Lester Shulman

Characterization of Human Metapneumovirus Infections in Israel

ABSTRACT Respiratory tract infections are a leading cause of morbidity and mortality worldwide. Even with the advancement of diagnostic tools, the causative agent of 20 to 30% of upper respiratory tract infections go undiagnosed. Recently, a newly identified human respiratory virus, human metapneumovirus (hMPV), was discovered in young children in The Netherlands. To study the prevalence of hMPV infections in Israeli children, respiratory specimens from 388 hospitalized children less than 5 years of age were evaluated for the presence of hMPV RNA, which was present in 42 (10.8%) of these samples. All hMPV-positive samples were negative for respiratory syncytial virus (RSV), influenza viruses (Flu) A and B, adenovirus, and parainfluenza viruses 1, 2, and 3. Conversely, hMPV RNA was not detected in 76 RSV-positive and 38 Flu A- or B-positive samples. Most hMPV activity was between the months February and April. Sequence analysis of 20 positive samples revealed that both of the hMPV genotypes (groups 1 and 2) have circulated in central Israel during the study period. Moreover, three of the four known hMPV subgroups (1A, 1B, and 2B) were detected among the tested samples. Seroprevalence of hMPV in 204 patients from the central part of Israel revealed that 100% of the children are hMPV seropositive by the age of 5 years old. We conclude that hMPV is a common respiratory pathogen in Israel, while mixed infections of hMPV with RSV or Flu in hospitalized children are apparently rare.

Mosquito Vectors of West Nile Fever in Israel

Abstract West Nile fever (WNF) is endemic in Israel. In 1999, country-wide adult mosquito surveys were initiated and intensified after the 2000 country-wide outbreak of WNF in humans. In 8 consecutive yr, groups of male and female specimens of different species and from different locations were tested for infection with West Nile virus (WNV). Three species made up >87% of the total catch: Culex pipiens L. (52%), with an infection rate (IR) of 0.5; Cx. perexiguus Theobald (20%), with an IR of 2.7; and Aedes caspius Pallas (15%), with an IR of 0.6. The geographical and temporal distribution of WNV-infected mosquitoes was similar but was not parallel to the seasonal abundance of the populations. The seasonal occurrence of human cases is in correlation with the finding of WNV-positive mosquito specimens reaching a peak 1 mo later than the mosquito peak. The relative importance of the mosquito species in the epidemiology of WNF is discussed. Cx. perexiguus is considered the major vector of WNF in Israel.

Characterization of Large Mumps Outbreak among Vaccinated Palestinian Refugees

During a large mumps virus (MuV) outbreak which occurred in the Palestinian refugee camps of the West Bank, 68.1% (2,636/3,871) of the cases were vaccinated with one dose of trivalent measles, mumps, and rubella (MMR) vaccine. Attack rates by camp ranged from less than 1 case per 1,000 people in the population to 43/1,000 (overall, 11/1,000). The outbreak lasted from December 2003 to June 2005, with two peaks, one from April to May 2004 and the other from March to April 2005. To control the outbreak, a mass MMR vaccination campaign was conducted in May 2005. Evaluation of the immune status of cases (n = 59) and healthy controls (n = 51) revealed high levels of mumps immunoglobulin G (IgG) and a low MuV-specific IgM in clinical cases indicative of a booster immune response. This suggested a secondary rather than a primary infection due to the insufficient protection conferred by the single vaccine dose included in the vaccination program. This prediction was further confirmed by the low seroprevalence (68.6%) found in the healthy control group, which was below the threshold level required for MuV herd immunity. Mumps diagnosis was established mainly by reverse transcription-PCR in clinical samples obtained within 48 h from the onset of disease. Of the parotid fluids and nasopharyngeal aspirates analyzed, 92% were positive for MuV RNA, while only 33% of the urine samples were positive. Phylogenetic analysis of the MuV SH gene identified the outbreak strain as the H genotype, which has been in circulation worldwide at least since 1989.

Abrogation of B16 melanoma metastases by long-term low-dose interleukin-6 therapy

We investigated the antitumor effects of human recombinant interleukin-6 (hrIL-6) on the highly metastatic B16 melanoma clone F10.9. These tumor cells were found to have very low levels of IL-6 receptors and in vitro IL-6 had no effect on cell proliferation or on the expression of MHC class I antigens. However, in vivo IL-6 was active against the metastatic growth of this tumor in mice, presumably through indirect immune effects. Low-dose IL-6 (1-10 micrograms/day), in three daily injections, 4 days a week, for 3 weeks, strongly inhibited the formation of experimental lung metastases following intravenous tumor cell inoculation. IL-6 therapy could be started even 10 days after tumor injection, when metastases are already established. Moreover, IL-6 treatment of mice bearing F10.9 tumors in the footpads resulted in complete protection against pulmonary spontaneous metastasis and in long-term survival. Histology confirmed the absence of micrometastases in most of the IL-6-treated animals. Analysis of the cytolytic activity of splenocytes at different times during therapy of tumor-bearing mice revealed significant lysis (up to 42%) of the melanoma F10.9 cells in the mice receiving IL-6 but not in the control mice.

High Frequency and Diversity of Rearrangements in Polyomavirus BK Noncoding Regulatory Regions Cloned from Urine and Plasma of Israeli Renal Transplant Patients and Evidence for a New Genetic Subtype

ABSTRACT Polyomavirus BK (BKV) establishes latent infection in various human tissues, including the kidney. Reactivation following renal transplantation (RT) may cause BKV-associated nephropathy, leading to graft loss. BKV reactivation is often associated with extensive rearrangements in the BKV noncoding regulatory region (NCRR). We explored the formation and predominance of the rearrangements versus the diversity of the rearrangements by cloning and characterizing PCR-amplified NCRR sequences from six Israeli RT patients. We found a high frequency and a high degree of diversity of rearrangements: NCRRs that contained major rearrangements (mrNCRRs), including large insertions and deletions, were detected in 0 to 100% of the clones from individual samples (mean, 50% and 67% in plasma and urine, respectively). In addition, we found a high frequency of mrNCRRs that contained single-nucleotide variations (snvNCRRs) among identical mrNCRRs and archetype clones. mrNCRRs were present in plasma and in concomitantly collected urine samples, but for each patient, only a subset of the mrNCRRs and snvNCRRs were present in both compartments at the same time and/or in subsequent samples from the same compartment. Some mrNCRRs were observed over several months, indicating the continuous replication of the viral genomes carrying them. Phylogenetic analysis based on the snvNCRR in the archetype clones grouped isolates from four of the patients into a new subgroup of genotype IV. Genotypes Ib-1 and Ib-2 were also found. Isolates from two patients had NCRRs from two genotypes, one concurrently with a RT and one after a second RT. Our study prompts further investigation of the functional consequences of NCRR rearrangements to assess their biological significance and their putative role in disease progression and prognosis.

Hemophagocytic lymphohistiocytosis associated with parechovirus 3 infection.

Hemophagocytic lymphohistiocytosis (HLH) denotes the common final pathway of a potentially fatal hyperinflammatory condition of diverse etiologies. We describe the first case of documented HLH associated with human parechovirus 3. A monoallelic Ala91Val mutation was found in the PRF1 gene, but the contribution of this mutation to HLH remains controversial. The diagnosis, based on accepted criteria, was established early in the course of the disease and led to successful treatment and complete recovery. The awareness of this new association is clinically important in facilitating early treatment, preventing organ damage, and increasing the likelihood of complete recovery.

Insight into the intrinsic sensitivity of the PCR assay used to detect CMV infection in amniotic fluid specimens

BACKGROUND PCR detection of human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) is the most sensitive tool for diagnosis of fetal infection, but has sub-optimal sensitivity. It has been suggested that inhibition by AF reduces the sensitivity of this assay, however this assumption has never been thoroughly studied. Several PCR assays have been shown to improve sensitivity, but comparative studies are insufficient to choose the optimal approach. OBJECTIVES To assess the effect of AF inhibition on PCR sensitivity and to determine the most sensitive assay for diagnosing fetal infection. STUDY DESIGN Plasmid containing HCMV DNA was tested by PCR, in water and in non-infected AF, to assess the inhibitory effect of AF. Twenty-three AF-infected samples were tested by various PCR protocols. AF supernatant, with or without DNA extraction, and AF cells, were assayed by single-round and semi-nested PCR. Viral load was measured in the supernatant by a commercial quantitative PCR kit. RESULTS The plasmid model demonstrated that single-round PCR was 2000-fold less sensitive in AF compared with water. Semi-nested PCR was only 10-fold less sensitive. Single-round PCR was 30% sensitive in HCMV-infected AF supernatants, and detected viral loads higher than 2.3 x 10(6) viral copies/ml. Extraction of DNA from the supernatant increased the sensitivity of this assay to 89% and the detection limit to 5.2 x 10(4) copies/ml. Semi-nested PCR performed on supernatant, with and without DNA extraction, was 96% and 100% sensitive, respectively, with a detection threshold of 3.8 x 10(3) copies/ml. Single-round and semi-nested PCR were 89% and 100% sensitive, respectively, in cells. The commercial quantitative PCR assay was 100% sensitive. CONCLUSIONS AF supernatant is inhibitory to PCR. The two most sensitive assays were semi-nested PCR performed on DNA extracted from the supernatant and the commercial quantitative PCR kit. Of these two, the latter is standardized, non-labor-intensive, and allows minimal opportunity for contamination, thereby making it the preferred method for diagnosis.

Involvement of Interleukin-6 in the Autocrine Stimulation of Chronic Lymphocytic Leukemia B Cells by Tumor Necrosis Factor

Tumor necrosis factor α (TNFα) acts as an autocrine growth factor in chronic B cell malignancies. TNF also induces production of interleukin 6 (IL-6) which stimulates B cell growth and differentiation. We have previously demonstrated increased TNFα production by (Rai) stage 0 chronic lymphocytic leukemia (B-CLL) cells and the absence of TNF production by cells from stage IV patients. In an attempt to elucidate a possible role for TNF in the malignant progression of B-CLL we investigated the possibility of IL-6 involvment in the stimulatory action of TNF on B-CLL cells. We observed that: (1) the in vitro proliferative response of B-CLL cells to recombinant human (rh)THFα was consistently inhibited by a monoclonal antibody (MoAb) against IL-6, (2) the release of IL-6 by B-CLL cells could be augmented by rhTNFα, (3) no differences were detected in the foregoing parameters between stage 0 and stage IV-derived cells and (4) despite the inhibitory action of an anti-IL-6 MoAb on the TNF-induced proliferative response of B-CLL cells, IL-6 receptor expression was undetectable in these cells. Although these findings are suggestive of an autocrine or paracrine mechanism involving TNF and IL-6, the importance of the release and action of these cytokines in the regulation of B-CLL cell growth and malignant progression still remains to be elucidated.

Genotyping Rotavirus RNA from Archived Rotavirus-Positive Rapid Test Strips

Genotyping circulating rotaviruses before and after introduction of rotavirus vaccine is useful for evaluating vaccine-associated changes in genotype distribution. We determined frequency of rotavirus genotypes among 61 rotavirus-positive children hospitalized in Israel during the 2005–06 rotavirus season. Accurate molecular epidemiologic data were recovered from affinity-concentrated rotavirus immobilized in rotavirus-positive bands from air-dried, diagnostic rotavirus rapid test strips (dipstick) stored at room temperature from 1 week to 5 years. G genotypes were identical for 21 paired dipsticks and suspensions, whereas dipsticks or suspensions detected an additional G genotype in 2 samples. RNA sequences from 7 pairs were identical. Phylogenetic analysis suggested previously unreported G2 sublineages and G9 lineages. The ease with which dipsticks can be stored at local facilities and transported to central reference laboratories can reverse increasing difficulties in obtaining geographically representative stool samples and expand surveillance to regions lacking adequate laboratory facilities.