Effects of co-infections on Marek's disease in poultry and development of novel recombinant Marek's disease virus vector vaccines

马立克氏病（MD）导致鸡的瘫痪和肿瘤。它是由马立克氏病病毒（MDV-1）的血清型1株引起的，这种病毒从受感染的鸡的皮肤上脱落，并在受污染的禽舍的灰尘中持续数月。它具有高度传染性，并通过吸入传播给其他鸡。MD是影响家禽健康、福利和生产力的主要疾病，全球家禽业每年估计损失20亿美元。MD在英国家禽中流行，但通过活疫苗病毒有效控制，活疫苗病毒是MDV-1的无害亲属，包括CVI 988、HVT和MDV血清型2（MDV-2）。MDV-2疫苗在美洲和亚洲广泛使用，但在英国没有使用。然而，通过检测从家禽养殖场收集的样本，我们发现MDV-2在英国广泛传播。MDV-2毒株以高水平自由和自然地传播，并在鸡群中长期存在，但人们对它们知之甚少;它们是来自进口家禽“逃逸”的疫苗毒株，还是天然存在的毒株？鸡可以同时感染MDV-1、MDV-2和疫苗病毒的任何组合（共感染）。我们已经在健康的鸡群中发现了MDV-2，以及患有MD的鸡群。我们想知道与MDV-2的共感染是否会影响鸡群健康和疾病，以及生产参数，如产蛋量和死亡率，以及某些MDV-2毒株是否可以用作有效的重组疫苗来对抗MD和其他家禽疾病。我们的目标是：（1）调查自然发生的MDV-2感染在田间的流行情况，以及它对鸡群生产力、免疫应答和疾病的影响，（2）鉴定MDV-2田间分离株，（3）开发新型MDV-2作为新型重组疫苗的潜在病毒载体。我们将选择两个MDV-2阳性和两个MDV-2阴性的鸡群，用于两个禽类（肉种鸡、蛋鸡）的定期采样，采集鸡的血液样本和鸡舍的灰尘样本。我们还将收集有关鸡群健康和生产力的数据。在Pirbright，我们将通过“聚合酶链反应”检测样本，以检测MDV-1、MDV-2和疫苗病毒的遗传物质，以显示MDV-2感染和散毒的动力学，以及MDV-1田间毒株和疫苗病毒的合并感染频率。利用数学模型，我们还将研究MDV-2在鸡群中的传播动力学。我们将通过对病毒遗传物质进行测序并与已知的MDV-2毒株进行比较来确定MDV-2毒株的变异性。我们将通过在细胞培养物中培养这些病毒，然后在受控的实验室条件下使用它们感染鸡来研究选定的MDV-2毒株的特征，以检查MDV-2的复制、持久性、临床体征和传播。大多数MDV-2毒株具有使其适合作为MD疫苗的特征：它们不引起疾病，它们在鸡中生长良好并持续数月，并且它们容易在鸡之间传播以维持鸡群对疫苗病毒的高水平暴露。此外，MDV-2可经遗传工程改造以携带来自其它重要家禽病毒病原体的基因，例如，传染性法氏囊病病毒（IBDV）和纽卡斯尔病病毒（NDV）;像这样的重组“载体疫苗”可能在单次接种中保护鸡抵抗IBD和ND以及MD。本研究将通过基因工程技术改造MDV-2毒株，构建“rMDV-2-IBD-ND”病毒，并在实验室条件下检测其对这三种疾病的保护作用。本研究对了解MDV-2广泛感染对商业家禽群健康和生产力的影响具有重要意义。一种新的重组MDV-2疫苗将是用于控制MD和其他家禽疾病的一组活病毒疫苗的有用补充。

Marek's disease (MD) causes paralysis and tumours in chickens. It is caused by serotype 1 strains of the Marek's disease virus (MDV-1) which is shed from skin of infected chickens and persists for many months in dust in contaminated poultry houses. It is highly contagious and spreads to other chickens by inhalation. MD is a major disease affecting poultry health, welfare, and productivity, with annual estimated loss to the global poultry industry of $2 billion. MD is endemic in UK poultry but is effectively controlled by live vaccine viruses, which are harmless relatives of MDV-1, and include CVI988, HVT, and MDV serotype 2 (MDV-2). MDV-2 vaccines are widely used in the Americas and Asia but not in the UK. However, by testing samples collected from poultry farms, we found MDV-2 is widespread in the UK. MDV-2 strains circulate freely and naturally at high levels and persist long-term in the flock, but little is known about them; are they derived from vaccine strains which 'escaped' from imported poultry, or are they naturally occurring strains? Chickens can be infected with any combination of MDV-1, MDV-2, and vaccine viruses at the same time (co-infection). We have found MDV-2 in healthy chicken flocks, as well as flocks that have MD. We would like to know whether co-infection with MDV-2 affects flock health and disease, and production parameters such as egg production and mortality, and whether certain MDV-2 strains could be used as effective recombinant vaccines against MD and other poultry diseases. Our objectives are to: (1) Investigate prevalence of naturally occurring MDV-2 infection in the field, and it's influence on flock productivity, immune responses and disease, (2) Characterise MDV-2 field isolates and (3) Exploit novel MDV-2 as potential viral vectors for novel recombinant vaccines.The project is a partnership with poultry industry vets. We will select two MDV-2-positive and two MDV-2-negative flocks for two bird types (broiler-breeder, layer) for regular sampling to collect blood samples from chickens and dust from the housing sheds. We will also collect data on flock health and productivity. At Pirbright, we will test the samples by 'polymerase chain reaction' to detect the genetic material of MDV-1, MDV-2 and vaccine viruses to show the kinetics of MDV-2 infection and shedding, and the frequency of co-infection with MDV-1 field strains and vaccine viruses. Using mathematical modelling, we will also investigate dynamics of transmission of MDV-2 within flocks. We will determine variability of MDV-2 strains by sequencing the virus genetic material and comparing with known MDV-2 strains. We will study the characteristics of selected MDV-2 strains by growing these viruses in cell culture then using them to infect chickens under controlled laboratory conditions to examine replication, persistence, clinical signs and transmission of MDV-2. Most MDV-2 strains have characteristics which make them suitable as vaccines against MD: they do not cause disease, they grow well in the chicken and persist for many months, and they are easily transmitted between chickens to maintain a high level of exposure of the flock to vaccine virus. Furthermore, MDV-2 can be genetically engineered to carry genes from other important poultry viral pathogens, e.g., infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV); a recombinant 'vectored vaccine' like this could potentially protect chickens against IBD and ND as well as MD in a single vaccination. We will engineer an appropriate MDV-2 strain to create a 'rMDV2-IBD-ND' virus, then test its ability to protect chickens against these three diseases under controlled laboratory conditions.This study is important to understand the effect of widespread MDV-2 infection on health and productivity of commercial poultry flocks. A new recombinant MDV-2 vaccine would be a useful addition to the set of live virus vaccines used to control MD and other poultry diseases.