Genome and Phylogenetic Analysis of Cucumber Green Mottle Mosaic Virus Global Isolates and Validation of a Highly Sensitive RT-qPCR Assay.

The last two decades have seen exponential growth in the international movement of seeds for annual food crops, from a gross U.S. import value of $349 million in 1999 to $1.05 billion in 2019. This has led to the proportionate growth of seedborne pathogens dispersed with seed stocks. One such viral pathogen is cucumber green mottle mosaic virus (CGMMV), a tobamovirus that infects cucurbit crops such as melon, watermelon, cucumber, pumpkin, and squash. The first CGMMV introduction to California occurred in 2013, with subsequent annual outbreaks or detections since then. Here, we describe the use of next-generation sequencing to characterize the full genomes of 25 CGMMV isolates collected between 2013 and 2020 in California, either from CGMMV field detections or seed lots identified as CGMMV positive. We sequenced an additional 31 CGMMV isolates collected in Europe, Israel, and southeast Asia that were provided by industry collaborators. We also performed an in silico nucleotide database search in GenBank for full genome CGMMV sequences to include in all in silico analyses. Based on conserved regions within the coat protein gene, we then developed a quantitative reverse-transcription PCR assay for the sensitive and specific detection of CGMMV in seed and plant samples. Finally, based on our sequence and phylogenetic analysis, our data support that CGMMV has been introduced multiple times into California.

First Report of Cucumber green mottle mosaic virus on Melon in the United States.

In July 2013, a melon (Cucumis melo var. Saski) field in Yolo County, California, was inspected as part of a phytosanitary inspection for seed production. The leaves of the plants showed mosaic, green mottle, and blotches. When plant sap was examined using a transmission electron microscope, rigid rod-shaped particles were observed. Melon plant samples were analyzed by both CDFA and USDA APHIS PPQ laboratories and tested positive using DAS-ELISA against Cucumber green mottle mosaic virus (CGMMV) (Agdia, Elkhart, IN). To confirm the presence of CGMMV, total RNA was analyzed by RT-PCR using primers CGMMV-F5370 5'-CTAATTATTCTGTCGTGGCTGCGGATGC-3' and CGMMV-R6390 5'-CTTGCAGAATTACTGCCCATA-3' designed by PPQ based on 21 genomic sequences of CGMMV found worldwide. The 976-bp amplicon was sequenced (GenBank Accession No. KJ453559) and BLAST analysis showed the sequence was 95% identical to MP and CP region of CGMMV isolates reported from Russia (GQ495274, FJ848666), Spain (GQ411361), and Israel (KF155231), and 92% to the isolates from China (KC852074), Korea (AF417243), India (DQ767631), and Japan (D12505). These analyses confirm the virus was CGMMV. To our knowledge, this is the first report of CGMMV in the United States. Based on our sequence data, a second set of primers (CGMMV-F5796 5'-TTGCGTTTAGTGCTTCTTATGT-3' and CGMMV-R6237 5'-GAGGTGGTAGCCTCTGACCAGA-3'), which amplified a 440-bp amplicon from CGMMV CP region, was designed and used for testing all the subsequent field and seed samples. Thirty-seven out of 40 randomly collected Saski melon samples tested positive for CGMMV, suggesting the virus was widespread in the field. All the melon samples also tested positive for Squash mosaic virus (SqMV) using DAS-ELISA (Agdia). Therefore, the symptoms observed likely resulted from a mixed infection. The melon field affected by CGMMV was immediately adjacent to fields of cucumber (Cucumis sativus var. Marketmore 76) and watermelon (Citrullus lanatus var. Sugar Baby) crops, both for seed production with no barrier between the crops. CGMMV was also detected from symptomatic plants from both fields. Seed lots used for planting all three crops were tested and only the melon seed was positive for CGMMV, suggesting the seed as the source of infection. The sequenced 440-bp RT-PCR amplicons from CGMMV-infected cucumber and watermelon plants and melon seeds were 99% identical to the CGMMV from the field melon. A cucumber plant infected with CGMMV but not SqMV was used for mechanical inoculation at the Contained Research Facility at University of California, Davis. Inoculated cucumber, melon, and watermelon plants showed green mottle and mosaic similar to that observed in the field. CGMMV is a highly contagious virus and damage by this virus on cucurbit crops has been reported in regions where CGMMV is present (2). CGMMV was detected on cucumber grown in greenhouses in Canada with 10 to 15% yield losses reported due to this virus (1). The three cucurbit crops in Yolo County were planted in an isolated area with no other cucurbits nearby. Measures, including destroying all the cucurbit plant material, have been taken to eradicate the virus. Use of CGMMV free cucurbit seed is necessary for prevention of this disease. References: (1) K.-S. Ling et al. Plant Dis. 98:701, 2014. (2) J. Y. Yoon et al. J. Phytopathol. 156:408, 2008.

The role of local steroid injections in an operational field hospital setting.

OBJECTIVES: To assess the safety and effectiveness of administering local steroid injections in an operational field hospital environment. METHOD: A prospective study of patients presenting to the physiotherapy department at the British Military Hospital (BMH) Shaibah, Iraq was undertaken from July 2006 - September 2006. Patients with a condition amenable to local steroid injection and who had not improved with conservative therapy were considered for the study. They then underwent local injection with steroid and local anaesthetic. RESULTS: During this period 12 patients were identified that fitted the criteria for local steroid injection. This represented 7% (12/179) of patients who were seen by the physiotherapy department in either an in or out-patient setting. All patients were injected with local anaesthetic and steroid injection with a single dose of intravenous antibiotic as antimicrobial cover. 10/12 (83.3%) were able to return to their unit within this theatre of operation. The only complication was one case of post injection flare of pain, which settled after 48 hours. CONCLUSION: The use of local steroid injection, as an adjunct to physical therapy, can enable service personnel to remain in the theatre of operations. These patients may have otherwise required aero medical evacuation. We consider the use of a local steroid injection to be a safe and effective intervention in certain patients where conservative measures alone do not work. This study highlights the safety of using steroid injections in an operational field hospital setting. Further large scale studies may help corroborate this conclusion.

Acute and chronic disability among U.S. farmers and pesticide applicators: the National Health Interview Survey (NHIS).

The National Health Interview Survey (NHIS) is a multipurpose household survey of the U.S. civilian non-institutionalized population conducted annually since 1957. From 1986 to 1994, over 450,000 U.S. workers, age 18 years and older, participated in a probability sampling of the entire non-institutionalized U.S. population; variables collected included a range of measures of acute and chronic disability. The objective of the present study was to assess predictors of health status, and acute and chronic disability for farmers and pesticide applicators (pesticide-exposed workers) compared to all other U.S. workers using the 1986-1994 NHIS. After adjustment for sample weights and design effects using SUDAAN, several measures of acute and chronic disability and health status were modeled with multiple logistic regression. Farmers (n = 9576) were significantly older compared to all other U.S. workers (n = 453,219) and pesticide applicators (n = 180). Farmers and pesticide applicators had a higher proportion of males, whites, and Hispanics and were less educated. After adjusting for age, gender, race-ethnicity, and education, compared to all other workers, farmers were significantly less likely to report acute and chronic disability and health conditions, while pesticide applicators were more likely to report chronic disability, health conditions, and poor health. Given the cross-sectional nature of the data and the significant job demands of farming, both leading to a relative healthy worker effect, the present results indicate that at any point in time, farmers report less acute and chronic disability, compared to other U.S. workers, whereas pesticide applicators report similar or poorer health.

Gestational herpes simplex virus hepatitis.

Hepatitis due to herpes simplex virus (HSV) is unusual in healthy individuals. To date, only 56 cases of HSV hepatitis in adult patients have been reported, including 21 pregnant patients. We describe a 25-year-old white woman in her 30th week of gestation who had progressive acute hepatitis. Histologic examination of the liver biopsy specimen showed diffuse microabscesses involving more than 50% of the hepatic parenchyma, with multiple hepatocytes containing Cowdry type A and ground-glass nuclear inclusions. The diagnosis of herpes hepatitis was confirmed by positive immunoreactivity to HSV antibodies in the tissue sections. Intravenous acyclovir therapy was immediately initiated, and the patient's condition improved dramatically. She then had a normal baby at term. Subsequently, the patient had a second pregnancy and an uncomplicated vaginal delivery without recurrence of the disease. Even though alterations of the humoral and cell-mediated immunity occur during pregnancy, herpes hepatitis is rare in pregnant women. Since the prompt administration of antiviral drugs is a lifesaving measure, we recommend including HSV hepatitis in the differential diagnosis of acute hepatitis in pregnancy.

Fetal macrosomia: does antenatal prediction affect delivery route and birth outcome?

OBJECTIVE: Our purpose was to determine whether clinical or ultrasonographic prediction of fetal macrosomia influences subsequent delivery route and birth outcome in a clinical setting where macrosomia is not considered an indication for cesarean delivery. STUDY DESIGN: The hospital records of 504 patients delivered of infants weighing > or = 4200 gm between October 1989 and March 1994 were reviewed. Statistical comparisons were made between patients in whom fetal macrosomia was predicted before delivery (n = 102) and those in whom it was not (n = 402). Cesarean delivery, shoulder dystocia, and birth trauma rates were the variables of interest. RESULTS: Cesarean sections were performed in 52% of the "predicted" group deliveries and in 30% of the "not predicted" group (p < 0.01). The increased cesarean delivery rate in the predicted group appeared to be related to an increased incidence of labor inductions (42.5% vs 26.6%, p = 0.005) and a greater proportion of failed inductions. The proportion of patients delivered by cesarean section without a trial of labor was similar in the predicted and not predicted groups (14.7% vs 10.2%, p = 0.21). There was no significant differences in the incidence of shoulder dystocia or the occurrence of birth trauma. CONCLUSIONS: The antenatal prediction of fetal macrosomia is associated with a marked increase in cesarean deliveries without a significant reduction in the incidence of shoulder dystocia or fetal injury. Ultrasonography and labor induction for patients at risk for fetal macrosomia should be discouraged.

Alloantibodies to PHA-activated lymphocytes detect human Qa-like antigens.

Platelet-absorbed sera were obtained from placental clots after delivery by multiparous women. These sera contained antibodies that react with PHA-activated lymphocytes after the latter are separated from peripheral blood and expanded with interleukin 2. These alloantibodies did not react with resting T lymphocytes, but reacted with B lymphocytes, PHA-activated lymphocytes, or both types of cells obtained from some but not all of the T lymphocyte donors. Reactions against B lymphocytes were associated with anti-Ia-like antibodies on the basis of blockage by turkey antibodies against human Ia. Reactions against PHA-activated lymphocytes that were blocked by turkey anti-beta 2m were classified as 'HT'. Several antibodies were found to give reactions to HT determinants in separate panels of lymphocytes from Tel Aviv and Boston. The reproducibility of the cytotoxicity reactions was 89%. Altogether, 23 of 1100 sera were found to contain these reactions when screened by a panel of cells obtained from 30 individuals of known HLA phenotypes. Correlation coefficients were determined for all reactions, determining three clusters of significant reactivities: sera 965 and 1032 defined HT-2; sera SF48 and 1642 defined HT-3; and sera 1136, 1605, 1014, and 1227 defined HT-4. HT-2 was found to be inherited with HLA in 11 siblings from four families. Some of these antibodies react with antigens (non-HLA) containing beta 2m that were expressed on activated lymphocytes, but not on resting T lymphocytes, and did not react with thymocytes from the same donors of the peripheral lymphocytes. Our findings suggest that the HT alloantigens expressed on lectin-activated lymphocytes are class I differentiation antigens of a system analogous to the murine Qa system.

Platelet HLA typing and cross-matching by a direct microcytotoxic assay. A new method employing carboxyfluoresceindiacetate-stained platelets.

Carboxyfluoresceindiacetate-labelled platelets can be used in direct microcytotoxic assays for the purpose of typing platelets for a number of antigenic determinants, such as HLA and beta-2 microglobulin. The assay when used in direct platelet cross-matching, where thrombocytopenic patient sera were screened, resulted in the uncovering of many extrapositive and weakly positive cross-matches not identified by the lymphocyte cross-match. The simplicity and rapidity of this new method makes it an attractive alternative for future use in research and clinical studies.