Mismatched hemagglutinin and neuraminidase specificities in recent human H3N2 influenza viruses

The hemagglutinin (HA) of influenza viruses initiates infection by binding to sialic acid on the cell surface via alpha2,6 (human) or alpha2,3 (avian) linkage. The influenza neuraminidase (NA) can cleave both alpha2,3- and alpha2,6-linked sialic acids, but all influenza NAs have a marked preference for the non-human alpha2,3 linkage. Recent H3N2 influenza viruses have lost the ability to agglutinate chicken red blood cells. To determine if changes in HA specificity or affinity correlate with NA specificity or activity, we examined red cell binding and elution of a series of H3N2 viruses. We found that the NA activity of many influenza viruses does not release binding by their HA. In some egg-adapted strains, lack of elution correlates with low levels of viral NA activity, and these elute rapidly when bacterial NA is added. However, a Fujian-like virus, A/Oklahoma/323/03, does not elute by its own NA or with Vibrio cholerae sialidase, and it binds to red cells pre-treated with V. cholerae sialidase. It elutes after addition of the broad specificity Micromonospora viridifaciens sialidase. Human glycophorin inhibits A/Oklahoma/323/03 hemagglutination 6-fold better than fetuin. We conclude that specific forms of sialic acid are used as receptor by recent human H3N2 influenza viruses, perhaps involving branched alpha2,6 sialic acid or alpha2,8 sialic acid structures on O-linked carbohydrates. The virus itself has no O-linked glycans, so even though the NA is not able to cleave receptors on cells, the viruses will not self-aggregate. It will be important to monitor efficacy of neuraminidase inhibitors in case there are NA-resistant receptors in the human respiratory tract that allow the viruses to be less dependent on NA activity.     

Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses

Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attractive candidate as an oncolytic virus for tumor therapy. Previous studies have suggested that VSV selectively infects tumor cells due to defects in their antiviral responses making them more susceptible to VSV infection than normal cells. We tested this hypothesis in the prostate tumor system by comparing LNCaP and PC-3 prostate tumor cells to benign human prostatic epithelial cells from patient prostatectomy specimens. We compared the cell killing ability of a recombinant virus containing a wild-type (wt) M protein (rwt) and an isogenic M protein mutant virus (rM51R-M) that induces interferon (IFN) in infected cells and should display a greater selectivity for tumor cells. Our results showed that in single-cycle infection experiments, LNCaP cells were sensitive to killing by both wt and mutant viruses, while PC-3 cells were highly resistant to VSV-induced cell killing. LNCaP and benign prostate cells were similarly susceptible to both viruses, indicating that normal prostate cells are not inherently resistant to killing by VSV. In each of the cell lines, the rM51R-M virus induced similar levels of apoptosis to rwt virus, showing that the M protein does not play a significant role in apoptosis induction by VSV in these cells. In multiple-cycle infection experiments, LNCaP cells were more sensitive than benign prostatic epithelial cells to virus-induced cell killing by rM51R-M virus, but not rwt virus. Both viruses were equally effective at reducing LNCaP tumor volume in vivo following intratumoral and intravenous inoculation in nude mice, while PC-3 tumors were resistant to VSV treatment. None of the mice treated with rM51R-M virus died as a result of virus infection, while 50-71% of mice treated with rwt virus succumbed to virus infection. Similarly, when inoculated by the more sensitive intranasal route, the rM51R-M virus was less pathogenic than the rwt virus from which it was derived. These results indicate that M protein mutant viruses are superior candidates as oncolytic viruses for therapies of prostate tumors, but future strategies for use of VSV will require testing individual tumors for their susceptibility to virus infection.     

Processing of Norwalk virus nonstructural proteins by a 3C-like cysteine proteinase

Expression of Norwalk virus nonstructural polyprotein precursor in vitro resulted in rapid cotranslational cleavage at specific sites. The cleavage products were similar to those previously identified for Southampton virus, a highly related virus. We inactivated the virally encoded proteinase responsible for cleavage of the nonstructural polyprotein by mutation of the putative catalytic cysteine residue, which resulted in production of full-length polyprotein precursor. NV proteinase was expressed in Escherichia coli as a glutathione S-transferase fusion and purified by GST-affinity chromatography. Activity of the purified proteinase was demonstrated by incubation with the full-length precursor protein. trans cleavage of the nonstructural protein precursor resulted in cleavage products similar to those observed during cotranslational cleavage, however, at lesser efficiency. NV proteinase displayed sensitivities to cysteine and serine protease inhibitors similar to poliovirus 3C proteinase, suggesting that NV proteinase is a member of the viral cysteine proteinase family. We propose that the proteinase may play a regulatory role in viral replication.     

Biclonal expansion of T cells infected with monoclonal Epstein-Barr virus (EBV) in a patient with chronic,active EBV infection

Recent studies have suggested that a high percentage of Epstein-Barr virus (EBV)-infected lymphocytes in peripheral blood of patients with chronic, active EBV infection (CAEBV) is of T cell origin. Although T cells are expanded oligoclonally in CAEBV, it is not clear whether the restricted diversity of T cells arise from immune reaction against EBV-related antigens or from proliferation of EBV-infected cells. We experienced a patient with CAEBV who had biclonal expansion of peripheral blood T cells. We identified clonotypes of these two T cell clones in detail and purified the T cell clones. EBV infected mainly the two T cell clones, whereas the viral loads in peripheral blood cells other than these T cell clones were low or undetectable. The EBV strains infecting the two T cells clones were indistinguishable from each other by a series of genotype analyses of the virus. These results suggest that the two T cell clones infected with the same monoclonal EBV proliferated in peripheral blood of the patient.     

V3 sequence analysis and biological characterization of HIV-1 isolates from asymptomatic and early symptomatic Tanzanian individuals

The aim of this study was to determine HIV-1 V3 sequences, in vitro biological characteristics and co-receptor usage of virus isolates from Tanzania. Virus was isolated from 14 of 17 samples investigated. Four of the isolates induced syncytia in MT-2 cells and used the CXCR4 co-receptor, while the remaining 10 isolates used the CCR5 co-receptor characteristic of non-MT-2 tropic viruses. One of the four MT-2 tropic isolates also used the CCR5 and CCR3 co-receptors. Proviral DNA was detected in all 14 isolates and PCR products were subjected to DNA sequencing. Unambiguous V3 amino acid sequences were obtained from 11 amplificates. Phylogenetic analysis indicated that these sequences were divergent and clustered in HIV-1 subtypes A, C or D. Sequences from the viruses that induced syncytia in MT-2 cells presented characteristic V3 phenotype-associated amino acids. Results of co-receptor analysis are in concordance with the isolate phenotype as determined by replication and induction of syncytia in MT-2 cells. The considerable diversity illustrated by a limited number of isolates from Tanzania is in accordance with reports from other regions of Africa.     

High variability in viral load in cerebrospinal fluid from patients with herpes simplex and varicella-zoster infections of the central nervous system

Infections of the central nervous system (CNS) caused by herpes viruses can result in severe diseases, often with a fatal outcome. In this study, the viral load in the cerebrospinal fluid (CSF) of patients with herpes simplex or varicella-zoster infections of the CNS was measured using a quantitative real-time PCR. The results suggest a high variability in viral load, with relatively mild disease associated with a high viral load in CSF and vice versa. Determination of the viral load in CSF does not therefore seem to be useful in assessing the prognosis of disease caused by these viruses.     

Herpes Simplex Virus Type 1 (HSV-1) Strain HSZP Host Shutoff Gene: Nucleotide Sequence and Comparison with HSV-1 Strains Differing in Early Shutoff of Host Protein Synthesis

The UL41 gene of the HSZP strain of herpes simplex virus type 1 (HSV-1) defective with respect to the early shutoff of host protein synthesis was sequenced and compared with the corresponding HSV-1 strain KOS and 17 gene sequences. In comparison with strain 17, nine mutations (base changes) were HSZP specific, five KOS specific and four were common for both strains. Nine mutations caused codon changes. Three of these mapped to the nonconserved regions and the others to the conserved regions of the functional map of UL4l gene. One KOS specific mutation mapped to the region responsible for the binding of the virion host shutoff (vhs) protein to the alpha-transinducing factor (VP16). The possible relationship between mutations and host shutoff function is discussed. The nucleotide sequence data of the UL41 gene of HSZP and KOS have been submitted to the Genbank nucleotide database and have been assigned the accesion numbers Z72337 and Z72338. This revised version was published online in July 2006 with corrections to the Cover Date.     

Incidence of latent infection of Epstein–Barr virus in lung cancers—an analysis of EBER1 expression in lung cancers by in situ hybridization

To evaluate the presence of Epstein–Barr virus (EBV) in lung cancers of Japanese patients, 81 lung cancers were examined using a highly sensitive in situ hybridization (ISH) method, employing an antisense oligonucleotide probe for EBV-encoded small nuclear RNA-1 (EBER). EBER1 expression was demonstrated in one poorly differentiated squamous cell carcinoma associated with marked lymphoid stroma (PDSCC-LS), two well differentiated adenocarcinomas, and two moderately differentiated squamous cell carcinomas, but was not detectable in other lung cancers, including small cell carcinomas. Unlike lymphoepithelioma-like undifferentiated carcinoma (LELC) of the lung, the PDSCC-LS consisted of poorly differentiated cells with distinct cell borders and nuclei with a coarse chromatin pattern and some prominent nucleoli. Most of the cancer cells expressed intense EBER1 signals. Although small to moderate numbers of cells positive for EBER1 were present in two adenocarcinomas and two squamous cell carcinomas, EBER1 signals varied in intensity and number in these four cases. Although polymerase chain reaction (PCR) and Southern blot hybridization with a ³²P-labelled probe internal to the primers were conducted to detect the EBV genome in 24 lung cancers, including five EBER1-positive cases, the genome was found to be positive in the five cases with EBER1-positive staining, including the PDSCC-LS, two adenocarcinomas and two squamous cell carcinomas, but not in the other cases. This study indicates that the morphological features of EBV-associated lung cancers are not restricted to the typical LELC type.     

High level expression of recombinant mumps nucleoprotein in Saccharomyces cerevisiae and its evaluation in mumps IgM serology.

To develop improved reagents for mumps serology a high-level yeast expression system was employed to produce recombinant mumps nucleoprotein (rNP). The rNP was purified by CsCl gradient centrifugation and yielded approximately 15 mg/l of yeast culture. Electron microscopy of the rNP revealed characteristic herring-bone structures. The electrophoretic mobility of rNP in yeast cells was similar to native NP in SDS-PAGE. Monoclonal antibodies to rNP reacted with native mumps virus nucleoprotein by immunofluorescence assay. A monoclonal antibody to native mumps virus NP reacted with rNP by Western blot assay. The rNP was investigated as antigen in an IgM capture enzyme immunoassay (EIA) using a horseradish peroxidase conjugate of monoclonal antibody to the rNP. Eighteen sera previously found to be positive by IgM capture radioimmunoassay (MACRIA) and 30 sera that were mumps IgM negative by MACRIA were tested by mumps IgM capture EIA. The results for the two test were concordant. In addition, 26 rheumatoid factor positive sera and 35 sera that were IgM positive for measles, rubella or parvovirus B19 were tested. Fifty-nine sera were negative by mumps IgM capture EIA but two sera collected from two infants 3 and 6 weeks after mumps, measles and rubella vaccination were positive. Mumps MACRIA confirmed these results. Compared to MACRIA the overall sensitivity was 100% (20/20) and specificity was 96.8% (30/31). The yeast expressed rNP was highly immunogenic and suitable for use in IgM capture EIA for the diagnosis of mumps.