Pacific states to poultry outbreaks in Minnesota, Missouri and Arkansas.
“The frequency of inter-hemispheric dispersal events of avian influenza viruses by migratory birds may be higher than previously recognized,” said Ramey.
While some of the samples for the project came from bird fecal samples collected from beaches at Izembek National Wildlife Refuge, most of the samples came from sport hunters.
“For the past several years, we’ve worked closely with sport hunters in the fall to obtain swab samples from birds and that has really informed our understanding of wildlife disease in this area,” said Bruce Casler, formerly a biologist with the USFWS Izembek National Wildlife Refuge and a co-author of the study. Non
H5N8 virus dutch outbreak (2014) linked to sequences of strains from asiaHarm Kiezebrink
Genetic analysis of influenza A(H5N8) virus from the Netherlands indicates that the virus probably was spread by migratory wild birds from Asia, possibly through overlapping flyways and common breeding sites in Siberia. In addition to the outbreak in the Netherlands, several other outbreaks of HPAI (H5N8) virus infections were reported in Europe at the end of 2014 after exponentially increasing deaths occurred in chicken and turkey flocks.
Genetic sequences submitted to the EpiFlu database indicated that the viruses from Europe showed a strong similarity to viruses isolated earlier in 2014 in South Korea, China, and Japan. An H5N8 virus isolated from a wigeon in Russia in September 2014 is located in the phylogenetic tree near the node of all sequences for H5N8 viruses from Europe.
In regard to time, this location fits the hypothesized route of H5N8 virus introduction into Europe. Furthermore, for several reasons, it is highly likely that the introduction of HPAI (H5N8) virus into the indoor-layer farm in the Netherlands occurred via indirect contact.
First, despite intensive monitoring, H5N8 viruses have never been detected in commercial poultry or wild birds in the Netherlands.
Second, when the virus was detected, the Netherlands had no direct trade contact with other European countries or Asia that might explain a route of introduction.
Third, because of the severity of disease in galliforms, outbreaks of H5N8 in the Netherlands before November 2014 would have been noticed.
An example of critical analysis of a Scientific Article.
Article Analysis. Scientific skills.
COVID-19 Chadox1 Vaccination.
Clinical trial in Rhesus macaque monkeys.
Corona-virus vaccine research paper analysis.
Spatio temporal dynamics of global H5N1 outbreaks match bird migration patternsHarm Kiezebrink
The global spread of highly pathogenic avian influenza H5N1 in poultry, wild birds and humans, poses a significant pandemic threat and a serious public health risk.
An efficient surveillance and disease control system relies on the understanding of the dispersion patterns and spreading mechanisms of the virus. A space-time cluster analysis of H5N1 outbreaks was used to identify spatio-temporal patterns at a global scale and over an extended period of time.
Potential mechanisms explaining the spread of the H5N1 virus, and the role of wild birds, were analyzed. Between December 2003 and December 2006, three global epidemic phases of H5N1 influenza were identified.
These H5N1 outbreaks showed a clear seasonal pattern, with a high density of outbreaks in winter and early spring (i.e., October to March). In phase I and II only the East Asia Australian flyway was affected. During phase III, the H5N1 viruses started to appear in four other flyways: the Central Asian flyway, the Black Sea Mediterranean flyway, the East Atlantic flyway and the East Africa West Asian flyway.
Six disease cluster patterns along these flyways were found to be associated with the seasonal migration of wild birds. The spread of the H5N1 virus, as demonstrated by the space-time clusters, was associated with the patterns of migration of wild birds. Wild birds may therefore play an important role in the spread of H5N1 over long distances.
Disease clusters were also detected at sites where wild birds are known to overwinter and at times when migratory birds were present. This leads to the suggestion that wild birds may also be involved in spreading the H5N1 virus over short distances.
Dossier transmission: Transmission of Avian Influenza Virus to DogsHarm Kiezebrink
Avian influenza was found in a dog on a farm in South Gyeongsang Province amid growing concerns that the disease could spread to other animals, officials the Ministry of Agriculture, Food and Rural Affairs said. The dog ― one of three at a duck farm in Goseong-gun, South Gyeongsang Province ― had antigens for the highly pathogenic H5N8 strain of bird flu, the Ministry of Agriculture, Food and Rural Affairs said. The disease affected the farm on Jan. 23.
Since the first case of a dog being infected with the poultry virus in March 2014, there have been 55 dogs found with antibodies to the bird flu virus. The antibody means the immune system of the dogs eliminated the virus. This is the first time bird flu has been found in a dog in Korea through the detection of antigens.
“None of these dogs had shown symptoms. No antigens or antibodies for the virus were found in the two other dogs, which means that dog-to-dog transmission is unlikely to have happened,” quarantine officials said.
The ministry suspected that the dog may have eaten infected animals at the farm. All poultry and dogs at the concerned farm were slaughtered as part of the preventive measures right after the farm was reported to have been infected with the disease, officials said.
Meanwhile, quarantine officials rejected the possibility of viral transmission to humans. According to the ministry’s report, about 450 workers at infected farms across the country had been given an antigen test, with none showing signs of infection. None of Korea’s 20,000 farm workers have reported any symptoms so far, officials added.
“It is thought that infected dogs do not show symptoms of the disease as they are naturally resistant to bird flu,” the ministry said. Meanwhile, the Agriculture Ministry has toughened the quarantine measures in Goseong-gun. The region is a frequented by migratory birds, which are suspected to have spread the viral disease.
Reseach on H9N2: evidence that link outbreaks in Eurasia, China, South Korea,...Harm Kiezebrink
In this study, scientists from the U.S. Geological Survey and U.S. Fish and Wildlife Service harnessed a new type of DNA technology to investigate avian influenza viruses in Alaska. Using a “next generation” sequencing approach, which identifies gene sequences of interest more rapidly and more completely than by traditional techniques, scientists identified low pathogenic avian influenza viruses in Alaska that are nearly identical to viruses found in China and South Korea.
The viruses were found in an area of western Alaska that is known to be a hot spot for both American and Eurasian forms of avian influenza.
“Our past research in western Alaska has shown that 70 percent of avian influenza viruses isolated in this area were found to contain genetic material from Eurasia, providing evidence for high levels of intercontinental viral exchange,” said Andy Ramey, a scientist with the USGS Alaska Science Center and lead author of the study. “This is because Asian and North American migratory flyways overlap in western Alaska.”
The new study, led by the USGS, found low pathogenic H9N2 viruses in an Emperor Goose and a Northern Pintail. Both of the H9N2 viruses were nearly identical genetically to viruses found in wild bird samples from Lake Dongting, China and Cheon-su Bay, South Korea.
“These H9N2 viruses are low pathogenic and not known to infect humans, but similar viruses have been implicated in disease outbreaks in domestic poultry in Asia,” said Ramey.
There is no commercial poultry production in western Alaska and highly similar H9N2 virus strains have not been reported in poultry in East Asia or North America, so it is unlikely that agricultural imports influenced this result.
The finding provides evidence for intercontinental movement of intact avian influenza viruses by migratory birds. The USGS recently released a publication about the detection of a novel highly pathogenic H5N8 virus in the U.S. that is highly similar to the Eurasian H5N8 viruses. This suggests that the novel re-assortment may be adapted to certain waterfowl species, enabling it to survive long migrations. That virus, and associated strains, have now spread from early detections in wild and domestic birds in Pacific states to poultry outbreaks in Minnesota, Missouri and Arkansas.
“The frequency of inter-hemispheric dispersal events of avian influenza viruses by migratory birds may be higher than previously recognized,” said Ramey.
While some of the samples for the project came from bird fecal samples collected from beaches at Izembek National Wildlife Refuge, most of the samples came from sport hunters.
“For the past several years, we’ve worked closely with sport hunters in the fall to obtain swab samples from birds and that has really informed our understanding of wildlife disease in this area,” said Bruce Casler, formerly a biologist with the USFWS Izembek National Wildlife Refuge and a co-author of the study. Non
H5N8 virus dutch outbreak (2014) linked to sequences of strains from asiaHarm Kiezebrink
Genetic analysis of influenza A(H5N8) virus from the Netherlands indicates that the virus probably was spread by migratory wild birds from Asia, possibly through overlapping flyways and common breeding sites in Siberia. In addition to the outbreak in the Netherlands, several other outbreaks of HPAI (H5N8) virus infections were reported in Europe at the end of 2014 after exponentially increasing deaths occurred in chicken and turkey flocks.
Genetic sequences submitted to the EpiFlu database indicated that the viruses from Europe showed a strong similarity to viruses isolated earlier in 2014 in South Korea, China, and Japan. An H5N8 virus isolated from a wigeon in Russia in September 2014 is located in the phylogenetic tree near the node of all sequences for H5N8 viruses from Europe.
In regard to time, this location fits the hypothesized route of H5N8 virus introduction into Europe. Furthermore, for several reasons, it is highly likely that the introduction of HPAI (H5N8) virus into the indoor-layer farm in the Netherlands occurred via indirect contact.
First, despite intensive monitoring, H5N8 viruses have never been detected in commercial poultry or wild birds in the Netherlands.
Second, when the virus was detected, the Netherlands had no direct trade contact with other European countries or Asia that might explain a route of introduction.
Third, because of the severity of disease in galliforms, outbreaks of H5N8 in the Netherlands before November 2014 would have been noticed.
An example of critical analysis of a Scientific Article.
Article Analysis. Scientific skills.
COVID-19 Chadox1 Vaccination.
Clinical trial in Rhesus macaque monkeys.
Corona-virus vaccine research paper analysis.
Spatial, temporal and genetic dynamics of H5N1 in chinaHarm Kiezebrink
The spatial spread of H5N1 avian influenza, significant ongoing mutations, and long-term persistence of the virus in some geographic regions has had an enormous impact on the poultry industry and presents a serious threat to human health.
This study revealed two different transmission modes of H5N1 viruses in China, and indicated a significant role of poultry in virus dissemination. Furthermore, selective pressure posed by vaccination was found in virus evolution in the country.
Phylogenetic analysis, geospatial techniques, and time series models were applied to investigate the spatiotemporal pattern of H5N1 outbreaks in China and the effect of vaccination on virus evolution.
Results showed obvious spatial and temporal clusters of H5N1 outbreaks on different scales, which may have been associated with poultry and wild-bird transmission modes of H5N1 viruses. Lead–lag relationships were found among poultry and wild-bird outbreaks and human cases. Human cases were preceded by poultry outbreaks, and wild-bird outbreaks were led by human cases.
Each clade has gained its own unique spatiotemporal and genetic dominance. Genetic diversity of the H5N1 virus decreased significantly between 1996 and 2011; presumably under strong selective pressure of vaccination. Mean evolutionary rates of H5N1 virus increased after vaccination was adopted in China.
Human-to-Human transmission of H7H7 in Holland 2003Harm Kiezebrink
The outbreak of highly pathogenic avian influenza A virus subtype H7N7 started at the end of February, 2003, in commercial poultry farms in the Netherlands. In this study, published in The Lancet in 2004, it is noted that an unexpectedly high number of transmissions of avian influenza A virus subtype H7N7 to people directly involved in handling infected poultry, providing evidence for person-to-person transmission.
Although the risk of transmission of these viruses to humans was initially thought to be low, an outbreak investigation was launched to assess the extent of transmission of influenza A virus subtype H7N7 from chickens to humans.
453 people had health complaints—349 reported conjunctivitis, 90 had influenza-like illness, and 67 had other complaints. We detected A/H7 in conjunctival samples from 78 (26·4%) people with conjunctivitis only, in five (9·4%) with influenza-like illness and conjunctivitis, in two (5·4%) with influenza-like illness only, and in four (6%) who reported other symptoms. Most positive samples had been collected within 5 days of symptom onset. A/H7 infection was confirmed in three contacts (of 83 tested), one of whom developed influenza-like illness. Six people had influenza A/H3N2 infection. After 19 people had been diagnosed with the infection, all workers received mandatory influenza virus vaccination and prophylactic treatment with oseltamivir. More than half (56%) of A/H7 infections reported here arose before the vaccination and treatment programme.
El coronavirus, relacionado con el virus que causa el SARS (síndrome respiratorio agudo severo), ha desencadenado un renovado debate sobre si las variantes de laboratorio de ingeniería de virus con posible potencial pandémico valen los riesgos.
En un artículo publicado en Nature Medicine 1 el 9 de noviembre, los científicos investigaron un virus llamado SHC014, que se encuentra en murciélagos de herradura en China. Los investigadores crearon un virus quimérico, compuesto por una proteína de superficie de SHC014 y la columna vertebral de un virus del SARS que se había adaptado para crecer en ratones e imitar una enfermedad humana. La quimera infectó las células de las vías respiratorias humanas, lo que demuestra que la proteína de superficie de SHC014 tiene la estructura necesaria para unirse a un receptor clave en las células e infectarlas. También causó enfermedades en ratones, pero no los mató.
----------------------
Engineered bat virus stirs debate over risky research
Lab-made coronavirus related to SARS can infect human cells.
12 November 2015
An experiment that created a hybrid version of a bat coronavirus — one related to the virus that causes SARS (severe acute respiratory syndrome) — has triggered renewed debate over whether engineering lab variants of viruses with possible pandemic potential is worth the risks.
In an article published in Nature Medicine 1 on 9 November, scientists investigated a virus called SHC014, which is found in horseshoe bats in China. The researchers created a chimaeric virus, made up of a surface protein of SHC014 and the backbone of a SARS virus that had been adapted to grow in mice and to mimic human disease. The chimaera infected human airway cells — proving that the surface protein of SHC014 has the necessary structure to bind to a key receptor on the cells and to infect them. It also caused disease in mice, but did not kill them
Deadly H5N1 birdflu needs just five mutations to spread easily in peopleHarm Kiezebrink
Reference: Phys.org. 15 Apr 2014. Dutch researchers have found that the virus needs only five favorable gene mutations to become transmissible through coughing or sneezing, like regular flu viruses.
World health officials have long feared that the H5N1 virus will someday evolve a knack for airborne transmission, setting off a devastating pandemic. While the new study suggests the mutations needed are relatively few, it remains unclear whether they're likely to happen outside the laboratory.
The 3 P’s of avian influenza Prevent, Plan, PracticeHarm Kiezebrink
Avian Influenza has become endemic in many parts of the word. In it's current form it has been around since 1997 and although thy virus types have changed, emergency response, management & control are still a hot issue. In this article published in 2006 in the US magazine Poultry Perspectives, the subject what to do during crisis situations is presented. The conclusions are still valid today and may help to prevent large-scale outbreaks
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specifi c, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant.Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specifi c primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplifi ed from duck’s tissue lesions whereas Map were amplifi ed from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplifi cation of other pathogenic bacterial species. This technique is sensitive, specifi c, rapid and does not require fl uorescent probes or post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Unusual Features of the SARS-CoV-2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route
https://zenodo.org/record/4028830#.X2EiXWhKiUn
Medcrave - MERS coronavirus - current statusMedCrave
CDC: Centers for Disease Control; MERS-CoV: Middle
East Respiratory Syndrome Coronavirus; RT-PCR: Reverse
Transcriptase Polymerase Chain Reaction; VLP: Virus Like
Particles.
Recently, a new virus started to infect certain individuals in the Middle-East. It was soon identified as a previously unknown coronavirus that caused severe respiratory disease with a high rate of mortality. This virus, MERS-CoV, is still closely watched by health authorities as it has the potential to evolve and cause a major epidemic.
Modelling wind-borne spread of HPAI between farms (2012)Harm Kiezebrink
To understand the risks of spreading contaminated materials caused by stable gassing, a quantitative understanding of the spread of contaminated farm dust between locations is a prerequisite for obtaining much-needed insight into one of the possible mechanisms of disease spread between farms.
The researchers Amos Ssematimba, Thomas J. Hagenaars, Mart C. M. de Jong of the Dutch Department of Epidemiology, Crisis Organization and Diagnostics, Central Veterinary Institute (CVI) part of Wageningen University and Research Centre, Lelystad, The Netherlands, and Quantitative Veterinary Epidemiology, Department of Animal Sciences, Wageningen University, Wageningen, The Netherland developed a model to calculate the quantity of contaminated farm-dust particles deposited at various locations downwind of a source farm and apply the model to assess the possible contribution of the wind-borne route to the transmission of Highly Pathogenic Avian Influenza virus (HPAI) during the 2003 epidemic in the Netherlands.
The model is obtained from a Gaussian Plume Model by incorporating the dust deposition process, pathogen decay, and a model for the infection process on exposed farms.
Using poultry- and avian influenza-specific parameter values we calculate the distance-dependent probability of between-farm transmission by this route.
A comparison between the transmission risk pattern predicted by the model and the pattern observed during the 2003 epidemic reveals that the wind-borne route alone is insufficient to explain the observations although it could contribute substantially to the spread over short distance ranges, for example, explaining 24% of the transmission over distances up to 25 km.
Spatial, temporal and genetic dynamics of H5N1 in chinaHarm Kiezebrink
The spatial spread of H5N1 avian influenza, significant ongoing mutations, and long-term persistence of the virus in some geographic regions has had an enormous impact on the poultry industry and presents a serious threat to human health.
This study revealed two different transmission modes of H5N1 viruses in China, and indicated a significant role of poultry in virus dissemination. Furthermore, selective pressure posed by vaccination was found in virus evolution in the country.
Phylogenetic analysis, geospatial techniques, and time series models were applied to investigate the spatiotemporal pattern of H5N1 outbreaks in China and the effect of vaccination on virus evolution.
Results showed obvious spatial and temporal clusters of H5N1 outbreaks on different scales, which may have been associated with poultry and wild-bird transmission modes of H5N1 viruses. Lead–lag relationships were found among poultry and wild-bird outbreaks and human cases. Human cases were preceded by poultry outbreaks, and wild-bird outbreaks were led by human cases.
Each clade has gained its own unique spatiotemporal and genetic dominance. Genetic diversity of the H5N1 virus decreased significantly between 1996 and 2011; presumably under strong selective pressure of vaccination. Mean evolutionary rates of H5N1 virus increased after vaccination was adopted in China.
Human-to-Human transmission of H7H7 in Holland 2003Harm Kiezebrink
The outbreak of highly pathogenic avian influenza A virus subtype H7N7 started at the end of February, 2003, in commercial poultry farms in the Netherlands. In this study, published in The Lancet in 2004, it is noted that an unexpectedly high number of transmissions of avian influenza A virus subtype H7N7 to people directly involved in handling infected poultry, providing evidence for person-to-person transmission.
Although the risk of transmission of these viruses to humans was initially thought to be low, an outbreak investigation was launched to assess the extent of transmission of influenza A virus subtype H7N7 from chickens to humans.
453 people had health complaints—349 reported conjunctivitis, 90 had influenza-like illness, and 67 had other complaints. We detected A/H7 in conjunctival samples from 78 (26·4%) people with conjunctivitis only, in five (9·4%) with influenza-like illness and conjunctivitis, in two (5·4%) with influenza-like illness only, and in four (6%) who reported other symptoms. Most positive samples had been collected within 5 days of symptom onset. A/H7 infection was confirmed in three contacts (of 83 tested), one of whom developed influenza-like illness. Six people had influenza A/H3N2 infection. After 19 people had been diagnosed with the infection, all workers received mandatory influenza virus vaccination and prophylactic treatment with oseltamivir. More than half (56%) of A/H7 infections reported here arose before the vaccination and treatment programme.
El coronavirus, relacionado con el virus que causa el SARS (síndrome respiratorio agudo severo), ha desencadenado un renovado debate sobre si las variantes de laboratorio de ingeniería de virus con posible potencial pandémico valen los riesgos.
En un artículo publicado en Nature Medicine 1 el 9 de noviembre, los científicos investigaron un virus llamado SHC014, que se encuentra en murciélagos de herradura en China. Los investigadores crearon un virus quimérico, compuesto por una proteína de superficie de SHC014 y la columna vertebral de un virus del SARS que se había adaptado para crecer en ratones e imitar una enfermedad humana. La quimera infectó las células de las vías respiratorias humanas, lo que demuestra que la proteína de superficie de SHC014 tiene la estructura necesaria para unirse a un receptor clave en las células e infectarlas. También causó enfermedades en ratones, pero no los mató.
----------------------
Engineered bat virus stirs debate over risky research
Lab-made coronavirus related to SARS can infect human cells.
12 November 2015
An experiment that created a hybrid version of a bat coronavirus — one related to the virus that causes SARS (severe acute respiratory syndrome) — has triggered renewed debate over whether engineering lab variants of viruses with possible pandemic potential is worth the risks.
In an article published in Nature Medicine 1 on 9 November, scientists investigated a virus called SHC014, which is found in horseshoe bats in China. The researchers created a chimaeric virus, made up of a surface protein of SHC014 and the backbone of a SARS virus that had been adapted to grow in mice and to mimic human disease. The chimaera infected human airway cells — proving that the surface protein of SHC014 has the necessary structure to bind to a key receptor on the cells and to infect them. It also caused disease in mice, but did not kill them
Deadly H5N1 birdflu needs just five mutations to spread easily in peopleHarm Kiezebrink
Reference: Phys.org. 15 Apr 2014. Dutch researchers have found that the virus needs only five favorable gene mutations to become transmissible through coughing or sneezing, like regular flu viruses.
World health officials have long feared that the H5N1 virus will someday evolve a knack for airborne transmission, setting off a devastating pandemic. While the new study suggests the mutations needed are relatively few, it remains unclear whether they're likely to happen outside the laboratory.
The 3 P’s of avian influenza Prevent, Plan, PracticeHarm Kiezebrink
Avian Influenza has become endemic in many parts of the word. In it's current form it has been around since 1997 and although thy virus types have changed, emergency response, management & control are still a hot issue. In this article published in 2006 in the US magazine Poultry Perspectives, the subject what to do during crisis situations is presented. The conclusions are still valid today and may help to prevent large-scale outbreaks
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specifi c, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant.Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specifi c primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplifi ed from duck’s tissue lesions whereas Map were amplifi ed from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplifi cation of other pathogenic bacterial species. This technique is sensitive, specifi c, rapid and does not require fl uorescent probes or post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Unusual Features of the SARS-CoV-2 Genome Suggesting Sophisticated Laboratory Modification Rather Than Natural Evolution and Delineation of Its Probable Synthetic Route
https://zenodo.org/record/4028830#.X2EiXWhKiUn
Medcrave - MERS coronavirus - current statusMedCrave
CDC: Centers for Disease Control; MERS-CoV: Middle
East Respiratory Syndrome Coronavirus; RT-PCR: Reverse
Transcriptase Polymerase Chain Reaction; VLP: Virus Like
Particles.
Recently, a new virus started to infect certain individuals in the Middle-East. It was soon identified as a previously unknown coronavirus that caused severe respiratory disease with a high rate of mortality. This virus, MERS-CoV, is still closely watched by health authorities as it has the potential to evolve and cause a major epidemic.
Modelling wind-borne spread of HPAI between farms (2012)Harm Kiezebrink
To understand the risks of spreading contaminated materials caused by stable gassing, a quantitative understanding of the spread of contaminated farm dust between locations is a prerequisite for obtaining much-needed insight into one of the possible mechanisms of disease spread between farms.
The researchers Amos Ssematimba, Thomas J. Hagenaars, Mart C. M. de Jong of the Dutch Department of Epidemiology, Crisis Organization and Diagnostics, Central Veterinary Institute (CVI) part of Wageningen University and Research Centre, Lelystad, The Netherlands, and Quantitative Veterinary Epidemiology, Department of Animal Sciences, Wageningen University, Wageningen, The Netherland developed a model to calculate the quantity of contaminated farm-dust particles deposited at various locations downwind of a source farm and apply the model to assess the possible contribution of the wind-borne route to the transmission of Highly Pathogenic Avian Influenza virus (HPAI) during the 2003 epidemic in the Netherlands.
The model is obtained from a Gaussian Plume Model by incorporating the dust deposition process, pathogen decay, and a model for the infection process on exposed farms.
Using poultry- and avian influenza-specific parameter values we calculate the distance-dependent probability of between-farm transmission by this route.
A comparison between the transmission risk pattern predicted by the model and the pattern observed during the 2003 epidemic reveals that the wind-borne route alone is insufficient to explain the observations although it could contribute substantially to the spread over short distance ranges, for example, explaining 24% of the transmission over distances up to 25 km.
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This project hopes to integrate typical guitar audio effects unto a single IC. Using analog
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Similar to Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013
Objective: To generate preliminary information about of enteroviruses and Enterovirus 71 (EV71) in patients with aseptic meningitis in Khartoum State, Sudan.
Method: Cerebrospinal fluid specimens were collected from 89 aseptic meningitis patients from different Khartoum Hospitals
(Mohammed Alamin Hamid Hospital, Soba Teaching Hospital, Omdurman Military Hospital, Alban Gadeed Teaching Hospital and Police Hospital) within February to May 2015. Among these 89 patients, 43 (48%) were males and 46 (52%) were females. The patient’s age ranged between 1 day and 30 years old. The collected specimens were assayed to detect enteroviruses and EV71 RNA using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique
Serological evidence of MERS-CoV antibodies in dromedary camels (Camelus drom...ILRI
Presented by Eric M. Fèvre, Sharon L. Deem, Margaret Kinnaird, Springer Browne, Dishon Muloi, Gert-Jan Godeke, Marion Koopmans and C.B.E.M. Reusken at the 4th Medical and Veterinary Virus Research Symposium in Kenya (MVVR), Nairobi, Kenya, 15-16 October 2015.
MERS virus is a virus that related to the SARS virus, this virus known as Middle East Respiratory Syndrome Virus, because this virus has caused several death of humans in Middle East, especially in Saudi Arabia.
Two main animal pathogenic subspecies of Mycobacterium avium are M. avium avium (Maa) and M. avium paratuberculosis (Map). Their pathogenicity is host-specific, Maa causing avian tuberculosis in poultry whereas Map commonly cross-infects to ruminant. Veterinary diagnosis of M. avium infections is microscopic examination of acid-fast bacilli or culture in Löwenstein-Jensen medium,which are time-consuming and low sensitivity. This present study aimed to apply real-time PCR coupled with High-Resolution Melting (HRM) analysis for differential detection of Maa in Thai domestic ducks. Specific primer targeting host-expression dependent (hed) region
was designed, PCR product of Maa were amplified from duck’s tissue lesions whereas Map were amplified from cow and deer. HRM real-time PCR was performed and analyzed. Different HRM patterns were showed and melting temperature were analyzed at 83.26 ± 0.12°C for Maa and 84.04 ± 0.09°C for Map. This technique can detect as few as 102 DNA copies and present high specifi city by negative amplification of other pathogenic bacterial species. This technique is sensitive, specific, rapid and does not require fluorescent probes or
post-PCR electrophoresis. Our technique is a possible new tool for the detection of Maa and Map infection in tissue specimens.
Dynamic Aspects of Schistosoma Haematobium Infection as Experimental Model.pdfAlim A-H Yacoub Lovers
Abdul-Hussein H Awad, Alim A-H Yacoub, Sabeeh H Al-Mayah. Dynamic Aspects of Schistosoma Haematobium Infection as Experimental Model. Medical Journal of Basra University 1995;13(1&2):21-30
Determination of baseline Widal titre among apparently healthy population in ...IOSR Journals
Present study was conducted to determine the baseline widal titer of healthy population of Dehradun city. A total of 300 serum samples were collected from healthy individual with no history of fever and who had not received any vaccination for enteric fever. Tube agglutination test was done with commercially available antigens which contained the Salmonella enterica serovar typhi O and H antigens, the Salmonella enterica serovar paratyphi AH antigen and paratyphi BH antigen. In the present study an agglutination titer for TO – 1:20 is 28%, for 1:40 is 24%, followed by 1:80 and 1: 160 which is 10%, 4% respectively. The highest sample with an anti-H titre found with 1:20 (22%) followed by 1:40(17%). Based upon the results of the study it has been recommended that a single Widal can be significant in an endemic region when higher titre (1:160) is obtained.
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The Gram stain is a fundamental technique in microbiology used to classify bacteria based on their cell wall structure. It provides a quick and simple method to distinguish between Gram-positive and Gram-negative bacteria, which have different susceptibilities to antibiotics
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
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Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
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Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Pharma Pcd Franchise in Jharkhand - Yodley Lifesciences
Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in Egypt, June 2013
1. 1www.eurosurveillance.org
Research articles
Seroepidemiology for MERS coronavirus using
microneutralisation and pseudoparticle virus
neutralisation assays reveal a high prevalence of
antibody in dromedary camels in Egypt, June 2013
R A Perera1,2
, P Wang2,3,4
, M R Gomaa5
, R El-Shesheny5
, A Kandeil5
, O Bagato5
, L Y Siu3
, M M Shehata5
, A S Kayed5
, Y Moatasim5
,
M Li3
, L L Poon1
, Y Guan1
, R J Webby6
, M A Ali5
, J S Peiris (malik@hku.hk)1
, G Kayali (ghazi.kayali@stjude.org)6
1. Centre of Influenza Research, School of Public Health, The University of Hong Kong, Hong Kong, China
2. These authors contributed equally to the work and are joint first authors
3. Hong Kong University-Pasteur Research Pole, The University of Hong Kong, Hong Kong, China
4. Key Laboratory of Protein and Peptide Pharmaceuticals, Chinese Academy of Sciences - University of Tokyo Joint Laboratory of
Structural Virology and Immunology, Beijing, China
5. Division of Environmental Research, National Research Centre, Giza, Egypt
6. Division of Virology, Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis, United States
Citation style for this article:
Perera RA, Wang P, Gomaa MR, El-Shesheny R, Kandeil A, Bagato O, Siu LY, Shehata MM, Kayed AS, Moatasim Y, Li M, Poon LL, Guan Y, Webby RJ, Ali MA, Peiris JS,
Kayali G. Seroepidemiology for MERS coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in
dromedary camels in Egypt, June 2013.
Euro Surveill. 2013;18(36):pii=20574. Available online: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20574
Article submitted on 26 August 2013 / published on 5 September 2013
We describe a novel spike pseudoparticle neutralisa-
tion assay (ppNT) for seroepidemiological studies on
Middle East respiratory syndrome coronavirus (MERS-
CoV) and apply this assay together with conventional
microneutralisation (MN) tests to investigate 1,343
human and 625 animal sera. The sera were collected
in Egypt as a region adjacent to areas where MERS has
been described, and in Hong Kong, China as a control
region. Sera from dromedary camels had a high preva-
lence of antibody reactive to MERS-CoV by MERS NT
(93.6%) and MERS ppNT (98.2%) assay. The antibody
titres ranged up to 1,280 and higher in MN assays
and 10,240 and higher in ppNT assays. No other
investigated species had any antibody reactivity to
MERS-CoV. While seropositivity does not exclude the
possibility of infection with a closely related virus, our
data highlight the need to attempt detection of MERS-
CoV or related coronaviruses in dromedary camels. The
data show excellent correlation between the conven-
tional MN assay and the novel ppNT assay. The newly
developed ppNT assay does not require Biosafety Level
3 containment and is thus a relatively high-throughput
assay, well suited for large-scale seroepidemiology
studies which are needed to better understand the
ecology and epidemiology of MERS-CoV.
Introduction
A novel lineage C beta-coroanvirus was isolated from
a patient with fatal viral pneumonia in Saudi Arabia in
2012 and termed Middle East respiratory syndrome cor-
onavirus (MERS-CoV) [1]. As of 3 September 2013, 108
human cases have been confirmed, 50 of which were
fatal [2]. Locally acquired cases have been reported
from Jordan, Qatar, Saudi Arabia and the United Arab
Emirates, and imported index cases, sometimes with
secondary local transmission, have been reported in
France, Germany, Italy, Tunisia and the United Kingdom
[2-4]. Clusters of cases suggestive of limited human-
to-human transmission have been reported; the larg-
est cluster of cases to date occurred at a healthcare
facility in Al-Hasa, Saudi Arabia [5]. The epidemiology
of the disease so far is suggestive of multiple zoonotic
transmissions from an animal reservoir leading to
human infection, sometimes with secondary transmis-
sion events in humans.
Phylogenetically closely related, although not identi-
cal, viruses have been found in insectivorous bats in
Africa and Europe [6,7]. More recently, a very short
fragment (181 bp) of the RNA-dependent RNA polymer-
ase gene that was genetically identical to MERS-CoV
has been detected in a Taphozous perforatus bat cap-
tured in the vicinity of the residence of a human case
with MERS [8]. These findings remain to be confirmed
with more definitive sequence data. Even if MERS-CoV
is found in bats, the possibility of an intermediate peri-
domestic host remains important to explore.
Since antibody responses following coronavirus infec-
tion remain detectable for many years [9], seroepidemi-
ology of potential animal species for MERS-CoV-specific
antibody is a logical approach to identify candidate
species for further investigation. A recent report sug-
gests that MERS-CoV antibody was detected in drom-
edary camels in the Arabian peninsula [10]. While a
number of serological tests, including ELISA assays,
immunoflourescence assays and immunoassays using
recombinant viral proteins have been used for detect-
ing serological responses in infected humans [11,12],
virus neutralisation is the most specific serological
2. 2 www.eurosurveillance.org
test and currently considered the gold-standard.
However, virus neutralisation requires the handling of
live virus and requires Biosafety Level 3 containment.
We have therefore developed a pseudoparticle neutral-
isation (ppNT) assay where the spike protein of MERS-
CoV is expressed by a replication-incompetent human
immunodeficiency (HIV) virus that contains a luciferase
reporter gene. Similar pseudotype viruses have been
used successfully in serological tests for severe acute
respiratory syndrome coronavirus (SARS-CoV) and
influenza viruses such as the highly pathogenic avian
influenza A(H5N1) virus [13]. Pseudotyped MERS-CoV
has been used to study the mechanisms of virus entry,
and it has been shown that cell transduction by such
particles is blocked by neutralising antibodies specific
for MERS-CoV [14].
The geographical distribution of MERS-CoV in its ani-
mal reservoir is not defined. Being a Middle Eastern
country with an ecology and domestic livestock prac-
tices fairly similar to some countries where human
MERS infections have occurred, we reasoned that
Egypt would be a relevant geographical location for
seroepidemiological studies. We have used both the
ppNT assay and conventional microneutralisation (MN)
tests to carry out seroepidemiological surveillance in
humans and livestock in Egypt. Human and animal sera
collected in Hong Kong were used as controls.
Methods
Sera from dromedary camels (n=110), water buffaloes
(n=8) and cows (n=25) were collected from two abat-
toirs, one located in Cairo and the second located in
the Qalyubia governorate in the Nile Delta region. The
dromedary camels were mostly imported from Sudan
for slaughter and were five to seven years-old. Upon
import, they were held on Egyptian farms for four to
five months before transport to the abattoirs in open
trucks. Sera from sheep (n=5) and goats (n=13) were
collected from backyard animals from a village in the
Nile Delta. All sera were collected in June 2013.
Human sera (n=815) were collected in 2012–13 as part
of an ongoing community-based seroepidemiological
study on influenza virus among healthy subjects in
Cairo and the Nile Delta region. The age range of the
subjects was between two and 79 years-old (median:
29 years). Fifty-eight per cent of the study subjects
were female.
Sera collected in Hong Kong served as un-exposed
controls. These included archived age-stratified
human sera (n=528) collected in Hong Kong in 2011 and
2012, with more than 50 sera from each decade of age
(range: <10 to 80 years-old). Swine sera (n=260) were
collected from an abattoir in Hong Kong in 2011 and
2012. Sera (n=204) from wild northern pintails (Anas
acuta) and Eurasian widgeons (Anas penelope) were
collected in December 2010 from the Mai Po wetlands
nature reserve in Hong Kong.
As positive controls, we used a convalescent serum
from a human patient with MERS, kindly provided by
Dr C Drosten (Institute of Virology, University of Bonn
Medical Centre, Bonn, Germany), and sera from two
experimentally infected macaques and a non-infected
control macaque kindly provided by Bart Haagmans
(Erasmus University Medical Center, Rotterdam, the
Netherlands).
An acute and convalescent serum from a patient with
SARS was used as a further negative control. The MN
antibody titre was <10 to SARS-CoV in the acute serum,
and 160 in the convalescent serum.
The study was approved by the institutional review
boards of the University of Hong Kong and St Jude
Children’s Research Hospital and the Ethics Committee
of the National Research Centre, Egypt.
Viruses and virus titration
MERS-CoV (strain EMC) virus was obtained from
Dr R Fouchier (Erasmus University Medical Center,
Rotterdam, the Netherlands). SARS-CoV (strain HKU-
39849) was taken from the virus repository at Hong
Kong University. Virus stock for MERS-CoV was pre-
pared in Vero cell culture (ATCC CCL-81) in minimal
essential medium containing 2% fetal bovine serum,
100 units/mL penicillin and 100 μg/mL streptomycin.
Virus aliquots were stored at -80 °C. Virus was titrated
in serial half-log10
dilutions (from 0.5 log to 7 log) to
obtain 50% tissue culture infectious dose (TCID50
) on
96-well tissue culture plates of Vero cells. The plates
were observed in a phase contrast microscope for cyto-
pathic effect (CPE) daily for three days. The endpoint of
viral dilution leading to CPE in 50% of inoculated wells
was estimated by using the Reed Muench method and
designated as one TCID50
. SARS-CoV was grown and
titrated in the same manner with the exception that
Vero E6 cells (ATCC CRL-1586) were used.
Microneutralisation tests
Serial two-fold dilutions of heat-inactivated sera (56 °C
for 30 minutes) were made, starting with a dilution of
1:10. The serum dilutions were mixed with equal vol-
umes of 200 TCID50
of MERS-CoV or SARS-CoV as indi-
cated. After 1 h of incubation at 37 °C, 35 µL of the
virus–serum mixture was added in quadruplicate to
Vero or Vero-E6 cell monolayers for MERS-CoV and
SARS-CoV, respectively, in 96-well microtiter plates.
After 1 h of adsorption, an additional 150 µL of cul-
ture medium were added to each well and the plates
incubated for three more days at 37 °C in 5% CO2
in a
humidified incubator. A virus back-titration was per-
formed without immune serum to assess input virus
dose. CPE was read at three days post infection. The
highest serum dilution that completely protected the
cells from CPE in half of the wells was taken as the
neutralising antibody titre and was estimated using
the Reed-Muench method. Positive and negative con-
trol sera were included to validate the assay.
3. 3www.eurosurveillance.org
MERS-CoV spike pseudoparticle
neutralisation assay
A codon-optimised spike gene was designed according
to published MERS-CoV genome sequence (GenBank
accession number: JX869059.1), synthesised by
GeneCust (Luxembourg) and subcloned into pcDNA3.1+
vector to generate pcDNA-S. To produce HIV/MERS
spike pseudoparticles, 10 µg pNL Luc E-
R-
and 10 µg
pcDNA-S were co-transfected into 4x106
293T cells [13].
Supernatants of transfected cells were harvested 48 h
later and quantified for HIV p24 viral protein using a
p24 ELISA Kit (Cell Biolabs, San Diego, United States).
For the ppNT assay, HIV/MERS pseudoparticles con-
taining 5 ng p24 were used to infect Vero E6 cells
(ATCC CRL-1586) in a single well (96-well plate format;
1x104
cells/well). Infected cells were lysed in 20 µl
lysis buffer and 100 µl of luciferase substrate at two
days postinfection (Promega Corporation, Madison,
United States). Luciferase activity was measured in a
Microbeta luminometer (PerkinElmer, Waltham, United
States).
For the ppNT, HIV/MERS pseudoparticles (5 ng of p24)
were pre-incubated with serially diluted sera for 30 min
at 4 °C and then added to cells in triplicate. Residual
virus replication was assayed at two days post infec-
tion, as described above. The highest serum dilu-
tion giving a 90% reduction of luciferase activity was
regarded as the ppNT antibody titre.
Results
Overall, 976 human and animal sera from Egypt and 992
human and animal sera from Hong Kong were tested by
MN at a screening dilution of 1:10 and 1:20 (Table 1).
None of the age-stratified human sera (n=528), swine
sera (n=260) or wild bird sera (n=204) collected in
Hong Kong had any neutralising activity for MERS-
CoV in the MN tests. Similarly, none of the sera from
humans (n=815), water buffaloes (n=8), cows (n=25),
sheep (n=5) and goats (n=13) collected in Egypt were
positive in the screening MN tests. In contrast, 103 of
110 sera collected in Egypt from dromedary camels
neutralised MERS-CoV at the screening dilution of 1:20
or higher.
Entry of MERS pseudoparticles was shown to be inhib-
ited by increasing concentrations of 0–20 mM NH4
Cl
(data not shown), demonstrating pH dependent entry of
the MERS pseudoparticles as previously reported [14].
The MERS ppNT assay was evaluated using two sera
from experimentally infected macaques, one negative
control serum from an uninfected macaque, a human
convalescent serum from a MERS patient and five neg-
ative human control sera from Hong Kong (Figure 1).
The MERS ppNT assay was then used to screen 115
human sera from Hong Kong and 100 randomly selected
human sera from Egypt which were all serologically
negative for MERS-CoV. Sixteen dromedary camel sera
that were positive in the MN screening assay were all
found to have a high neutralising activity in the ppNT
assay. In addition, five of six sera that were negative
in the MN assay had a weak, but detectable, activity
in the ppNT test (Table 1, Table 2, Figure 2). The camel
sera that were found to be positive at a screening dilu-
tion of 1:20 in the MN test had antibody titres in the
MERS NT screen ranging from 40 to 1,280 and higher,
and MERS ppNT titres ranging from 640 to 10,240 and
higher. One of the five MERS MN-negative sera was
negative in the MERS ppNT assay, while the other four
had low MERS ppNT titres ranging from 40 to 160.
Table 1
Screening results for MERS-CoV microneutralisation and MERS-CoV spike protein pseudoparticle neutralisation, human
and animal samples from Egypt and Hong Kong, 2012–2013 (n=1,968)
Sera Source of sera
MERS-CoV micro-neutralisation titre ≥1:20 MERS-CoV spike pseudotype antibody titre ≥1:20
Total tested % Positive (n) Total tested % Positive (n)
Humana
Egypt
815 0 (0/815) 100 0 (0/100)
Goatb
13 0 (0/13) ND ND
Sheepb
5 0 (0/5) ND ND
Water buffalob
8 0 (0/8) ND ND
Cowb
25 0 (0/25) ND ND
Camelb
110 93.6 (103/110) 110 98.2 (108/110)
Human
Hong Kong
528 0 (0/528) 115 0 (0/115)
Swine 260 0 (0/260) ND ND
Wild bird 204 0 (0/204) ND ND
MERS-CoV: Middle East respiratory syndrome coronavirus; ND: not done.
a
Collected in 2012–13.
b
Collected in June 2013.
Details of sera collected in Hong Kong as given in Methods.
4. 4 www.eurosurveillance.org
The correlation of the MERS MN and MERS ppNT titres
are shown in Figure 3 (Pearson’s correlation coef-
ficient: R=0.88). The MERS ppNT test appears to be
more sensitive than the MERS MN test, and thus some
of the apparently MN-negative camel sera give low
titre-positive results in the MERS ppNT assay. However,
none of the human sera from Egypt (n=100) or Hong
Kong (n=115) had any detectable antibody in the MERS
ppNT test. None of the camel sera with high antibody
titres to MERS-CoV had any cross-neutralising activity
to SARS-CoV (Table 2).
Discussion
Of 1,968 human and animal sera tested by MERS-CoV
MN and 325 human and animal sera tested by MERS-
CoV ppNT assays, only sera from dromedary camels
had any neutralising antibody activity to the MERS-
CoV. Of the 110 camel sera, 93.6% were seropositive
by MERS-CoV MN test and 98.2% were seropositive
by MERS-CoV ppNT test. The antibody titres were very
high in MN as well as ppNT, suggesting that the virus
infecting these camels was MERS-CoV virus itself or a
very closely related virus.
It is known that dromedary camels host bovine corona-
viruses (BCoV) which are lineage A beta-coronaviruses.
However cross-neutralisation between MERS-CoV (lin-
eage C beta-coronavirus) and BCoV was excluded by
Reusken and colleagues in their study of sera from
dromedary camels [10]. Furthermore, BCoV is antigeni-
cally closely related to the human coronavirus OC43.
Human beta-coronavirus lineage A viruses OC43 and
Figure 1
MERS-CoV spike protein pseudoparticle neutralisation, human and animal samples from Egypt and Hong Kong, 2012–13
(n=9)
CPS: counts per second; MERS-CoV: Middle East respiratory syndrome coronavirus;
As positive controls, we used a convalescent human serum (CHS) from a patient with MERS, kindly provided by Dr C Drosten (Institute of
Virology, University of Bonn Medical Centre, Bonn, Germany) and sera from two experimentally infected macaques (MAC1, MAC2), kindly
provided by Bart Haagmans (Erasmus University Medical Center, Rotterdam, the Netherlands). As negative controls we used serum from a
non-infected control macaque (NMS) and five human sera (NHS 1–5) from Hong Kong. The horizontal dotted line represents the 90% reduction
in luciferase activity which represents the cut-off for positivity in the assay. Each batch of assays had the cut-off determined with reference to
a serum-free negative control, and the data represented here are a compilation of two experiments. Thus the cut-off line is a representative
indication based on an average of cut-offs used in seperate experiments.
MAC 1
MAC 2
CHS
100
1,000
10,000
100,000
1,000,000
1:20 1:40 1:80 1:160 1:320 1:640 1:1,280 1:2,560 1:5,120
Luciferaseactivity(CPS)
Serum dilution
NMS
MAC 1
MAC 2
NHS 1
NHS 2
NHS 3
NHS 4
NHS 5
CHS
Cut-off
5. 5www.eurosurveillance.org
HKU1, and alpha-coronaviruses (229E and NL63) are
ubiquitous respiratory viruses infecting humans and
the panel of human sera of different ages tested can
be expected to have varying levels of antibody to these
viruses. The lack of any MERS-neutralising activity
in the human sera we studied also indicates that the
MN and ppNT assays are specific for MERS-CoV. The
lack of cross-reactivity with convalescent serum from
patients with SARS provides additional evidence of
the lack of cross-reactivity in the MERS-CoV serology
assays. Furthermore, it is notable that the camel sera
with high antibody titres to MERS-CoV did not cross-
react with SARS-CoV, a beta-coronavirus of lineage B.
Taken together these data indicate that a MERS-CoV or
a highly related virus is endemic in dromedary camels
imported for slaughter in Egypt. These findings pro-
vide independent confirmation of the results recently
reported by Reusken et al. who found very high anti-
body titres to MERS-CoV in dromedary camels [10].
The dromedary camels sampled in our study were
those brought to abattoirs for slaughter in Cairo and
in the Qalyubia governorate in the Nile Delta region.
These animals were sourced from other East African
countries such as Sudan and held in Egypt for some
time prior to slaughter. Thus it is unclear where the ani-
mals originally acquired the infection. Considering the
similar data from dromedary camels in Oman and the
Canary Islands [10], it is likely that this coronavirus is
widespread in North and East Africa and the Arabian
peninsula.
There is substantial movement of people between
Egypt and Saudi Arabia and other states on the Arabian
peninsula, and thus it is possible that people may get
infected, either as part of their travel to endemic areas
or through zoonotic transmission within the coun-
try. There is also much movement of livestock across
these Middle Eastern countries. The lack of antibody to
MERS-CoV in sera of people resident in Egypt indicates
Figure 2
MERS-CoV spike protein pseudoparticle neutralisation on selected sera from dromedary camels, Egypt, June, 2013 (n=21)
CPS: counts per second; MERS-CoV: Middle East respiratory syndrome coronavirus; MN: microneutralisation; ppNT: pseudoparticle
neutralisation.
Sixteen sera found to be positive and five sera found to be negative in the MERS-CoV MN screening assay were titrated in the MERS-CoV ppNT
assay. The sera used are shown in Table 2. The horizontal dotted line represents the 90% reduction in luciferase activity which represents the
cut-off for positivity in the assay.Each batch of assays had the cut-off determined with reference to a serum-free negative control and the data
represented here are a compilation of two experiments. Thus the cut-off line is a representative indication based on an average of cut-offs
used in seperate experiments.
10
100
1,000
10,000
100,000
1,000,000
1:20 1:40 1:80 1:160 1:320 1:640 1:1,280 1:2,560 1:5,120 1:10,240
Luciferaseactivity(CPS)
Serum dilution
C29
C101
C107
C108
C109
C110
C111
C112
C113
C115
C116
C117
C118
C119
C120
C121
C127
C132
C144
C147
C585
Cut-off
6. 6 www.eurosurveillance.org
that this infection is not common in Egypt, either as an
infection acquired through travel or as an occasional
zoonotic infection.
The MERS-CoV ppNT assay described here is a safe
and specific assay for large scale seroepidemiological
studies in a range of animal species, and such studies
are urgently needed in regions where MERS-CoV cases
have been detected as well as other regions. The HIV
backbone used for pseudoparticle production is not
replication-competent and the MERS-CoV pseudoparti-
cles can therefore be produced and used in Biosafety
Level 2 containment; in contrast, MN assays involve
handling of the live MERS-CoV and require Biosafety
Level 3 containment which is not always available in
affected regions. Unlike immunoassays, there is no
requirement for finding and optimising an enzyme-
labelled anti-Ig conjugate for each species to be
investigated. Furthermore, the MERS-CoV ppNT assay
appears around 10 times more sensitive than the con-
ventional MN assay (Figure 3, Table 2). The MN assay
is a neutralisation assay based on TCID50
rather than
a plaque reduction assay, which perhaps makes it less
sensitive than a plaque neutralisation assay. In any
event, experience with influenza virus serology using
pseudoparticle assays has shown that they are more
sensitive than conventional MN assays for detecting
neutralising antibodies. Thus MERS-CoV ppNT can be
used as a screening assay, and positive sera can be
retested for confirmation in a MERS MN tests.
Serological data does not provide proof that the virus
infecting dromedary camels is the MERS CoV, and
infection by a closely related coronavirus or a chimeric
virus with a MERS-CoV-like spike protein cannot be
ruled out until the dromedary camel virus is detected
and genetically sequenced. However, it provides a
strong impetus to attempt to seek the virus in speci-
mens from these animals and to identify the MERS-
related virus that appears to be infecting them. These
serological studies also need to be extended to other
domestic animals species to define the circulation of
MERS-CoV or related viruses in animals in close con-
tact with humans. Such studies should also include
humans exposed to dromedary camels. It is important
to note that waning antibody levels may result in false-
negative serology results, and this is particularly rel-
evant in mild or asymptomatic episodes of infection
where the peak antibody titre may be lower and drop
more quickly.
Table 2
Antibody titres of selected sera from dromedary camels
tested by microneutralisation for MERS-CoV and
SARS-CoV and by MERS spike protein pseudoparticle
neutralisation, Egypt, June, 2013 (n=21)
Camel sera
Antibody titres
MERS-CoV MN
test
SARS-CoV MN
test
MERS-CoV
ppNT test
C101 <10 Negative <10 Negative 40
C127 <10 Negative <10 Negative 160
C132 <10 Negative <10 Negative 40
C144 <10 Negative <10 Negative 160
C585 <10 Negative <10 Negative <20 Negative
C29 320 <10 Negative 2,560
C107 160 <10 Negative 5,120
C108 160 <10 Negative 5,120
C109 640 <10 Negative ≥10,240
C110 ≥1,280 <10 Negative ≥10,240
C111 320 <10 Negative 5,120
C112 320 <10 Negative 5,120
C113 320 <10 Negative 2,560
C115 160 <10 Negative 1,280
C116 320 <10 Negative 5,120
C117 640 <10 Negative 5,120
C118 640 <10 Negative 5,120
C119 80 <10 Negative 640
C120 40 <10 Negative 1,280
C121 160 <10 Negative 2,560
C147 ≥1,280 <10 Negative ≥10,240
MERS-CoV: Middle East respiratory syndrome coronavirus; MN:
microneutralisation; ppNT: pseudoparticle neutralisation; SARS-
CoV: severe acute respiratory syndrome coronavirus.
Figure 3
Correlation of MERS-CoV antibody titres determined by
MERS-CoV microneutralisation and MERS-CoV spike
protein pseudoparticle neutralisation in selected sera from
dromedary camels, Egypt, June, 2013 (n=21)
MERS-CoV: Middle East respiratory syndrome coronavirus; MN:
microneutralisation; ppNT: pseudoparticle neutralisation.
The data used as those shown in Table 2. In the event of
overlapping dots, their MN titre (X axis) was increased or
decreased by 0.05% to slightly offset the overlap for ease of
observation. The limit of detection in the MN and ppNT assays
were titres of 10 and 20 respectively; and thus these values on the
Y and X axis correspond to <10 and <20, respectively.
100
1,000
10,000
100 1,000 10,000 100,000<20
<10
Neutralising titre by MERS-CoV ppNT
NeutralisingtitrebyMERS-CoVMN
7. 7www.eurosurveillance.org
If the detection of MERS-CoV in insectivorous bats is
confirmed [8] and if indeed the coronavirus we and oth-
ers demonstrated to be common in dromedary camels
is confirmed to be MERS-CoV, we will have a scenario
of a virus reservoir in bats with a peridomestic animal
such as the camel as intermediate host, which may in
fact be the immediate source of human infection. It is
notable that a number of index cases with MERS-CoV
had a history of exposure to camels, although this is by
no means universally the case. Given that the MERS-
like coronavirus in camels appears to be ubiquitous, it
remains to be explained why MERS in humans appears
relatively rare. Coronaviruses are well known to mutate
to markedly change virulence or host range. Examples
are the emergence of the less pathogenic porcine res-
piratory coronavirus from virulent transmissible gas-
troenteritis virus of pigs, or virulent feline infectious
peritonitis viruses emerging from low pathogenic feline
coronaviruses [15]. Furthermore, the SARS-like virus
detected in civets and other small mammals in live
animal markets in southern China in 2002–03 initially
appeared to infect humans, who appear to have sero-
converted, but with minimal disease and onward trans-
mission [16], while a few amino acid changes in the
SARS-CoV spike protein allowed that virus to acquire
efficient transmissibility and virulence in humans [17].
Thus, previous experience with animal and human
coronaviruses highlights the public health urgency of
investigations of MERS-CoV and MERS-CoV-like viruses
in domestic and wild animals.
Acknowledgements
We thank Dr CYH Leung for providing wild bird sera from
Hong Kong. This study was supported in part by research
contract from the National Institute of Allergy and Infectious
Diseases (NIAID) contract HHSN266200700005C and a
grant from the European Community Seventh Framework
Programme (FP7/2007-2013) under project European man-
agement Platform for Emerging and Re-emerging Disease
entities (Grant agreement No. 223498) (EMPERIE).
Conflict of interest
None declared.
Authors’ contributions
Pei-gang Wang developed the MERS-CoV pseudotype assay
and carried out the tests. Ranawaka AMP Perera developed
the MERS-CoV microneutralisation test and carried out the
tests in BSL3 containment. Leo LLM Poon and Yi Guan pro-
vided advice on laboratory methods. Lewis YL Siu and Ming-
yuan Li carried out the MERS-CoV pseudoparticle assays.
Mokhtar R. Gomaa, Rabeh El-Shesheny, Ahmed Kandeil, Ola
Bagato, Mahmoud M. Shehata, Ahmed S. Kayed and Yassmin
Moatasim collected human and animal sera in Egypt. Richard
J. Webby and Mohamed A. Ali provided advice on field study
design. Joseph SM Peiris and Ghazi Kayali designed and co-
ordinated the study and wrote the manuscript. All authors
reviewed and commented on the manuscript.
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