Patients with Cancer Appear More Vulnerable to SARS-CoV-2: A Multicenter Study during the COVID-19 Outbreak
2020

Mengyuan Dai
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Dianbo Liu
Computational Health Informatics Program
Boston Chil-dren's HospitalBostonMassachusetts

Department of Pediatrics
Har-vard Medical SchoolBostonMassachusetts

Miao Liu
Department of Pathology, Brigham and Women's Hospital
Department of Oncology
Harvard Medical School
Boston, Mas-sachusetts. 7

Zhongnan Hospital of Wuhan Uni-versity
WuhanHubeiChina

Fuxiang Zhou
Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Guiling Li
Cancer Center, Union Hospital affi liated to Tongji Medical College of Huazhong University of Science and Technology
Wuhan, HubeiChina

Zhen Chen
Department of Emergency
The Central Hospital of Wuhan affi liated to Tongji Medical College of Huazhong University of Sci-ence and Technology
Wuhan, HubeiChina

Zhian Zhang
Department of Gynecology
11 Affi liated Cancer Hospital and Institute of Guangzhou Medical University
The Central Hospital of Huanggang
Huanggang, Hubei, Guang-zhou, Guangdong ProvinceChina, China

Hua You
Meng Wu
Department of Ultrasound
Zhongnan Hospital of Wuhan University
WuhanChina

Qichao Zheng
Department of Ultrasound
Zhongnan Hospital of Wuhan University
WuhanChina

Yong Xiong
Department of Infectious Disease
Zhongnan Hospital of Wuhan University
WuhanChina

Huihua Xiong
Cancer Center, Tongji Hospital affi liated to Tongji Medical College of Huazhong University of Science and Technology
Wuhan, HubeiChina

Chun Wang
Department of Oncology
The Central Hospital of Wuhan affi liated to Tongji Medi-cal College of Huazhong University of Science and Technology
Wuhan, HubeiChina

Changchun Chen
Department of Radiology
Hubei Cancer Hospital
Wuhan, HubeiChina

Fei Xiong
Department of Thoracic Surgery
Hubei Cancer Hospital
Wuhan, HubeiChina

Yan Zhang
Department of Obstetrics and Gynecology
Renmin Hospital of Wuhan University
WuhanChina

Yaqin Peng
Department of Obstetrics and Gynecology
Renmin Hospital of Wuhan University
WuhanChina

Siping Ge
Department of Oncology
Wuhan Puren Hospital
Wuhan, HubeiChina

Bo Zhen
Department of Obstetrics and Gynecology
The Central Hospital of Xianning
XianningHubeiChina

Tingting Yu
Department of Oncology
The Central Hospital of Xiaogan
XiaoganChina

Ling Wang
Department of Obstetrics and Gynecology
The People's Hospital of Huangmei
HuangmeiHubeiChina

Hua Wang
Department of Obstetrics and Gynecology
Xiangyang First People's Hospital affi liated to Hubei Univer-sity of Medicine
XiangyangHubeiChina

Yu Liu
Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Yeshan Chen
Cancer Center, Union Hospital affi liated to Tongji Medical College of Huazhong University of Science and Technology
Wuhan, HubeiChina

Junhua Mei
Department of Gynecology
11 Affi liated Cancer Hospital and Institute of Guangzhou Medical University
The Central Hospital of Huanggang
Huanggang, Hubei, Guang-zhou, Guangdong ProvinceChina, China

Xiaojia Gao
Department of Oncology
The Central Hospital of Wuhan affi liated to Tongji Medi-cal College of Huazhong University of Science and Technology
Wuhan, HubeiChina

Zhuyan Li
Department of Obstetrics and Gynecology
The People's Hospital of Shiyan
ShiyanHubeiChina

Lijuan Gan
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Can He
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Zhen Li
Yuying Shi
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Yuwen Qi
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Jing Yang
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Daniel G Tenen
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Hubei Cancer Clinical Study Center
WuhanChina

Har-vard Stem Cell Institute
Cancer Science Institute of Singapore
Harvard Medical School
BostonMassachusetts. 26

School of Public Health
National University of Singapore
Singapore. 27 Harvard T.H. Chan, Dana-Farber, Har-vard Cancer CenterBostonMassachusetts

Zhongnan Hospital of Wuhan University
Wuhan CityChina

Bos-ton Children's Hospital
Zhongnan Hospital of Wuhan Uni-versity
430071WuhanHubeiChina

Department of Pathology, Brigham and Women's Hospital
Harvard Medical School
Harvard Medical School
Boston, BosMA

Li Chai
Department of Pathology, Brigham and Women's Hospital
Department of Oncology
Harvard Medical School
Boston, Mas-sachusetts. 7

Zhongnan Hospital of Wuhan Uni-versity
WuhanHubeiChina

Lorelei A Mucci
Mauricio Santillana
Computational Health Informatics Program
Boston Chil-dren's HospitalBostonMassachusetts

Department of Pediatrics
Har-vard Medical SchoolBostonMassachusetts

Hongbing Cai
Department of Gynecological Oncology
Zhongnan Hospital of Wuhan University
WuhanHubeiChina

Hubei Key Laboratory of Tumor Bio-logical Behaviors
WuhanChina

Dai
Hubei Cancer Clinical Study Center
WuhanChina

M Dai
D Liu
M Liu
Hongbing Cai
Miao Liu

RESEARCH BRIEF

Patients with Cancer Appear More Vulnerable to SARS-CoV-2: A Multicenter Study during the COVID-19 Outbreak

Cancer Discov
10202010.1158/2159-8290.CD-20-0422june 2020 CANCER DISCOVERY | 783 note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). Cancer Research. on November 18 RESEARCH BRIEF 784 | CANCER DISCOVERY june 2020 contributed equally to this article. Current address for H. Cai:
The novel COVID-19 outbreak has affected more than 200 countries and territories as of March 2020. Given that patients with cancer are generally more vulnerable to infections, systematic analysis of diverse cohorts of patients with cancer affected by COVID-19 is needed. We performed a multicenter study including 105 patients with cancer and 536 age-matched noncancer patients confi rmed with COVID-19. Our results showed COVID-19 patients with cancer had higher risks in all severe outcomes. Patients with hematologic cancer, lung cancer, or with metastatic cancer (stage IV) had the highest frequency of severe events. Patients with nonmetastatic cancer experienced similar frequencies of severe conditions to those observed in patients without cancer. Patients who received surgery had higher risks of having severe events, whereas patients who underwent only radiotherapy did not demonstrate signifi cant differences in severe events when compared with patients without cancer. These fi ndings indicate that patients with cancer appear more vulnerable to SARS-CoV-2 outbreak. SIGnIFICAnCE: Because this is the fi rst large cohort study on this topic, our report will provide muchneeded information that will benefi t patients with cancer globally. As such, we believe it is extremely important that our study be disseminated widely to alert clinicians and patients.

INTRODUCTION

A new acute respiratory syndrome coronavirus, named SARS-CoV-2 by the World Health Organization (WHO), has rapidly spread around the world since its first reported case in late December 2019 from Wuhan, China (1). As of March 2020, this virus has affected more than 200 countries and territories, infecting more than 800,000 individuals and causing more than 40,000 deaths (2).

With more than 18 million new cases per year globally, cancer affects a significant portion of the population. Individuals affected by cancer are more susceptible to infections due to coexisting chronic diseases, overall poor health status, and systemic immunosuppressive states caused by both cancer and anticancer treatments (3). As a consequence, patients with cancer who are infected by the SARS-CoV-2 coronavirus may experience more difficult outcomes than other populations. Until now, there is still no systematic evaluation of the effects that the SARS-CoV-2 coronavirus has of patients with cancer in a representative population. A recent study reported a higher risk of severe events in patients with cancer when compared with patients without cancer (4); however, the small sample size of SARS-CoV-2 patients with cancer used in the study limited how representative it was of the whole population and made it difficult to conduct more insightful analyses, such as comparing clinical characteristics of patients with different types of cancer, as well as anticancer treatments (5,6).

Using patient information collected from 14 hospitals in Hubei Province, China, the epicenter of the 2019-2020 COVID-19 outbreak, we describe the clinical characteristics and outcomes [death, intensive care unit (ICU) admission, development of severe/critical symptoms, and utilization of invasive mechanical ventilation] of patients affected by the SARS-CoV-2 coronavirus for 105 hospitalized patients with cancer and 536 patients without cancer. We document our findings for different cancer types and stages, as well as different types of cancer treatments. We believe the information and insights provided in this study will help improve our understanding of the effects of SARS-CoV-2 in patients with cancer.

RESULTS

Patients Characteristics

In total, 105 COVID-19 patients with cancer were enrolled in our study for the time period January 1, 2020, to February 24, 2020, from 14 hospitals in Wuhan, China. COVID-19 patients without cancer matched by the same hospital, hospitalization time, and age were randomly selected as our control group. Our patient population included 339 females and 302 males. Patients with cancer [median = 64.00, interquar-tile range (IQR) = 14.00], when compared with those without cancer (median = 63.50, IQR = 14.00) had similar age distributions (by design), experienced more in-hospital infections [20 (19. 1A). We also conducted survival analysis on occurrence of any severe condition which included death, ICU admission, having severe symptoms, and utilization of invasive mechanical ventilation (see cumulative incidence curves in Fig. 1B). In general, patients with cancer deteriorated more rapidly than those without cancer. These observations are consistent with logistic regression results ( Supplementary Fig. S1), after adjusting for age, sex, smoking, and comorbidities including diabetes, hypertension, and chronic obstructive pulmonary disease (COPD). According to our multivariate logistic regression results, patients with cancer still had an excess OR of 2.17 (P = 0.06) for death ( Supplementary Fig. S1A Table  S1). The consistency of observed ORs between the multivariate regression model and unadjusted calculation reassures the association between cancer and severe events even in the presence of other factors such as age differences.

Cancer Types

Information regarding potential risks of severe conditions in SARS-CoV-2 associated with each type of cancer was calculated. We compared different conditions among cancer types (

Timeline of Severe Events

To evaluate the time-dependent evolution of the disease, we conducted the timeline of different events for COVID-19 patients with cancer ( Fig. 2A ) and COVID-19 patients without cancer ( Fig. 2B ) with death and other severe events marked in the fi gure. COVID-19 patients with cancer had a mean length of stay of 27.01 days (SD 9.52) and patients without cancer had a mean length of stay of 17.75 days (SD 8.64); the difference is signifi cant (Wilcoxon test, P < 0.01). To better clarify the contributing factors that might infl uence outcomes, we also included logistic regression of COVID-19 patients with cancer adjusted by immunosuppression levels in Supplementary Table S5. However, no signifi cant association between immunosuppression and severe outcomes was observed from the analysis (with all P > 0.05).

DISCUSSION

The fi ndings in this study suggest that patients with cancer infected with SARS-CoV-2 tend to have more severe outcomes when compared with patients without cancer. Patients with hematologic cancer, lung cancer, and cancers in metastatic stages demonstrated higher rates of severe events compared with patients without cancer. In addition, patients who underwent cancer surgery showed higher death rates and higher chances of having critical symptoms.

The SARS-CoV-2 virus has spread rapidly globally; thus, many countries have not been ready to handle the large volume of people affected by this outbreak due to a lack of knowledge about how this coronavirus affects the general population. To date, reports on the general population infected with SARS-CoV-2 suggest elderly males have a higher incidence and death rate ( 7,8 ). Limited information is known about the outcome of patients with cancer who contract this highly communicable disease. Cancer is among the top causes of death. Asia, Europe, and North America have the highest incidence of cancer in the world ( 9 ), and at the moment of the writing of this study the SARS-CoV-2 virus is mainly spreading in these three areas (referred from https://www.cdc.gov/ media/releases/2020/s0226-Covid-19-spread.html ; https://www. nytimes.com/2020/02/27/world/coronavirusnews.html ). Although COVID-19 patients with cancer may share some epidemiologic features with the general population with this disease, they may also have additional clinical characteristics. Therefore, we conducted this study on patients with cancer with coexisting COVID-19 disease to evaluate the potential effect of COVID-19 on patients with cancer.

On the basis of our analysis, COVID-19 patients with cancer tend to have more severe outcomes when compared with the noncancer population. Although COVID-19 is reported to have a relatively low death rate of 2% to 3% in the general population ( 10 ), patients with cancer and COVID-19 not only have a nearly 3-fold increase in the death rate than that Cancer Research.

on November 18, 2021. © 2020 American Association for cancerdiscovery.aacrjournals.org Downloaded from of COVID-19 patients without cancer, but also tend to have much higher severity of their illness. Altogether, these findings suggest that patients with cancer are a much more vulnerable population in the current COVID-19 outbreak. Our findings are consistent with those presented in a previous study based on 18 patients with cancer (4). Because of the limited number of patients with cancer in the previous study, the authors concluded that among patients with cancer, age is the only risk factor for the severity of the illness. On the basis of our data on 105 patients with cancer, we have discovered additional risk factors, including cancer types, cancer stage, and cancer treatments, which may contribute to the severity of the disease among patients with cancer.

Our data demonstrate that the severity of SARS-CoV-2 infection in patients is significantly affected by the types of tumors. From our analysis, patients with hematologic cancer have the highest severity and death rates among all patients with cancer, and lung cancer follows second. Patients with hematologic cancer in our study include patients with leukemia, myeloma, and lymphoma, who have a more compro-mised immune system than patients with solid tumors (11). These patients all had a rapidly deteriorating clinical course once infected with COVID-19. Because malignant or dysfunctional plasma cells, lymphocytes, or white blood cells in general in hematologic malignancies have decreased immunologic function (12)(13)(14), this could be the main reason why patients with hematologic cancer have very high severity and death rates. All patients with hematologic cancer are prone to the complications of serious infection (12)(13)(14), which can exacerbate the condition which could have worsened in patients with COVID-19. In our study, 55.56% of patients with hematologic cancer had severe immunosuppression, which may be the main reason for deteriorated outcomes. Although the small sample size limits representativity of the observation, we believe our finding can serve as an informative starting point for further investigation when a larger cohort from a wide range of healthcare providers becomes available. Among solid tumors, lung cancer is the highest risk category disease in patients with SARS-CoV-2 infection (Fig.  1C). Decreased lung function and severe infection in patients  Cancer Research.

on November 18, 2021. © 2020 American Association for cancerdiscovery.aacrjournals.org Downloaded from with lung cancer could contribute to the worse outcome in this subpopulation (15,16).

In our analysis, we classified the SARS-CoV-2 infectionrelated high risk factors based on death, severe or critical illness, ICU admission, and the utilization of invasive mechanical ventilation. Using these parameters, we detected a multi-fold increase in risk in the cancer population, in contrast to the noncancer population. If there were primary or metastatic tumors in the lungs, patients were more prone to a deteriorated course in a short time. Intriguingly, when patients with cancer had only early-stage disease without metastasis, we did not observe any difference between the cancer and noncancer population in terms of COVID-19related death rate or severity (Fig. 1E). The stage of cancer diagnosis seemed to play a significant role in the severity and death rate of COVID-19.

Patients with cancer received a wide range of treatments, and we also found that different types of treatments had different influences on severity and death when these patients contracted COVID-19. Recently, immunotherapy has assumed a very important role in treating tumors, which aids in treatment of cancer by blocking the immune escape of cancer cells. But in our study, in contrast to patients with cancer with other treatments, patients with immunotherapy had the highest death rate and the highest severity of illness, a very puzzling finding. According to pathologic studies on the patients with COVID-19, there were desquamation of pneumocytes and hyaline membrane formation, implying that these patients had acute respiratory distress syndrome (ARDS; ref. 17). ARDS induced by cytokine storm is reported to be the main reason for death of SARS-CoV-2-infected patients (18). It is possible that in this setting, immunotherapy induces the release of a large amount of cytokines, which can be toxic to normal cells, including lung epithelial cells (19)(20)(21), and therefore lead to a more severe illness. However, in this study the number of patients with immunotherapy was too small; further research with a large case population needs to be conducted in the future.

In addition, COVID-19 patients with cancer who are under active treatment or not under active treatment do not show differences in their outcomes, and there is a significant difference between COVID-19 patients with cancer but not with active treatment and patients without cancer (Supplementary Table S2). These results indicate that COVID-19 patients with both active treatment and just cancer history have a higher risk of developing severe events than noncancer COVID-19 patients. The possible reasons could be due to some known cancer-related complications, for example, anemia, hypoproteinaemia, or dyspnea in early phase of COVID-19 (22). We considered that cancer had a lifetime effect on patients and that cancer survivors always need routine follow-up after primary resection. Therefore, in clinical COVID-19 patient management, equivalent attention needs to be paid to those with cancer whether they are under active therapeutics or not during the outbreak of COVID-19.

This study has several limitations. Although the cohort of COVID-19 patients with cancer is one of the largest in Hubei province, China, the epicenter of the initial outbreak, a larger cohort from the whole country or even from multiple countries will be more representative. Large-scale national and international research collaboration will be necessary to achieve this. At the initial stage of the outbreak, data collection and research activities were not a priority of the hospitals. Therefore, it was not possible to record and collect some data that are potentially informative for our analysis in a timely manner. In addition, due to the urgency of clinical treatment, medical data used in this study were largely disconnected from the patients' historical electronic medical records, which are mostly stored with a different healthcare provider than the medical center providing COVID-19 care. This left us with limited information about each patient.

Our study is the midsize cohort study on this topic and will provide much-needed information on risk factors of this population. We hope that our findings will help countries better protect patients with cancer affected by the ongoing COVID-19 pandemic.

METHODS

Study Design and Patients

We conducted a multicenter study focusing on the clinical characteristics of confirmed cases of COVID-19 patients with cancer in 14 hospitals in Hubei province, China; all of the 14 hospitals served as government-designated hospitals for patients diagnosed with COVID-19 (listed in the Supplementary Data). COVID-19 was diagnosed according to the WHO interim guidance (23). Patients with cancer confirmed with COVID-19 who were hospitalized from January 1, 2020, to February 24, 2020, were enrolled. SARS-CoV-2-infected patients without cancer matched by the same hospital and hospitalization time were randomly selected as our control group. In addition, as age is one of the major predictors of severity of respiratory diseases like COVID-19 (4), we excluded from our analysis 117 younger COVID-19 patients without cancer so that median ages of patients with cancer (median = 64.0, IRQ = 14.00) and patients without cancers (median = 63.5, IQR = 14.00) would be comparable. Four clinical outcomes were monitored up to February 24, 2020, the final date of follow-up. This case series was approved by the Institutional Ethics Board of Zhongnan Hospital of Wuhan University (Wuhan, Hubei, China, No. 2020029). It is worth pointing out that because of limited resources and information available in early stages of this outbreak, sample sizes of some subgroups were small and collection of data for patients with better matched clinical characteristics was not possible. This limited this study design.

Procedures

Medical records of patients were acquired by the data collection team of the above 14 hospitals. A team of physicians who had been taking care of patients with SARS-CoV-2 infection reviewed the data. We used a standardized case-report form to collect clinical data. Primary cancer characteristics and detailed treatment information were extracted from past medical records by oncologists in the Hubei Anti-Cancer Association. Data were entered into a computerized database. Cases with insufficient records of previous disease history were excluded. Information collected included demographic data, medical history, comorbidities, symptoms, vital signs, routine blood test, and chest CT scans. Only cancer treatments within 40 days before the onset of COVID-19 symptoms were considered for this study. Each patient's medical record was reviewed by at least two oncologists. Our institutional ethics review board approved the study and waived the need for informed consent.

End Points and Assessments

There were four primary outcomes analyzed in this study: death, admission into the ICU, development of severe or critical symptoms, and utilization of invasive mechanical ventilation. The clinical definition of severe/critical symptoms follows the 5th edition of the 2019 Novel Coronavirus Disease (COVID-19) Diagnostic Criteria published by the National Health Commission in China, including septic shock, ARDS, acute kidney injury, disseminated intravascular coagulation, and rhabdomyolysis.

Statistical Analysis

The aim of this study was to report clinical outcomes of COVID-19 patients with cancer. For categorical data, percentages of patients in each category were calculated. The Wilcoxon rank sum test was used to compare continuous data, and the Fisher exact test was used to compare categorical data from different categories without multitest adjustment. Multivariate logistic regression was used to estimate ORs and 95% CIs of each factor of interest with outcomes after data was normalized using Z-scores. The ORs were adjusted by age, sex, diabetes, hypertension, smoking, and COPD at admission. Time from onset of symptoms to severe outcomes was investigated using survival analysis, with follow-up from initial onset of symptoms until February 24, 2020. Multivariate Cox regression was conducted to estimate the HRs and their corresponding 95% CI. Similar to logistic regression, Cox regression was adjusted by age, sex, diabetes, hypertension, smoking, and COPD at admission. The Kaplan-Meier product-limit estimator was used to conduct survival analysis. All survival analyses were conducted using Lifelines 0.24.0 in Python environment.

), 1.99 (P < 0.01) for experiencing any severe symptoms (Supplementary Fig. S1B), 3.13 (P < 0.01) for ICU admission (Supplementary Fig. S1C), and 2.71 (P = 0.04) for utilization of invasive mechanical ventilation (Supplementary Fig. S1D; Supplementary

Figure 1 .
1Severe conditions in patients with and without cancer, and patients with different types, stages, and treatments of cancer. Severe conditions include death, ICU admission, having severe/critical symptoms, and usage of invasive mechanical ventilation. Incidence and survival analysis of severe conditions among COVID-19 patients with cancer and without cancer (A and B), among patients with different types of cancer (C and D), among patients with metastatic and nonmetastatic cancers (E and F), (continued on following page) Cancer Research.

Figure 1 .
1(Continued) among patients with lung cancer, other cancers than lung with lung metastasis, and other cancers than lung without lung metastasis (G and H), and patients receiving different types of cancer treatments (I and J). P values indicate differences between cancer subgroups versus patients without cancer. For A, C, E, G, I, *, P < 0.05; **, P < 0.01. OR, 95% CI, and P values between different subgroups are listed in Supplementary

Figure 2 .
2Timeline of events for COVID-19 patients. A, Timeline of events in COVID-19 patients with cancer. B, Timeline of events in COVID-19 patients without cancer. For visualization purposes, patients without timeline information are excluded and only 105 COVID-19 patients without cancer are shown.

//cancerdiscovery.aacrjournals.org/content/suppl/2020/04/24/2159-8290.CD-20-0422.DC Access the most recent supplemental material at: E-mail alerts related to this article or journal. Sign up to receive free emailreprints of this article or to subscribe to the journal, contact the AACR Publications Permissions Rightslink site. (CCC) Click on "Request Permissions" which will take you to the Copyright Clearance Center's . http://cancerdiscovery.aacrjournals.org/content/10/6/783 To request permission to re-use all or part of this article, use this link Cancer Research. on November 18, 2021. © 2020 American Association for cancerdiscovery.aacrjournals.org Downloaded from

04%) of 105 patients vs. 8 (1.49%) of 536 patients; P < 0.01], and had more smoking history [36 (34.28%) of 105 patients vs. 46 (8.58%) of 536 patients; P < 0.01], but had no significant differences in sex, other baseline symptoms, and other comorbidities (Table 1). With respect to signs and symptoms upon admission, COVID-19 patients with cancer were similar to those without cancer except for a higher prevalence of chest distress [15 (14.29%) of 105 patients vs. 36 (6.16%) of 536 patients; P = 0.02]. Compared with COVID-19 patients without cancer, patients with cancer had higher observed death rates [OR, 2.34; 95% confidence interval (CI), (1.15-4.77); P = 0.03], higher rates of ICU admission [OR, 2.84; 95% CI (1.59-5.08); P < 0.01], higher rates of having at least one severe or critical symptom [OR, 2.79; 95% CI, (1.74-4.41); P < 0.01], and higher chances of needing invasive mechanical ventilation (Fig.Clinical Outcomes

Table 2 )Table 1 .
21. Lung cancer was the most frequent cancer type [22 (20.95%) of 105 patients], followed by gastrointestinal cancer [13 (12.38%) of 105 patients], breast cancer [11 (10.Characteristics of COVID-19 patients with and without cancer48%) of

P < 0.01], and use of invasive mechanical ventilation [OR, 55.42; 95% CI (13.21-232.47); P < 0.01]. In contrast, patients with nonmetastatic cancer did not demonstrate statistically significant differences compared with patients without cancer, with all P > 0.05 (Fig. 1E and F; SupplementaryTables S2 and S3). In addition, when compared with patients without cancer, patients with lung cancer or other cancers with lung metastasis also showed higher risks of death, ICU admission rates, higher critical symptoms, and use of invasive mechanical ventilation, with all P values below 0.01, but other cancers without lung metastasis had no statistically significant differences (all P values > 0.05;Fig. 1Gand H; Supplementary Table S3) when compared with patients without cancer. Among the 105 COVID-19 patients with cancer in our study, 13 (12.26%) had radiotherapy, 17 (14.15%) received chemotherapy, 8 (7.62%) received surgery, 4 (3.81%) had targeted therapy, and 6 (5.71%) had immunotherapy within 40 days before the onset of COVID-19 symptoms. All of Other cancers without lung metastasis Other cancers with lung metastasis Lung cancer No cancer Other cancers without lung metastasis Other cancers with lung metastasis Lung canceron November 18, 2021. © 2020 American Association for
cancerdiscovery.aacrjournals.org
Downloaded from
in Fig. 1C and D and Supplementary Table S2, patients
with hematologic cancer including leukemia, lymphoma, and
myeloma have a relatively high death rate [3 (33.33%) of 9
patients], high ICU admission rate [4 (44.44%) of 9 patients],
high risks of severe/critical symptoms [6 (66.67%) of 9
patients], and high chance of utilization of invasive mechani-
cal ventilation [2 (22.22%) of 9 patients]. Patients with lung
cancer had the second-highest risk levels, with death rate [4
(18.18%) of 22 patients], ICU admission rate [6 (27.27%) of 22
patients], risks of severe/critical symptoms [11 (50.00%) of 22
patients], and the chance of utilization of invasive mechanical
ventilation [4 (18.18%) of 22 patients; Table 2].

Cancer Stage

We found that patients with metastatic cancer (stage IV)
had even higher risks of death [OR, 5.58; 95% CI (1.71-18.23);
P = 0.01], ICU admission [OR, 6.59; 95% CI (2.32-18.72);
P < 0.01], having severe conditions [OR, 5.97; 95% CI (2.24-
15.91); Cancer Treatments

**

**

**
**

*

*
*

*

*

**

**
**

**
**

*

No cancer

No cancer
Targeted therapy
Radiotherapy
Chemotherapy
Surgery
Immunotherapy

No cancer
Targeted therapy
Radiotherapy
Chemotherapy
Surgery
Immunotherapy

Table S2 .Table 2 .
S22For B, D, F, H, J, HR, 95% CI, and P values are listed in Supplementary Table S3.AACRJournals.org Severe events in 105 patients with cancer for each type of cancerCancer Research.

on November 18, 2021. © 2020 American Association for
cancerdiscovery.aacrjournals.org
Downloaded from
Cancer types

Total
number

Death
n %

Average time
to death

ICU
admission
n %

Average
time to ICU

Critical
symptom
n %

Average time
to critical

IMV
n %

Average
time to IMV

Lung cancer
22/20.95%
18.18 16.75/8.17
27.27
10.00/6.82
50.00
8.55/5.71
18.18
14.50/6.98

Gastrointestinal
cancer

13/12.38%
7.69
24.0/NA
23.08
9.05/6.16
30.77
8.50/5.85
7.69
22.00/NA

Breast cancer
11/10.48%
0.00
NA/NA
0.00
NA/NA
18.18
12.00/7.00 0.00
NA/NA

Thyroid cancer
11/10.48%
0.00
NA/NA
0.00
NA/NA
9.09
8.00/NA
0.00
NA/NA

Blood cancer
9/8.57%
33.33 19.33/1.89
44.44
2.82/1.80
66.67
3.83/3.08
22.22
9.00/NA

Cervix cancer
6/5.71%
0.00
NA/NA
16.67
4.00/NA
33.33
7.00/3.00
0.00
NA/NA

Esophagus
cancer

6/5.71%
16.67 28.00/NA
33.33
9.52/0.50
50.00
7.33/2.05
16.67
16.00/NA

All cancer
105/100%
11.43 19.92/6.13
19.05
6.51/4.16
34.29
7.56/5.2
9.52
14.56/5.68

Note: Only cancer types with more than 5 patients are listed. Numbers are presented as n /%; average time to events are presented as mean/SD
(days), from initial onset of COVID-19 symptoms to death/ICU admission/critical symptom/IMV.

Abbreviations: NA, not applicable; IMV, invasive mechanical ventilation.

the targeted therapeutic drugs were EGFR-tyrosine kinase
inhibitors for treatment of lung cancer, and all of the immu-
notherapy drugs were PD-1 inhibitors for the treatment of
lung cancer. A patient with cancer may have more than one
type of therapy. Our observation suggested that patients
who received immunotherapy tended to have high rates of
death [2 (33.33%) of 6 patients] and high chances of devel-
oping critical symptoms [4 (66.67%) of 6 patients]. Patients
who received surgery demonstrated higher rates of death [2
(25.00%) of 8 patients], higher chances of ICU admission [3
(37.50%) of 8 patients], higher chances of having severe or
critical symptoms [5 (62.50%) of 8 patients], and higher use
of invasive ventilation [2 (25.00%) of 8 patients] than other
treatments excluding immunotherapy. However, patients
with cancer who received radiotherapy did not show statisti-
cally signifi cant differences in having any severe events when
compared with patients without cancer, with all P values >
0.10 ( Fig. 1I and J ). Clinical details on the cancer diagnoses
and cancer treatments are summarized in Supplementary
Table S4.

Cancer Research. on November 18, 2021. © 2020 American Association for cancerdiscovery.aacrjournals.org Downloaded from june 2020 CANCER DISCOVERY | 791
Published OnlineFirst April 28, 2020; DOI: 10.1158/2159-8290.CD-20-0422
Acknowledgments
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases. M Dai, D Liu, M Liu, L Wang, L Gan, L Chai, M Santillana, H Cai Development Of Methodology, : M Dai, D Liu, M Liu, F Xiong, L Gan, M Santillana, H Cai, ; M Dai, M Liu, F Zhou, Z Zhang, M Wu, Q Zheng, Y Xiong, H Xiong, C Wang, C Chen, Y Zhang, Y Peng, S Ge, B Zhen, T Yu, H Wang, Y Liu, J Mei, X Gao, Zhuyan Li, L Gan, C He, Y Li, Y Shi, J Qi, H Yang, ; M Cai, D Dai, M Liu, C Liu, Y Wang, Zhen Zhang, M D Li, M Liu, G Liu, C Li, C Wang, Y Chen, Y Chen Zhang, W J Study ; Guan, Z Y Ni, Y Hu, W H Liang, C Q Ou, J X He, Santillana Writing, review, and/or revision of the manuscript. M. Liu, Z. Chen, C. Wang, M. Santillana, H. Cai REFEREnCES 1382Acquisition of data (provided animals, acquired and managed patients, provided facilities. et al. Clinical characteristics of coronavirus disease 2019 in Chinadesign: M. Dai, D. Liu, M. Liu, L. Wang, L. Gan, L. Chai, M. Santillana, H. Cai Development of methodology: M. Dai, D. Liu, M. Liu, F. Xiong, L. Gan, M. Santillana, H. Cai Acquisition of data (provided animals, acquired and man- aged patients, provided facilities, etc.): M. Dai, M. Liu, F. Zhou, Z. Zhang, M. Wu, Q. Zheng, Y. Xiong, H. Xiong, C. Wang, C. Chen, Y. Zhang, Y. Peng, S. Ge, B. Zhen, T. Yu, H. Wang, Y. Liu, J. Mei, X. Gao, Zhuyan Li, L. Gan, C. He, Zhen Li, Y. Shi, Y. Qi, J. Yang, H. Cai Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Dai, D. Liu, M. Liu, C. Wang, Y. Zhang, Zhen Li, M. Santillana Writing, review, and/or revision of the manuscript: M. Dai, D. Liu, M. Liu, F. Zhou, Z. Chen, H. You, M. Wu, Q. Zheng, C. Wang, B. Zhen, D.G. Tenen, L. Chai, L.A. Mucci, M. Santillana, H. Cai Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Liu, M. Liu, G. Li, C. Wang, C. Chen, Y. Zhang, Y. Chen Study supervision: M. Liu, Z. Chen, C. Wang, M. Santillana, H. Cai REFEREnCES 1. Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al. Clinical char- acteristics of coronavirus disease 2019 in China. N Engl J Med 2020; 382:1708-20.

Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention. Z Wu, J M Mcgoogan, JAMA. 323Wu Z, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: sum- mary of a report of 72314 cases from the Chinese Center for Disease Control and Prevention. JAMA 2020;323:1239-42.

Cancer statistics in China. W Chen, R Zheng, P D Baade, S Zhang, H Zeng, F Bray, CA Cancer J Clin. 66Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, et al. Cancer statistics in China, 2015. CA Cancer J Clin 2016;66:115-32.

Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China. W Liang, W Guan, R Chen, W Wang, J Li, K Xu, Lancet Oncol. 21Liang W, Guan W, Chen R, Wang W, Li J, Xu K, et al. Cancer patients in SARS-CoV-2 infection: a nationwide analysis in China. Lancet Oncol 2020;21:335-37.

Risk of COVID-19 for cancer patients. Y Xia, Jin R Zhao, J Li, W Shen, H , Lancet Oncol. 21180Xia Y, Jin R, Zhao J, Li W, Shen H. Risk of COVID-19 for cancer patients. Lancet Oncol 2020;21:e180.

Risk of COVID-19 for patients with cancer. H P Wang, L Zhang, Lancet Oncol. 21181Wang HP, Zhang L. Risk of COVID-19 for patients with cancer. Lancet Oncol 2020;21:e181.

Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. X Yang, Y Yu, J Xu, H Shu, J Xia, H Liu, Lancet Respir Med. Epub ahead of printYang X, Yu Y, Xu J, Shu H, Xia J, Liu H, et al. Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study. Lancet Respir Med 2020 Feb 24 [Epub ahead of print].

Clinical characteristics of 82 death cases with COVID-19. B Zhang, X Zhou, Y Qiu, F Feng, J Feng, Y Jia, 2020Zhang B, Zhou X, Qiu Y, Feng F, Feng J, Jia Y, et al. Clinical character- istics of 82 death cases with COVID-19. MediRxiv 2020.

Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. F Bray, J Ferlay, I Soerjomataram, R L Siegel, L A Torre, A Jemal, CA Cancer J Clin. 68Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality world- wide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68:394-424.

Report of cancer epidemiology in China. R S Zheng, K X Sun, S W Zhang, H M Zeng, X N Zou, R Chen, Zhonghua Zhong Liu Za Zhi. 41Zheng RS, Sun KX, Zhang SW, Zeng HM, Zou XN, Chen R, et al. Report of cancer epidemiology in China, 2015. Zhonghua Zhong Liu Za Zhi 2019;41:19-28.

Adult haemophagocytic syndrome. M Ramos-Casals, P Brito-Zerón, A López-Guillermo, M A Khamashta, X Bosch, Lancet. 383Ramos-Casals M, Brito-Zerón P, López-Guillermo A, Khamashta MA, Bosch X. Adult haemophagocytic syndrome. Lancet 2014;383:1503-16.

Multiple myeloma. M S Raab, K Podar, I Breitkreutz, P G Richardson, K C Anderson, Lancet. 374Raab MS, Podar K, Breitkreutz I, Richardson PG, Anderson KC. Mul- tiple myeloma. Lancet 2009;374:324-39.

EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3 in acute myeloid leukemia cells. E Lainey, A Wolfromm, A Q Sukkurwala, J B Micol, P Fenaux, L Galluzzi, Cell Cycle. 12Lainey E, Wolfromm A, Sukkurwala AQ, Micol JB, Fenaux P, Galluzzi L, et al. EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3 in acute myeloid leu- kemia cells. Cell Cycle 2013;12:2978-91.

What does this mutation mean? The tools and pitfalls of variant interpretation in lymphoid malignancies. Y Guillermin, J Lopez, K Chabane, S Hayette, C Bardel, G Salles, Int J Mol Sci. 191251Guillermin Y, Lopez J, Chabane K, Hayette S, Bardel C, Salles G, et al. What does this mutation mean? The tools and pitfalls of variant interpretation in lymphoid malignancies. Int J Mol Sci 2018;19:1251.

Lung pathology of fatal severe acute respiratory syndrome. J M Nicholls, L L Poon, K C Lee, W F Ng, S T Lai, C Y Leung, Lancet. 361Nicholls JM, Poon LL, Lee KC, Ng WF, Lai ST, Leung CY, et al. Lung pathology of fatal severe acute respiratory syndrome. Lancet 2003; 361:1773-8.

Clinical evidence does not support corticosteroid treatment for 2019-nCoV lung injury. C D Russell, J E Millar, J K Baillie, Lancet. 395Russell CD, Millar JE, Baillie JK. Clinical evidence does not support corticosteroid treatment for 2019-nCoV lung injury. Lancet 2020; 395:473-5.

Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Z Xu, L Shi, Y Wang, J Zhang, L Huang, C Zhang, Lancet Respir Med. 8Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological findings of COVID-19 associated with acute respiratory distress syn- drome. Lancet Respir Med 2020;8:420-2.

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. N Chen, M Zhou, X Dong, J Qu, F Gong, Y Han, Lancet. 395Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epide- miological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet 2020;395:507-13.

Improving immune-vascular crosstalk for cancer immunotherapy. Y Huang, Bys Kim, C K Chan, S M Hahn, I L Weissman, W Jiang, Nat Rev Immunol. 18Huang Y, Kim BYS, Chan CK, Hahn SM, Weissman IL, Jiang W. Improving immune-vascular crosstalk for cancer immunotherapy. Nat Rev Immunol 2018;18:195-203.

Severe cytokine release syndrome in a patient receiving PD-1-directed therapy. S J Rotz, D Leino, S Szabo, J L Mangino, B K Turpin, J G Pressey, Pediatr Blood Cancer. 6426642Rotz SJ, Leino D, Szabo S, Mangino JL, Turpin BK, Pressey JG. Severe cytokine release syndrome in a patient receiving PD-1-directed therapy. Pediatr Blood Cancer 2017;64:e26642.

A review of cancer immunotherapy toxicity. L B Kennedy, Aks Salama, CA Cancer J Clin. 70Kennedy LB, Salama AKS. A review of cancer immunotherapy toxic- ity. CA Cancer J Clin 2020;70:86-104.

Clinical characteristics of COVID-19-infected cancer patients: a retrospective case study in three hospitals within Wuhan, China. L Zhang, F Zhu, L Xie, C Wang, J Wang, R Chen, Ann Oncol. Epub ahead of printZhang L, Zhu F, Xie L, Wang C, Wang J, Chen R, et al. Clinical characteristics of COVID-19-infected cancer patients: a retrospec- tive case study in three hospitals within Wuhan, China. Ann Oncol 2020 Mar 26 [Epub ahead of print].

Clinical management of severe acute respiratory infection when novel coronavirus (2019-nCoV) infection is suspected: interim guidance. World Health OrganizationWorld Health Organization. Clinical management of severe acute respiratory infection when novel coronavirus (2019-nCoV) infection is suspected: interim guidance.


Molecular characterization and infectivity of a Tomato leaf curl New Delhi virus variant associated with newly emerging yellow mosaic disease of eggplant in India

Dharmendra Pratap
Ashwin R Kashikar
Sunil K Mukherjee
Molecular characterization and infectivity of a Tomato leaf curl New Delhi virus variant associated with newly emerging yellow mosaic disease of eggplant in India
R E S E A R C H Open Access
Background: Begomoviruses have emerged as serious problem for vegetable and fiber crops in the recent past, frequently in tropical and subtropical region of the world. The association of begomovirus with eggplant yellow mosaic disease is hitherto unknown apart from one report from Thailand. A survey in Nagpur, Central India, in 2009-2010 showed severe incidence of eggplant yellow mosaic disease. Here, we have identified and characterized a begomovirus responsible for the newly emerging yellow mosaic disease of eggplant in India. Results: The complete DNA-A and DNA-B genomic components of the causative virus were cloned and sequenced. Nucleotide sequence analysis of DNA-A showed that it shared highest 97.6% identity with Tomato leaf curl New Delhi virus-India[India:Udaipur:Okra:2007] and lowest 87.9% identity with Tomato leaf curl New Delhi virus-India[India:NewDelhi:Papaya:2005], while DNA-B showed highest 94.1% identity with ToLCNDV-IN[IN:UD:Ok:07] and lowest 76.2% identity with ToLCNDV-India[India:Lucknow]. Thus, it appears that this begomovirus is a variant of ubiquitous ToLCNDV and hence, we suggest the name ToLCNDV-India[India:Nagpur:Eggplant:2009] for this variant. The pathogenicity of ToLCNDV-IN[IN:Nag:Egg:09] isolate was confirmed by agroinfiltraion and dimeric clones of DNA-A and DNA-B induced characteristic yellow mosaic symptoms in eggplants and leaf curling in tomato plants.Conclusion: This is the first report of a ToLCNDV variant moving to a new agriculturally important host, eggplant and causing yellow mosaic disease. This is also a first experimental demonstration of Koch's postulate for a begomovirus associated with eggplant yellow mosaic disease.

Background

Geminiviruses (family Geminiviridae) possess monopartite or bipartite circular, single-stranded DNA genomes encapsidated in geminate (18-22 nm diameters) particles. They infect a wide range of monocotyledonous and dicotyledonous plants but each member of the Geminiviridae family has its own limited host range. They are classified into four genera: Mastrevirus, Curtovirus, Topocuvirus and Begomovirus, on the basis of their genome organization, host range and insect vector [1,2]. A large number of important diseases are caused by begomoviruses, which comprises of more than 180 species [1]. Begomoviruses are transmitted by whitefly Bemisia tabaci (Gennadius) predominantly in the tropical and subtropical regions of the world and cause severe disease in dicot plants including tomato, pepper, cassava, beans, cotton and cucurbits [3][4][5].

The majority of the begomoviruses are bipartite and the genomic components are referred as DNA-A and DNA-B [1,6]. DNA-A encodes the replication-associated protein (Rep) which is essential for viral replication; the replication enhancer protein (REn); the transactivator protein (TrAP) that controls late gene expression and involved in RNAi suppression; and the coat protein (CP) for encapsidation and insect transmission. The DNA-B encodes the nuclear shuttle protein (NSP) and the movement protein (MP), both of which are vital in systemic spread and symptom expression. The two genomic components share a common region (CR) that contains motifs required for the control of gene expression and initiation of replication. CR has conserved reiterated motifs and a putative stem-loop structure containing the highly conserved nonanucleotide TAA-TATT↓AC, which is nicked by the Rep protein to initiate the rolling circle replication [7,8]. However, a few Old World begomoviruses such as Tomato leaf curl virus (ToLCV) and Tomato yellow leaf curl virus (TYLCV) are monopartite and have single genomic component, homologous to DNA-A of the bipartite begomoviruses. These monopartite viruses require only DNA-A to cause systemic infection [9][10][11].

In the recent past, novel satellite molecules called betasatellites (formerly known as DNA-β), were found to be associated with some monopartite begomoviruses such as Cotton leaf curl Multan virus (CLCuMV), which is responsible in the latest disease epidemic of cotton in Pakistan [12]. Betasatellites (~1.4 kb) have sequences unrelated to those of their helper begomoviruses but intriguingly, they depend on helper virus for replication, transmission and spread [13,14]. Betasatellites encode a single gene βC1, which is known to be a gene silencing suppressor [15]. Betasatellite is also known to play an important role in determining the host range of its associated begomovirus [14,16,17]. Some of the betasatellite molecules have relaxed trans-replication characteristics [18] and it has also been established that association of betasatellite increases disease severity and reduces the period between inoculation and appearance of symptoms [19]. It has also been shown that betasatellite can replace the movement function of DNA-B in case of bipartite begomoviruses [20].

Eggplant (Solanum melongena L.) commonly known as brinjal in India, is an economically important vegetable crop. The production of eggplant is severely affected by a number of plant viruses, particularly the RNA viruses [21]. Begomovirus association with eggplant is so far unknown except for one report from Thailand [1,22].

The natural occurrence of eggplant yellow mosaic disease (EYMD) was observed in a survey conducted in and around Nagpur region of Central India during 2009-2010 and the disease incidence was found to be around 60-65%. The disease prevails across the year and there is no significant seasonal variation about the severity of disease incidence. Infected plants showed severe yellow mosaic and mottling of leaves at later stage of infection. As the presence of whiteflies in the infected fields was noticed, we wanted to explore the possible association of begomoviruses with the EYMD in central India. We employed rolling circle amplification (RCA) technique to identify the infecting begomovirus. Our results revealed the clear association of a ToLCNDV isolate with EYMD in India.

Furthermore, we constructed dimeric agroinfectious clones from isolated genomic components and observed successful infectivity of these agro-clones in eggplants as well as in tomato plants. We have also recorded the period of disease appearance, symptom severity and accumulation of virus in the agroinfiltrated plants. We were also interested to investigate the degree of disease severity when the non-cognate betasatellite is infiltrated along with both the genomic components of the virus and for the purpose, the dimeric clone of non cognate betasatellite (CLCuMV) was used. To the best of our knowledge, this is the first record of a ToLCNDV variant moving to a new host, eggplant and causing EYMD in India and we have artificially simulated the new emerging disease thereby satisfying Koch's postulates.

Methods

Genomic DNA isolation

Different leaf samples of eggplant showing yellow mosaic symptoms were collected from various locations of Nagpur in Central India. From the fields surveyed, infected leaf samples from many different plants were collected and used for genomic DNA isolation. Total genomic DNA was isolated from infected samples by Cetyltrimethylammonium bromide (CTAB) method [23].

Full length genomic amplification

Full length viral genomes (DNA-A and DNA-B) were amplified from the DNA isolated above using RCA [24] based TempliPhi™ DNA amplification kit (GE Healthcare). RCA reaction was performed as per manufacturer's instruction. The concatemers produced in the reaction were monomerized by restriction digestion with suitable restriction enzymes.

Cloning and sequencing

Aliquots of 1 μl of the above RCA products were digested independently with various restriction enzymes: BamHI, ClaI, EcoRI, HindIII, EcoRV, SacI, and NcoI. Digested products were resolved on 1% agarose gel and the bands corresponding to~2.7 kb genomes were purified using Hi Yield™Gel/PCR DNA Mini Kit. The 2.7 kb monomers (A or B) were cloned into the respective sites of pGreen0029 vector and henceforth, designated as pGreen-1.0A (SacI) or pGreen-1.0B (XbaI). Monomeric full length clones were purified using HiYield™ Plasmid Mini Kit. Sequences of the recombinant clones were determined commercially by TCGA Company, India. We also investigated for any possible presence DNA-β satellite molecule in infected samples by RCA and also by Polymerase chain reaction (PCR) using the universal betasatellite primers, namely, Beta 01 and Beta 02 [25].

Sequence analysis

Sequences of all the monomeric clones were assembled and analyzed using the software BIOEDIT version 7.0 programs [26]. Database searches with begomovirus sequences were carried out by NCBI-BLAST program (http://blast.ncbi.nlm.nih.gov). Nucleotide (nt) and amino acid (aa) sequence alignments were performed using CLUSTALW program using Mac Vector software (v11.1.2; MacVector Inc., USA). The phylogenetic tree was constructed using nucleotide sequences of complete DNA-A and DNA-B of ToLCNDV isolates and other selected begomovirus species reported from India and worldwide. Few other selected begomoviruses causing yellow mosaic disease in different plants were also taken into consideration so as to reveal the relationship of the virus isolate under study with its homologues. The phylogenetic tree was constructed with distance/neighborjoining method with 1000 bootstrap replications and viewed with the help of MacVector suite program 10.5 (Mac Vector Inc, USA).

Construction of agroinfectious clones and their infectivity

To check the infectivity of the above virus isolate, we used the high fidelity PCR based strategy for making dimeric clones of both DNA-A and DNA-B. For the purpose, we designed two different sets of abutting primers specific for amplifications of complete DNA-A and DNA-B genomic components of the virus isolate. For DNA-A, forward primer 5'-GAGCTCGTGCAGTTGTCCCCAT-3' and reverse primer 5'-AAGCTTCATAGGGGCTGTCGAAGTTGA-3' were synthesized commercially (IDT, USA). The nucleotides underlined represent the respective restriction sites. We incorporated one natural restriction site (SacI) of the virus in the forward primer and one introduced restriction site (HindIII) in the reverse primer for the ease of cloning. The PCR conditions were as follows: initial denaturation at 94°C, 30 cycles at 94°C for 1 min, annealing for 1 min at 58°C, and extension for 3 min at 72°C, followed by a final extension of 10 min at 72°C. The PCR product was subsequently cloned in pGEM-T Easy vector (Promega) and the clones were confirmed by colony PCR, restriction digestion and sequencing. The 2.7 kb band, released by digestion with HindIII and SacI from the recombinant pGEM-T Easy clone of DNA-A, was subsequently cloned at the same restriction sites of a binary vector pGreen 0029. This clone was named pG-A'. At the next step, the pGreen-1.0A plasmid DNA was digested with SacI and the 2.7 viral kb DNA was recloned at the SacI site of pG-A'. In this way, a complete head to tail DNA-A dimer was cloned in pGreen0029 (hereafter called as pGreen-2.0A). Insert integrity and orientation of the dimeric clones of DNA-A were confirmed by restriction digestion with DraI, a unique cutter in the viral DNA sequence. Two bands corresponding to 7.3 and 2.7 kb dropped out following digestion with DraI.

For construction of DNA-B dimeric clone, a similar strategy was adopted. The abutting primers were designed in a similar manner: forward primer 5'-TCTA-GAACTCATTTGGTGTC-3' and reverse primer 5'-CT GCAGGAG AGAAACTGCAACTTCT-3'. The nucleotides underlined represent the respective restriction sites. We incorporated one natural restriction site (XbaI) in the forward primer and one introduced restriction site (PstI) in the reverse primer. The PCR conditions were same as described above for DNA-A. The PCR products obtained were subsequently cloned in pGEM-T Easy vector (Promega) and the clones were confirmed by restriction digestion and sequencing. The 2.7 kb DNA released by digestion with XbaI and PstI from pGEM-T Easy clone of DNA-B was subsequently cloned at the same restriction sites of binary vector pGreen0029. The recombinant clone was named pG-B'. The 2.7 kb viral DNA of pGreen-1.0B plasmid was subsequently mobilized at the XbaI site of pG-B' and the final dimeric clone was named pGreen-2.0B. Insert integrity and orientation of clones were confirmed by digestion with the internal cutter ScaI.

pGreen-2.0A and pGreen-2.0B plasmid DNAs were mobilized into competent Agrobacterium tumefaciens strain EHA105 with the helper plasmid pSoup by electroporation method using a Gene Pulser Apparatus [27]. Agrobacterium colonies were confirmed by colony PCR. The primers were designed from the flanking sequences of the inserts in the pGreen vector. Empty binary vector pGreen0029 in Agrobacterium was used as negative control for mock inoculation on control plants. To study the effects by betasatellite in disease severity, we also used dimeric agroinfectious clone of non-cognate CLCuMB betasatellite originated from northwestern India-CLCuMB[IN:ND1:03] [28].

A. tumefaciens cultures were incubated with shaking (200 rpm) at 28°C for 48 h (OD 600 = 1) in yeast extract-Manitol (YEM) medium (pH 6.8) containing kanamycin (50 μg/ml), rifampicin (20 μg/ml) and chloramphenicol (34 μg/ml). Agrobacterium cells were harvested and resuspended in MES buffer [10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 10 mM Magnesium chloride (MgCl 2 )] and used for agroinfiltration on test plants.

Eggplant (Solanum melongena cv.Pusa Purple Long) and tomato (Solanum lycopersicum cv. Pusa Ruby) plants were grown in vermiculite inside a temperature controlled glasshouse maintained at 25 ± 2°C and a 16/8 h light/dark cycle. 5-6 leaf stage eggplant and tomato plants were used for agroinfiltration. All the agroinfectious constructs, pGreen-2.0A, pGreen-2.0B and pBin-2.0β were used for infiltration in tomato and eggplants either individually or in various combinations with equimolar concentrations as described earlier [29]. Five plants each of eggplant and tomato plants were used for various combinations of agro-constructs and agroinfiltrated plants were grown in an insect-free glasshouse and the plants were maintained at 16/8 h light/dark periods for 30 weeks to observe the symptoms periodically.

Analysis of viral DNA in inoculated plants

Total genomic DNA isolated from agroinfiltrated plants was used as a template for RCA. The templified products were digested with DNA-A specific single cutter restriction enzyme and the digested products were analyzed on agarose gel. Semiquantitative-PCR was also performed using ToLCNDV eggplant isolate DNA-A specific abutting primers to examine the relative viral accumulation efficiency of the virus in infiltrated test plants. The band intensities were quantified using Image J software. The quantified intensity values were normalized with respect to the respective actin controls which was taken as negative control. The value obtained for A +B was arbitrarily assigned as 100%. The sequence of actin primers and PCR conditions were similar as described earlier [30].

Results

Field detection of the viral disease and cloning of viral DNA

Eggplants showing severe yellow mosaic symptoms were observed in fields during a survey carried out in 2009-2010 in central India ( Figure 1A, B). To characterize the presence of begomovirus in the infected plants, total DNA was isolated from the leaf samples of infected plants as well as from healthy eggplants (as negative control). RCA was performed with the isolated DNA and the RCA products were digested with different restriction enzymes. The results presented in Figure 1C clearly demonstrate the presence of~2.7 kb product in case of infected samples when digested with HindIII, EcoRI, BamH1, SacI and EcoRV, while no such band appeared on digestion with NcoI. The appearance of 2.7 kb amplified DNA suggested the presence of a begomovirus in the infected leaves. In healthy leaf samples no band corresponding to 2.7 kb was observed, as expected. The 2.7 kb fragments obtained from digestion with above restriction enzymes were directly cloned in pGreen0029 at their respective sites and sequenced. The DNA was extracted separately from the leaves of 15 different infected plants of same neighborhood and subjected to similar treatments of RCA followed by restriction digestion. In every case, similar results were obtained and the amplified and digested DNAs were cloned and sequenced. About twenty clones from the leaf DNA of each infected plant were sequenced.

Viral DNA sequence analysis

The end sequences (about 700 bases in total), obtained from each of the positive samples, showed the presence   Figure 2). Phylogenetic analysis of the DNA-B genome also showed that ToLCNDV-IN[IN:Nag:Egg:09] isolate has close relationship with ToLCNDV strains (Figure 3).

Infectivity of the cloned genomic components

To assess the infectivity of the present ToLCNDV isolate, agroinfectious constructs of both the genomes were introduced into the leaves of tomato as well as eggplant using the agro-infiltration technique. Agroinfiltration of various constructs were carried out either individually or in various combinations and the consequent disease expressions are summarized in Table 3. When both the genomic components (DNA-A and DNA-B) were coinfiltrated, typical yellow mosaic symptoms at 60 days post inoculation (dpi) was observed in eggplants (5/5) (Figure 4, panels C and G) and the same constructs evoked typical downward leaf curling in tomato plants (5/5) at 45 dpi ( Figure 5, panels C and G). The symptoms persisted even at 150 and 180 dpi in eggplants and tomato plants respectively.

To However, the severity of the symptoms was most pronounced when all of the three components, i.e., DNA-A/DNA-B/DNA-β, were co-transferred in plants. Figure  4 shows the severity of yellow mosaic disease in eggplants and the leaves curled with progression of the disease (panels D and H). The disease expression time also reduced to 45 days. Similar was the case with tomato and the curled leaves showed mosaic symptoms eventually due to virus infection ( Figure 5, panels D and H). The disease also expressed early, i.e., at about 30 dpi in tomato plants.

As expected, neither empty pGreen vector (negative control) nor DNA-B and DNA-β construct caused any The semi-qPCR was carried out to know the level of viral DNA accumulation in plants infiltrated with various constructs. The results revealed that the viral DNA accumulation in tomato plants was highest when the plants were infiltrated with all the three components, namely DNA-A, DNA-B and non cognate DNA-β. The accumulation of viral DNA was lowest when the DNA-A and DNA-β was used for co-infiltration ( Figure 6A, C). Similar results were also obtained with eggplants ( Figure 6B, D). However, the effects of non cognate β DNA was less pronounced in eggplants compared tomato.

The measured intensities of the 2.7 kb bands were normalized with respect to the corresponding actin controls. The average normalized intensity values (based on three independent studies) are presented as bar graphs in Figure 6C, D. Our data reflect that the accumulation of viral DNA increased to~95% on introduction of non cognate DNA β in tomato while there was a modest increase of~14% in eggplant. The accumulation of viral DNA was further reduced up to~27% when DNA-B was substituted by non cognate DNA-β in tomato plants whereas the same was~50% in eggplant ( Figure 6C, D).

Discussion

Eggplant is one of the preferred hosts for rearing of whiteflies and was known to be immune for begomovirus [31]    ToLCNDV is an economically important pathogen and found to be associated with various crop plants in India, Pakistan, Thailand and Bangladesh [1]. ToLCNDV has been reported for the first time in India from tomato and it required both DNA-A and DNA-B for symptom development [2]. Later on, it has been reported from various crops and weeds such as, chili [32], cucurbits [33], potato [34], papaya [35], bittergourd [36], cotton (GenBank Accession number, EF063145) and Solanum nigrum (GenBank Acc. No. AJ620187). As already known, virus isolated from a plant may or may not be the cause of disease unless it satisfies the Koch's postulates and despite a large host range and geographical distribution of ToLCNDV, very few cloned isolates have been shown to be infectious to either experimental plants or the hosts from which they were isolated.

Therefore  Interestingly, ToLCNDV-IN[IN:Nag:Egg:09] gave the typical leaf curling symptoms, not the yellow mosaic in tomato plants. Thus, these results also indicated that the nature of symptoms and disease is largely a host-driven process in this case. The successful virus infection is dependent on productive interactions between viral and host factors at each stage of the infection process and host-adaptation have a different basis in different plants [37,38].

On agroinfiltration with empty vector (taken as negative control) or only DNA-A of  [20]. However, the symptoms took longer time to develop and they were mild comparative to that when both the genomic components (DNA-A and DNA-B) were present. It indicated that DNA-β can substitute DNA-B up to certain extent but not completely. These results are also in accordance with the previous findings that, DNA-B plays the major role in symptom production and viral pathogenicity in bipartite begomovirus [39]. The DNA-β satellite molecules are also known for intensification of disease symptoms in a host dependent manner [27,40]. Co-infiltration of DNA-A and DNA-B of ToLCNDV-IN [IN:Nag:Egg:09] with DNA-β of CLCuMV resulted in enhanced severity of disease in both eggplant and tomato. The appearance of disease symptoms also took lesser time compared to that required by both DNA-A and DNA-B. Thus, our results promoted the role of DNA-β in increasing the disease severity [41].

The symptomatic variation with agroinfiltrated test plants which were infiltrated with various combinations of constructs were corroborated with molecular findings as the relative virus accumulation of virus increased to about~95% and~14% in tomato and eggplant respectively when DNA-β was infiltrated along with both DNA-A and DNA-B. The value reduced to~27% and~50% respectively in tomato and eggplant when DNA-B was replaced with DNA-β, where the value obtained for A+B was arbitrarily assigned as 100%. Furthermore, these results support the notion that DNA-β increases the disease severity by increasing the accumulation of the helper virus. The variation observed in tomato and eggplant may be due to the fact that DNA-β increased the symptoms severity in a host dependent manner [14,42].

Thus, we have identified and characterized the virus associated with EYMD as a variant of ubiquitous TOLCNDV, but presence of new begomoviruses species with the eggplant cultivation in India cannot be negated as different species of monopartite and bipartite begomoviruses exist across India and among them many monopartite begomoviruses with betasatellite molecules are already known to be prevalent in southern India [43]. The diversity of begomovirus may lead to adaptation of eggplants as a new host and as a result the eggplants in other part of India might be under severe threat. Therefore, some practical intervention measures, such as enforcement of quarantine regulations in the trade of live plant materials and development of resistant plants are urgently needed to curb the viral threat.

Conclusion

Taken together, on the basis of molecular characterization and infectivity test, we can conclude that ToLCNDV-IN[IN:Nag:Egg:09] is a newly emerging variant of ToLCNDV moving to a new economically important host, eggplant and subsequently posing severe constraint on eggplant production in India. This is also a first experimental demonstration of Koch's postulate for a begomovirus associated with eggplant yellow mosaic disease.

Figure 1
1(A) Naturally infected eggplants with yellow mosaic symptoms and mottling of leaves. (B) A magnified image of a part of the diseased leaf [encircled in red (A)]. (C) The profile of restriction digestions of the RCA products of DNA derived from the leaves of naturally infected plants. The DNA marker is shown at the extreme left of the figure. The enzymes used for digestion are indicated at the top of the figure.

investigate the possible role of non-cognate DNAβ satellite in the disease severity, we co-infiltrated the dimeric clone of betasatellite, CLCuMB[IN:ND1:03] along with both the genome-components of ToLCNDV-IN[IN:Nag:Egg:09] on the test plants (eggplant and tomato). The eggplants exhibited severe yellow mosaic symptoms at 45 dpi (Figure 4, panels D and H) while severe downward leaf curling was observed in tomato at 30 dpi (Figure 5, panels D and H). The DNA-A genomic component failed to develop disease either in eggplant or tomato when infiltrated alone (Figure 4, panels A and E; Figure 5, panels A and E). In addition, we were also interested to examine that the DNA-B component of the ToLCNDV-IN[IN:Nag: Egg:09] isolate could be replaced by non-cognate CLCuMV DNA-β for systemic disease development. As anticipated, co-infiltration of DNA-A and DNA-β resulted in successful systemic disease development. The symptoms were mild mosaic in eggplant (2/5) (Figure 4, panels B and F) and leaf curling in tomato plants (3/5) (Figure 5, panels B and F). Thus, non cognate CLCuMV DNA-β substituted DNA-B of ToLCNDV-IN[IN:Nag: Egg:09] for systemic viral movement in both eggplant and tomato plants, albeit in a reduced manner. However, the disease expression period was prolonged compared to when the B-component was administered with DNA-A. The symptom development periods were 90 and 80 dpi for eggplant and tomato respectively.

Figure 2
2Phylogenetic tree of complete DNA-A of ToLCNDV-IN[IN: Nag: Egg: 09]. The phylogenetic tree was constructed with distance/ neighbour-joining method with 1000 bootstrap replications and viewed with the help of MacVector suite program 10.5 (Mac Vector Inc, USA).

Figure 3
3Phylogenetic tree of complete DNA-B of ToLCNDV-IN[IN:Nag:Egg:09]. The phylogenetic tree was constructed with distance/ neighbour-joining method with 1000 bootstrap replications and viewed with the help of MacVector suite program 10.5 (Mac Vector Inc, USA).

, to satisfy the Koch's postulates, the infectivity of ToLCNDV-IN[IN:Nag:Egg:09] was established by infiltrating the agroinfectious clones in eggplant. The agroinfiltration in eggplants resulted in the disease symptoms that are similar to those occurred in the virus infected eggplant in fields. Hence, we fulfilled the Koch's postulates and showed for the first time that ToLCNDV-IN[IN:Nag:Egg:09] is responsible for the newly emerging EYMD in central India.

Figure 4
4The disease was induced by ToLCNDV-IN[IN:Nag:Egg:09] in eggplants that were inoculated with different constructs as labeled. The agro-inoculated plants are shown at the bottom panel and the corresponding representative leaves are shown in the top panel in the magnified form to reveal the patterns of disease symptoms at around 90 dpi.

Figure 5
5Plants were inoculated with different constructs as labeled. Rest of the condition is same as described inFigure 4.

Figure 6
6The plants (Tomato and Eggplant) were agro-infiltrated with various constructs as shown on the top of the figures, and the level of viral DNA accumulation was checked by semi-qPCR at 90 dpi using DNA-A specific abutting primers ToLCNDV-IN[IN:Nag:Egg: 09]. The amplification products (~2.7 kb) were separated on 1% agarose gel and are presented in panels (a) and (b) for tomato and eggplant respectively. The band intensities were quantified and the normalized values with respect to corresponding loading controls (ACTIN) are plotted as bar graphs in panels (c) and (d) respectively. The standard deviations shown are based on three independent experiments.

of a bipartite (DNA-A and DNA-B) begomovirus. All DNA-A clones were found to be more than 99% identical amongst themselves and all the DNA-B clones were also about 97% identical amongst themselves. Hence, for subsequent studies, only a few clones of each type, i.e., having either DNA-A or DNA-B, were chosen for further characterization of the virus. To sequence the remaining portion of DNA-A and DNA-B, primer walking was carried out by designing additional internal primers. Three clones of each of DNA-A and DNA-B were sequenced completely and one such clone of each DNA-A and DNA-B was used for characterization of the full genome. The restriction enzymes SacI and A of the virus isolate with the selected strains of TOLCNDV, TYLCV and other begomoviruses reported from India and worldwide (Table 1) showed highest identity (97.6%) with ToLCNDV-IN[IN: UD: Ok: 07] and lowest (87.9%) with ToLCNDV-IN[IN:ND:Pap:05] (Table 2). We thus suggest the name of the variant as ToLCNDV-India[India:Nagpur:Eggplant:2009] in accordance with the latest International Committee on Table 1 Name, acronym and GenBank accession numbers of the selected begomovirus genome sequences used for study The ToLCNDV variant under study is written in bold. The nucleotide sequence analysis of DNA-B of the ToLCNDV isolate under study with selected DNA-B sequences of begomovirus in NCBI database showed that it has highest sequence identity (94.1%) with ToLCNDV-IN[IN:UD:Ok:07] and lowest with ToLCNDV-IN[IN: Luck] (Table 2). The phylogenetic tree analysis on the basis of nucleotide sequence of DNA-A genome of the ToLCNDV-IN [IN:Nag:Egg:09] with the DNA-A sequences of other selected begomoviruses revealed that the ToLCNDV-IN [IN:Nag:Egg:09] isolate has close relationship with the strains of ToLCNDV. In contrast, DNA-A nucleotide sequences of begomovirus species associated with EYMD in Thailand, showed divergent relationship with ToLCNDV-IN[IN: Nag: Egg: 09] isolate (EcoRV for DNA-A and HindIII and XbaI for DNA-B
were found to be unique cutters. The complete DNA-A
and DNA-B sequences are available in GenBank with
the accession numbers HQ264185 and HQ264186
respectively. The DNA-A and DNA-B components are
comprised of 2741 nt and 2698 nt respectively and their
genome organizations are identical to those of the pre-
viously characterized bipartite begomoviruses. DNA-A
contains six open reading frames (ORFs) of which two
ORFs AV1 and AV2 are in the virion sense and the

other four ORFs, i.e., AC1, AC2, AC3 and AC4, are in
complementary sense. These ORFs are interspersed by
276 nt long intergenic region (IR) located between the
ORFs AC1 and AV2. The DNA-B component contains
two ORFs designated as BVI on the sense strand and
BC1 on the complementary strand. The DNA-A and
DNA-B genomes did not exhibit any sequence similar-
ity, except for approximately 160 nt common region
(CR) which showed~96.8% sequence identity with each
other. In the CR, a 34 bp potential stem-loop forming
region (5'-GCGGCCATTCGTATAATATTACCGAATG
GCCGCG-3') was identified which includes the con-
served nonanucleotide sequence TAATATTAC present
within the replication origin of almost all geminiviruses.
The nucleotide sequence comparison of complete
DNA-Begomoviruses
DNA-A*
DNA-B*
Acronym

Tomato leaf curl New Delhi virus-India [India: New Delhi:2005]
DQ169056
DQ169057
ToLCNDV-IN[IN:ND:05]

Tomato leaf curl New Delhi virus-India[India:Udaipur:Okra:2007]
EF035482
EF043394
ToLCNDV-IN-[IN:UD:Ok:07]

Tomato leaf curl New Delhi virus-Bangladesh
EF450316
NA
ToLCNDV-BD

Tomato leaf curl New Delhi virus -OM-Taiwan
GU180095
GU180096
ToLCNDV-[OM: Tai]

Tomato leaf curl New Delhi virus-Pakistan
EF620534
EF620535
ToLCNDV-Pak

Tomato leaf curl New Delhi virus -India-186b
GQ865546
NA
ToLCNDV-IN-186b

Tomato leaf curl New Delhi virus [India:Bangalore:OY135:2005]
GU112084
GU112085
ToLCNDV-IN[IN: Ban:05]

Tomato leaf curl New Delhi virus -India[Pakistan:Solanum nigrum:1997]
AJ620187
AJ620188
ToLCNDV-IN[PK: Sn: 97]

Tomato leaf curl New Delhi virus-India[India:New Delhi:Severe:1992]
U15015
U15017
ToLCNDV-IN[IN: ND: Svr:92]

Tomato leaf curl New Delhi virus-India [Bangladesh:Jessore: Severe:2005]
AJ875157
AJ875158
ToLCNDV-IN[BG:Jes:Svr:05]

Tomato leaf curl New Delhi virus-India [India: Lucknow]
Y16421
X89653
ToLCNDV-IN[IN: Luck]

Tomato leaf curl New Delhi virus -India [India: Happur: Potato: 2005]
EF043230
EF043233
ToLCNDV-IN[IN:Hap:Pot:05]

Tomato leaf curl New Delhi virus -India[India: Meerut: Potato: 2005]
EF043231
EF043232
ToLCNDV-IN[IN: Mer: Pot: 05]

Tomato leaf curl New Delhi virus-Thailand[Cucumber: Thailand]
AB330079
AB330080
ToLCNDV-TH[Cuc:Thai]

Tomato leaf curl New Delhi virus-India [India: New Delhi: Mild :1992]
U15016
NA
ToLCNDV-IN[IN: ND: Mld:92]

Tomato leaf curl New Delhi virus-Thailand [Luffa:Thailand]
AF102276
NA
ToLCNDV-TH[TH: Luf]

Tomato leaf curl New Delhi virus-India [India: New Delhi:Papaya:2005]
DQ989325
NA
ToLCNDV-IN[IN:ND:Pap:05]

Tomato leaf curl New Delhi virus [India:Aurangabad:OY164A:2006]
GU112088
GU112089
ToLCNDV-IN[IN: Aug: 06]

Tomato leaf curl New Delhi virus [India:Nagpur:Eggplant:2009]
HQ264185
HQ264186
ToLCNDV-IN[IN:Nag:Egg:09]

Tomato leaf curl Palampur virus-India
AM884015
AM992534
ToLCPMV

Squash leaf curl China virus [Pumpkin :Coimbatore]
AY184487
AY184488
SLCCV-IN[Pump:Coi]

Tomato yellow leaf curl Kanchanaburi virus[Thailand Kanchanaburi 2: Eggplant: 2001]
AF511530
AF511528
TYLCKaV-TH[TH:Kan2:Egg:01]

Tomato yellow leaf curl Kanchanaburi virus[Vietnam: Binhudong: Eggplant:2005]
DQ641702
NC_005811
TYLCKaV-TH[Vn:Bin:Egg:05]

Pratap et al. Virology Journal 2011, 8:305
http://www.virologyj.com/content/8/1/305
Taxonomy of Viruses (ICTV) guidelines for species
demarcation in the genus [1].

Table 2
2Percent identities (nucleotide # ) of ToLCNDV-IN [IN:Nag:Egg:09] with DNA-A and DNA-B of selected begomoviruses reported worldwide symptoms in test plants even at 180 dpi and no noticeable phenotypic change in eggplant and tomato plants were observed. In contrast, plants infiltrated with the agroinfectious constructs were always stunted in growth.Virus-Acronym
DNA-A
DNA-B

ToLCNDV-IN[IN:UD:Ok:07]
97.6
94.1

ToLCNDV-IN-186b
97.0
NA

ToLCNDV-IN[IN: Ban:05]
96.3
86.4

ToLCNDV-IN[IN: Aug: 06]
95.6
88.3

ToLCNDV-IN[IN:ND:05]
94.5
86.4
ToLCNDV-IN[PK: Sn: 97]
94.5
82.0

ToLCNDV-IN[IN: ND: Svr:92]
94.3
86.4

ToLCNDV-IN[BG:Jes:Svr:05]
94.3
86.4

ToLCNDV-IN[IN: Luck]
94.0
76.2

ToLCNDV-IN[IN: Mer: Pot: 05]
93.7
86.3

ToLCNDV-Pak
93.2
90.9

ToLCNDV-TH[Cuc:Thai]
92.7
81.4

ToLCNDV-IN[IN: ND: Mld:92]
92.7
NA
ToLCNDV-[OM: Tai]
92.1
80.6

ToLCNDV-IN[IN:Hap:Pot:05]
91.4
86.7

ToLCNDV-TH[TH: Luf]
91.1
NA

ToLCNDV-BD
88.8
NA

ToLCNDV-IN[IN:ND:Pap:05]
87.9
NA

SLCCV-IN[Pump:Coi]
86.8
63.3

TolCPMV
85.3
68.1

TYLCKaV-TH[Vn:Bin:Egg:05]
67.3
39.0
TYLCKaV-TH[TH:Kan2:Egg:01]
66.9
39.0

# Information about the sequences used for comparison is provided in Table1

NA-Sequence not available

Pratap et al. Virology Journal 2011, 8:305
http://www.virologyj.com/content/8/1/305

Table 3
3Infectivity and symptom induced by ToLCNDV-IN[IN:Nag:Egg:09] with or without non-cognate betasatellite CLCuMB-[IN:ND1:03] and the number of symptomatic plants as confirmed by RCAHost/Inoculated dimeric
agro-constructs

Symptomatic plants/Inoculated plants
Types of symptoms
(90dpi)

Eggplant
DNA-2A
0/5
No Symptoms

DNA-2A+DNA-2B
5/5
Yellow mosaic

DNA-2A+ DNA-2β
3/5
Mild yellow mosaic

DNA-2A+ DNA-2B + 2β
5/5
Severe yellow mosaic

DNA-2B
0/5
No Symptoms

DNA-2β
0/5
No Symptoms

DNA-2B+ CLCuMV-DNA-2β
0/5
No Symptoms

Tomato
DNA-2A
0/5
No Symptoms

DNA-2A+ DNA-2B
5/5
Leaf curling

DNA-2A+ DNA-2β
4/5
Mild leaf curling

DNA-2A+ DNA-2B + DNA-2β
5/5
Severe leaf curling with occasionally yellow mosaic

DNA-2B
0/5
No Symptoms

DNA2β
0/5
No Symptoms

2B+ 2β
0/5
No Symptoms

ToLCNDV-IN[IN:Nag: Egg:09], no symptoms were recorded even by 180 dpi. This showed that DNA-A alone of ToLCNDV-IN[IN: Nag:Egg:09] was neither able to sustain nor produce systemic infection in the host plants. This phenomenon is perhaps true for most of the geminiviruses with the bipartite genomes [2,15]. The association of a DNA-β molecule with the field infection of ToLCNDV-IN[IN:Nag: Egg:09] could not be detected by either PCR or RCR method. Nevertheless, we still suspect that in succeeding time, betasatellite associated monopartite begomovirus may be identified with EYMD in southern India. Consequently, we used the non-cognate CLCuMB[IN: ND1:03] molecule to examine its role in degree of virus infection. When CLCuMB[IN: ND1:03] was coinfiltrated with DNA-A of ToLCNDV-IN[IN:Nag: Egg:09], produced mild leaf curling in tomato plants. This result suggested that CLCuMV DNA-β can successfully substitute the DNA-B for systemic movement of ToLCNDV-IN[IN:Nag:Egg:09] and symptom development. These results are in conformity with the earlier findings that DNA-β can substitute DNA-B for systemic movement
Pratap et al. Virology Journal 2011, 8:305 http://www.virologyj.com/content/8/1/305
Pratap et al. Virology Journal 2011, 8:305 http://www.virologyj.com/content/8/1/305 Page 4 of 13
Pratap et al. Virology Journal 2011, 8:305 http://www.virologyj.com/content/8/1/305 Page 7 of 13
Pratap et al. Virology Journal 2011, 8:305 http://www.virologyj.com/content/8/1/305 Page 8 of 13
Pratap et al. Virology Journal 2011, 8:305 http://www.virologyj.com/content/8/1/305 Page 9 of 13
Pratap et al. Virology Journal 2011, 8:305 http://www.virologyj.com/content/8/1/305 Page 10 of 13
AcknowledgementsWe are grateful to Dr. Nirupam Roy Choudhury, ICGEB, New Delhi for helpful discussion and critical reading of the manuscript. We also thank Dr. V. G. Malathi, IARI, New Delhi for CLCuMV betasatellite dimeric clone and Dr. M. K. Reddy, ICGEB, New Delhi for pGreen 0029 and pSoup vector.Authors' contributions DP carried out the entire experiments. ARK carried out the survey and collected the infected samples. SKM conceived of the study, DP and SKM participated in designing the experiments. DP and SKM prepared the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
Geminivirus strain demarcation and nomenclature. C M Fauquet, R W Briddon, J K Brown, E Moriones, J Stanley, M Zerbini, X Zhou, Archives of Virology. 153Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X: Geminivirus strain demarcation and nomenclature. Archives of Virology 2008, 153:783-821.

Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. M Padidam, R N Beachy, C M Fauquet, J Gen Virol. 76Pt 1Padidam M, Beachy RN, Fauquet CM: Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity. J Gen Virol 1995, 76(Pt 1):25-35.

Current status of Bemisia tabaci as a plant pest and virus vector in agro-ecosystems worldwide. J K Brown, FAO Plant Protection Bulletin (FAO. Brown JK: Current status of Bemisia tabaci as a plant pest and virus vector in agro-ecosystems worldwide. FAO Plant Protection Bulletin (FAO);

. Bulletin Phytosanitaire de la FAO (FAO. Bulletin Phytosanitaire de la FAO (FAO);

Boletin Fitosanitario de la FAO (FAO). Boletin Fitosanitario de la FAO (FAO) 1994.

A worldwide survey of tomato yellow leaf curl viruses. H Czosnek, H Laterrot, Archives of Virology. 142Czosnek H, Laterrot H: A worldwide survey of tomato yellow leaf curl viruses. Archives of Virology 1997, 142:1391-1406.

Geminiviruses emerge as serious crop threat. A S Moffat, Science. 286Moffat AS: Geminiviruses emerge as serious crop threat. Science 1999, 286:1835-1835.

Infectivity of the cloned geminivirus genome requires sequences from both DNAs. J Stanley, Stanley J: Infectivity of the cloned geminivirus genome requires sequences from both DNAs. 1983.

Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. L Hanley-Bowdoin, S B Settlage, B M Orozco, S Nagar, D Robertson, Critical Reviews in Plant Sciences. 18Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D: Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Critical Reviews in Plant Sciences 1999, 18:71-106.

Molecular characterization of the Rep protein of the blackgram isolate of Indian mungbean yellow mosaic virus. V Pant, D Gupta, N R Choudhury, V G Malathi, A Varma, S K Mukherjee, J Gen Virol. 82Pant V, Gupta D, Choudhury NR, Malathi VG, Varma A, Mukherjee SK: Molecular characterization of the Rep protein of the blackgram isolate of Indian mungbean yellow mosaic virus. J Gen Virol 2001, 82:2559-2567.

Nucleotide sequence and genome organization of tomato leaf curl geminivirus. I B Dry, J E Rigden, L R Krake, P M Mullineaux, M A Rezaian, Journal of General Virology. 74Dry IB, Rigden JE, Krake LR, Mullineaux PM, Rezaian MA: Nucleotide sequence and genome organization of tomato leaf curl geminivirus. Journal of General Virology 1993, 74:147-147.

Tomato yellow leaf curl virus from sardinia is a whiteflytransmitted monoparatite geminivirus. A Kheyr-Pour, M Bendahmane, V Matzeit, G P Accotto, S Crespi, B Gronenborn, Nucleic Acids Research. 19Kheyr-Pour A, Bendahmane M, Matzeit V, Accotto GP, Crespi S, Gronenborn B: Tomato yellow leaf curl virus from sardinia is a whitefly- transmitted monoparatite geminivirus. Nucleic Acids Research 1991, 19:6763-6763.

Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. N Navot, E Pichersky, M Zeidan, D Zamir, H Czosnek, Virology. 185Navot N, Pichersky E, Zeidan M, Zamir D, Czosnek H: Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. Virology 1991, 185:151-161.

Cotton leaf curl virus disease. R W Briddon, P G Markham, Virus Research. 71Briddon RW, Markham PG: Cotton leaf curl virus disease. Virus Research 2000, 71:151-159.

Identification of DNA components required for induction of cotton leaf curl disease. R W Briddon, S Mansoor, I D Bedford, M S Pinner, K Saunders, J Stanley, Y Zafar, K A Malik, P G Markham, Virology. 285Briddon RW, Mansoor S, Bedford ID, Pinner MS, Saunders K, Stanley J, Zafar Y, Malik KA, Markham PG: Identification of DNA components required for induction of cotton leaf curl disease. Virology 2001, 285:234-243.

A unique virus complex causes Ageratum yellow vein disease. K Saunders, I D Bedford, R W Briddon, P G Markham, S M Wong, J Stanley, Proc Natl Acad Sci. 97Saunders K, Bedford ID, Briddon RW, Markham PG, Wong SM, Stanley J: A unique virus complex causes Ageratum yellow vein disease. Proc Natl Acad Sci USA 2000, 97:6890-6895.

A Begomovirus DNAbeta-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. X Cui, G Li, D Wang, D Hu, X Zhou, J Virol. 79Cui X, Li G, Wang D, Hu D, Zhou X: A Begomovirus DNAbeta-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. J Virol 2005, 79:10764-10775.

Bhendi yellow vein mosaic disease in India is caused by association of a DNA [beta] satellite with a Begomovirus. J Jose, R Usha, Virology. 305Jose J, Usha R: Bhendi yellow vein mosaic disease in India is caused by association of a DNA [beta] satellite with a Begomovirus. Virology 2003, 305:310-317.

Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus. K Saunders, N Salim, V R Mali, V G Malathi, R Briddon, P G Markham, J Stanley, Virology. 293Saunders K, Salim N, Mali VR, Malathi VG, Briddon R, Markham PG, Stanley J: Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus. Virology 2002, 293:63-74.

Replication promiscuity of DNA-beta satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-beta satellite localizes sequences involved in replication. K Saunders, R W Briddon, J Stanley, J Gen Virol. 89Saunders K, Briddon RW, Stanley J: Replication promiscuity of DNA-beta satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-beta satellite localizes sequences involved in replication. J Gen Virol 2008, 89:3165-3172.

Abdel-Salam AM, others: Diversity of DNA [beta], a satellite molecule associated with some monopartite begomoviruses. R W Briddon, S E Bull, I Amin, A M Idris, S Mansoor, I D Bedford, P Dhawan, N Rishi, S S Siwatch, Virology. 312Briddon RW, Bull SE, Amin I, Idris AM, Mansoor S, Bedford ID, Dhawan P, Rishi N, Siwatch SS, Abdel-Salam AM, others: Diversity of DNA [beta], a satellite molecule associated with some monopartite begomoviruses. Virology 2003, 312:106-121.

A monopartite begomovirusassociated DNA beta satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection. M Saeed, Y Zafar, J W Randles, M A Rezaian, J Gen Virol. 88Saeed M, Zafar Y, Randles JW, Rezaian MA: A monopartite begomovirus- associated DNA beta satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection. J Gen Virol 2007, 88:2881-2889.

Agrobacterium-mediated transformation of eggplant (Solanum melongena L.) using cotyledon explants and coat protein gene of Cucumber mosaic virus. D Pratap, S Kumar, S K Raj, A K Sharma, Indian Journal of Biotechnology. 10Pratap D, Kumar S, Raj SK, Sharma AK: Agrobacterium-mediated transformation of eggplant (Solanum melongena L.) using cotyledon explants and coat protein gene of Cucumber mosaic virus. Indian Journal of Biotechnology 2011, 10:19-24.

Molecular characterization of a new Begomovirus associated with tomato yellow leaf curl and eggplant yellow mosaic diseases in Thailand. S K Green, W S Tsai, S L Shih, M A Rezaian, U Duangsong, Plant Disease. 87Green SK, Tsai WS, Shih SL, Rezaian MA, Duangsong U: Molecular characterization of a new Begomovirus associated with tomato yellow leaf curl and eggplant yellow mosaic diseases in Thailand. Plant Disease 2003, 87:446-446.

Molecular cloning of Indian tomato leaf curl virus genome following a simple method of concentrating the supercoiled replicative form of viral DNA. K M Srivastava, V Hallan, R K Raizada, G Chandra, B P Singh, P V Sane, J Virol Methods. 51Srivastava KM, Hallan V, Raizada RK, Chandra G, Singh BP, Sane PV: Molecular cloning of Indian tomato leaf curl virus genome following a simple method of concentrating the supercoiled replicative form of viral DNA. J Virol Methods 1995, 51:297-304.

Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses. D Haible, S Kober, H Jeske, Journal of virological methods. 135Haible D, Kober S, Jeske H: Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses. Journal of virological methods 2006, 135:9-16.

Universal primers for the PCR-mediated amplification of DNA beta: a molecule associated with some monopartite begomoviruses. R W Briddon, S E Bull, S Mansoor, I Amin, P G Markham, Mol Biotechnol. 20Briddon RW, Bull SE, Mansoor S, Amin I, Markham PG: Universal primers for the PCR-mediated amplification of DNA beta: a molecule associated with some monopartite begomoviruses. Mol Biotechnol 2002, 20:315-318.

BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. T A Hall, Hall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. 1999, 95-98.

Characterization of DNAbeta associated with begomoviruses in China and evidence for coevolution with their cognate viral DNA-A. X Zhou, Y Xie, X Tao, Z Zhang, Z Li, C M Fauquet, J Gen Virol. 84Zhou X, Xie Y, Tao X, Zhang Z, Li Z, Fauquet CM: Characterization of DNAbeta associated with begomoviruses in China and evidence for co- evolution with their cognate viral DNA-A. J Gen Virol 2003, 84:237-247.

Molecular diversity of the DNA-beta satellites associated with tomato leaf curl disease in India. P N Sivalingam, V G Malathi, A Varma, Arch Virol. 155Sivalingam PN, Malathi VG, Varma A: Molecular diversity of the DNA-beta satellites associated with tomato leaf curl disease in India. Arch Virol 2010, 155:757-764.

. Pratap, Virology Journal. 8305Pratap et al. Virology Journal 2011, 8:305

Transient expression assay by agroinfiltration of leaves. M W Lee, Y Yang, Methods In molecular biology-Clifton Then Totowa. 323Lee MW, Yang Y: Transient expression assay by agroinfiltration of leaves. Methods In molecular biology-Clifton Then Totowa-2006, 323:225-225.

A geminiviral amplicon (VA) derived from Tomato leaf curl virus (ToLCV) can replicate in a wide variety of plant species and also acts as a VIGS vector. P Pandey, N R Choudhury, S K Mukherjee, Virol J. 6152Pandey P, Choudhury NR, Mukherjee SK: A geminiviral amplicon (VA) derived from Tomato leaf curl virus (ToLCV) can replicate in a wide variety of plant species and also acts as a VIGS vector. Virol J 2009, 6:152.

others: Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes. H Czosnek, A Kheyr-Pour, B Gronenborn, E Remetz, M Zeidan, A Altman, H D Rabinowitch, S Vidavsky, N Kedar, Y Gafni, Plant molecular biology. 22Czosnek H, Kheyr-Pour A, Gronenborn B, Remetz E, Zeidan M, Altman A, Rabinowitch HD, Vidavsky S, Kedar N, Gafni Y, others: Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes. Plant molecular biology 1993, 22:995-1005.

First report of Tomato leaf curl New Delhi virus affecting chilli pepper in Pakistan. M Hussain, S Mansoor, S Iram, Y Zafar, R W Briddon, Plant pathology. 53Hussain M, Mansoor S, Iram S, Zafar Y, Briddon RW: First report of Tomato leaf curl New Delhi virus affecting chilli pepper in Pakistan. Plant pathology 2004, 53:794-794.

Complete nucleotide sequence of a new isolate of tomato leaf curl New Delhi virus infecting cucumber, bottle gourd and muskmelon in Thailand. T Ito, P Sharma, K Kittipakorn, M Ikegami, Archives of Virology. 153Ito T, Sharma P, Kittipakorn K, Ikegami M: Complete nucleotide sequence of a new isolate of tomato leaf curl New Delhi virus infecting cucumber, bottle gourd and muskmelon in Thailand. Archives of Virology 2008, 153:611-613.

Potato leaf curl-a new disease of potato in northern India caused by a strain of Tomato leaf curl New Delhi virus. K S Usharani, B Surendranath, S M Paul-Khurana, I D Garg, V G Malathi, Plant pathology. 53Usharani KS, Surendranath B, Paul-Khurana SM, Garg ID, Malathi VG: Potato leaf curl-a new disease of potato in northern India caused by a strain of Tomato leaf curl New Delhi virus. Plant pathology 2004, 53:235-235.

Molecular evidence for association of Tomato leaf curl New Delhi virus with leaf curl disease of papaya (Carica papaya L.) in India. S K Raj, S K Snehi, M S Khan, R Singh, A A Khan, Australas Plant Dis Notes. 3Raj SK, Snehi SK, Khan MS, Singh R, Khan AA: Molecular evidence for association of Tomato leaf curl New Delhi virus with leaf curl disease of papaya (Carica papaya L.) in India. Australas Plant Dis Notes 2008, 3:152-155.

First report of Tomato leaf curl New Delhi virus infecting bitter gourd in Pakistan. M Tahir, M S Haider, Plant pathology. 54Tahir M, Haider MS: First report of Tomato leaf curl New Delhi virus infecting bitter gourd in Pakistan. Plant pathology 2005, 54:807-807.

Genetic determinants of hostspecificity in bipartite geminivirus DNA A components. W K Gillette, T J Meade, J L Jeffrey, Itd Petty, Virology. 251Gillette WK, Meade TJ, Jeffrey JL, Petty ITD: Genetic determinants of host- specificity in bipartite geminivirus DNA A components. Virology 1998, 251:361-369.

Complementable and noncomplementable host adaptation defects in bipartite geminiviruses. I T Petty, C G Miller, T J Meade-Hash, R L Schaffer, Virology. 212Petty IT, Miller CG, Meade-Hash TJ, Schaffer RL: Complementable and noncomplementable host adaptation defects in bipartite geminiviruses. Virology 1995, 212:263-267.

Both bipartite geminivirus movement proteins define viral host range, but only BL1 determines viral pathogenicity. D J Ingham, E Pascal, S G Lazarowitz, Virology. 207Ingham DJ, Pascal E, Lazarowitz SG: Both bipartite geminivirus movement proteins define viral host range, but only BL1 determines viral pathogenicity. Virology 1995, 207:191-204.

Tobacco curly shoot virus DNAβ is not necessary for infection but intensifies symptoms in a host-dependent manner. Z Li, Y Xie, X Zhou, Phytopathology. 95Li Z, Xie Y, Zhou X: Tobacco curly shoot virus DNAβ is not necessary for infection but intensifies symptoms in a host-dependent manner. Phytopathology 2005, 95:902-908.

Maintenance of an old world betasatellite by a new world helper begomovirus and possible rapid adaptation of the betasatellite. M S Nawaz-Ul-Rehman, S Mansoor, R W Briddon, C M Fauquet, J Virol. 83Nawaz-ul-Rehman MS, Mansoor S, Briddon RW, Fauquet CM: Maintenance of an old world betasatellite by a new world helper begomovirus and possible rapid adaptation of the betasatellite. J Virol 2009, 83:9347-9355.

Pathogenicity of a natural recombinant associated with ageratum yellow vein disease: implications for geminivirus evolution and disease aetiology. K Saunders, I D Bedford, J Stanley, Virology. 282Saunders K, Bedford ID, Stanley J: Pathogenicity of a natural recombinant associated with ageratum yellow vein disease: implications for geminivirus evolution and disease aetiology. Virology 2001, 282:38-47.

Diversity and distribution of begomoviruses infecting tomato in India. Chowda Reddy, R V Colvin, J Muniyappa, V Seal, S , 10.1186/1743-422X-8-305Archives of virology. 150Chowda Reddy RV, Colvin J, Muniyappa V, Seal S: Diversity and distribution of begomoviruses infecting tomato in India. Archives of virology 2005, 150:845-867. doi:10.1186/1743-422X-8-305

Molecular characterization and infectivity of a Tomato leaf curl New Delhi virus variant associated with newly emerging yellow mosaic disease of eggplant in India. Pratap, Virology Journal. 8305Cite this article asCite this article as: Pratap et al.: Molecular characterization and infectivity of a Tomato leaf curl New Delhi virus variant associated with newly emerging yellow mosaic disease of eggplant in India. Virology Journal 2011 8:305.