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Foot-and-Mouth Disease Virus Persists in the Light Zone of Germinal Centres
Nicholas Juleff<sup>1</sup><sup>,</sup><sup>2</sup><sup>*</sup>, Miriam Windsor<sup>1</sup>, Elizabeth Reid<sup>1</sup>, Julian Seago<sup>1</sup>, Zhidong Zhang<sup>1</sup>, Paul Monaghan<sup>1</sup>, Ivan W. Morrison<sup>2</sup>, Bryan Charleston<sup>1</sup>
1 Pirbright Laboratory, Institute for Animal Health, Woking, Surrey, United Kingdom, 2 Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, United Kingdom
Abstract
Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses of animals and is recognised as the most important constraint to international trade in animals and animal products. Two fundamental problems remain to be understood before more effective control measures can be put in place. These problems are the FMDV ?carrier state? and the short duration of immunity after vaccination which contrasts with prolonged immunity after natural infection. Here we show by laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborate by in situ hybridization that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of na?ve cattle. We propose that maintenance of non-replicating FMDV in these sites represents a source of persisting infectious virus and also contributes to the generation of long-lasting antibody responses against neutralising epitopes of the virus.
Citation: Juleff N, Windsor M, Reid E, Seago J, Zhang Z, et al. (2008) Foot-and-Mouth Disease Virus Persists in the Light Zone of Germinal Centres. PLoS ONE 3(10): e3434. doi:10.1371/journal.pone.0003434
Editor: Peter Sommer, Institut Pasteur Korea, Republic of Korea
Received: June 20, 2008; Accepted: September 23, 2008; Published: October 20, 2008
Copyright: ? 2008 Juleff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was funded by the Biotechnology and Biological Sciences Research Council, United Kingdom. B.C. is a Jenner Investigator. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: nicholas.juleff@bbsrc.ac.uk
Introduction
Foot-and-mouth disease virus (FMDV) causes an acute vesicular disease which is endemic throughout large parts of Asia, Africa and South America [1]. Although the disease has been eradicated in Europe and North America, introduction of infection, as occurred in the United Kingdom in 2001, can cause devastating outbreaks of disease [2]. Despite the availability of vaccines, control of such outbreaks continues to rely on detection and slaughter of affected herds [1]. One of the features of FMDV infection that has a major impact on control policies is the ?carrier state? [3]. A carrier of FMDV is defined as an animal from which live-virus can be recovered from scrapings of the oropharynx after 28 days following infection [4]. Over 50% of ruminants exposed to viral challenge, whether vaccinated or not, can become carriers [1]. Recovery of infectious virus from oropharyngeal scrapings of foot-and-mouth disease (FMD) recovered cattle is intermittent and the titre of virus recovered from carrier animals is low, often falling below the level thought to be necessary for successful transmission to susceptible animals [5]. Intermittent virus recovery may be related to the heterogeneous nature of oropharyngeal samples with saliva, mucus and cells present in varying quantities [3]. Although such carrier animals have never convincingly been directly demonstrated to transmit infection, they are perceived as a potential source of new infections and consequently there is a reluctance to use vaccination as a primary means of controlling outbreaks in disease-free countries [3]. Despite the potential epidemiological and immunological significance, very little is known about the mechanism by which the ?carrier state? is established or maintained. In one series of experiments, carriers were treated with dexamethasone in order to depress their immune systems, and kept in contact with susceptible cattle, but this had the reverse effect of causing the virus to disappear from oropharyngeal scrapings, only to reappear once the treatment was stopped [6]. There was no transmission between carrier and susceptible cattle. Despite the uncertainty concerning the capacity of the carrier animals to transmit virus, there is a requirement to identify and remove these animals before a country or region can declare freedom from infection and resume international animal trade. Hence the infection status of a country can have a profound impact on its economy [2].
Virus is cleared rapidly from blood during the acute stage of FMD, coinciding closely with the emergence of an antiviral antibody response. Viral RNA is detected in the blood of infected cattle, using real-time reverse transcription polymerase chain reaction (rRT-PCR), but becomes undetectable from as early as 3 to 5 days after onset of clinical signs. This is in contrast to pharyngeal tissue including the soft palate, nasopharynx, oropharynx, palatine tonsil and mandibular lymph node which have been shown to contain viral RNA for up to 72 days after infection [7]. The significance of continued detection of viral RNA has not been clear since FMDV proteins have not been detected, in previous studies in these tissues, following the resolution of vesicular lesions. Importantly, prior to this publication, FMDV proteins have not been detected previously in lymphoid tissue in vivo at any stage of infection and viral proteins have not been detected in any tissue following resolution of vesicular lesions.
Nicholas Juleff<sup>1</sup><sup>,</sup><sup>2</sup><sup>*</sup>, Miriam Windsor<sup>1</sup>, Elizabeth Reid<sup>1</sup>, Julian Seago<sup>1</sup>, Zhidong Zhang<sup>1</sup>, Paul Monaghan<sup>1</sup>, Ivan W. Morrison<sup>2</sup>, Bryan Charleston<sup>1</sup>
1 Pirbright Laboratory, Institute for Animal Health, Woking, Surrey, United Kingdom, 2 Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush Veterinary Centre, Roslin, Midlothian, United Kingdom
Abstract
Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses of animals and is recognised as the most important constraint to international trade in animals and animal products. Two fundamental problems remain to be understood before more effective control measures can be put in place. These problems are the FMDV ?carrier state? and the short duration of immunity after vaccination which contrasts with prolonged immunity after natural infection. Here we show by laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborate by in situ hybridization that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of na?ve cattle. We propose that maintenance of non-replicating FMDV in these sites represents a source of persisting infectious virus and also contributes to the generation of long-lasting antibody responses against neutralising epitopes of the virus.
Citation: Juleff N, Windsor M, Reid E, Seago J, Zhang Z, et al. (2008) Foot-and-Mouth Disease Virus Persists in the Light Zone of Germinal Centres. PLoS ONE 3(10): e3434. doi:10.1371/journal.pone.0003434
Editor: Peter Sommer, Institut Pasteur Korea, Republic of Korea
Received: June 20, 2008; Accepted: September 23, 2008; Published: October 20, 2008
Copyright: ? 2008 Juleff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was funded by the Biotechnology and Biological Sciences Research Council, United Kingdom. B.C. is a Jenner Investigator. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: nicholas.juleff@bbsrc.ac.uk
Introduction
Foot-and-mouth disease virus (FMDV) causes an acute vesicular disease which is endemic throughout large parts of Asia, Africa and South America [1]. Although the disease has been eradicated in Europe and North America, introduction of infection, as occurred in the United Kingdom in 2001, can cause devastating outbreaks of disease [2]. Despite the availability of vaccines, control of such outbreaks continues to rely on detection and slaughter of affected herds [1]. One of the features of FMDV infection that has a major impact on control policies is the ?carrier state? [3]. A carrier of FMDV is defined as an animal from which live-virus can be recovered from scrapings of the oropharynx after 28 days following infection [4]. Over 50% of ruminants exposed to viral challenge, whether vaccinated or not, can become carriers [1]. Recovery of infectious virus from oropharyngeal scrapings of foot-and-mouth disease (FMD) recovered cattle is intermittent and the titre of virus recovered from carrier animals is low, often falling below the level thought to be necessary for successful transmission to susceptible animals [5]. Intermittent virus recovery may be related to the heterogeneous nature of oropharyngeal samples with saliva, mucus and cells present in varying quantities [3]. Although such carrier animals have never convincingly been directly demonstrated to transmit infection, they are perceived as a potential source of new infections and consequently there is a reluctance to use vaccination as a primary means of controlling outbreaks in disease-free countries [3]. Despite the potential epidemiological and immunological significance, very little is known about the mechanism by which the ?carrier state? is established or maintained. In one series of experiments, carriers were treated with dexamethasone in order to depress their immune systems, and kept in contact with susceptible cattle, but this had the reverse effect of causing the virus to disappear from oropharyngeal scrapings, only to reappear once the treatment was stopped [6]. There was no transmission between carrier and susceptible cattle. Despite the uncertainty concerning the capacity of the carrier animals to transmit virus, there is a requirement to identify and remove these animals before a country or region can declare freedom from infection and resume international animal trade. Hence the infection status of a country can have a profound impact on its economy [2].
Virus is cleared rapidly from blood during the acute stage of FMD, coinciding closely with the emergence of an antiviral antibody response. Viral RNA is detected in the blood of infected cattle, using real-time reverse transcription polymerase chain reaction (rRT-PCR), but becomes undetectable from as early as 3 to 5 days after onset of clinical signs. This is in contrast to pharyngeal tissue including the soft palate, nasopharynx, oropharynx, palatine tonsil and mandibular lymph node which have been shown to contain viral RNA for up to 72 days after infection [7]. The significance of continued detection of viral RNA has not been clear since FMDV proteins have not been detected, in previous studies in these tissues, following the resolution of vesicular lesions. Importantly, prior to this publication, FMDV proteins have not been detected previously in lymphoid tissue in vivo at any stage of infection and viral proteins have not been detected in any tissue following resolution of vesicular lesions.