First isolation and characterization of a mosquito-borne orbivirus belonging to the species Umatilla virus in East Asia

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.     

A new cryptic virus belonging to the family Partitiviridae was found in watermelon co-infected with Melon necrotic spot virus

A novel virus was detected in watermelon plants (Citrullus lanatus Thunb.) infected with Melon necrotic spot virus (MNSV) using SOLiD next-generation sequence analysis. In addition to the expected MSNV genome, two double-stranded RNA (dsRNA) segments of 1,312 and 1,118 bp were also identified and sequenced from the purified virus preparations. These two dsRNA segments encode two putative partitivirus-related proteins, an RNA-dependent RNA polymerase (RdRP) and a capsid protein, which were sequenced. Genomic-sequence analysis and analysis of phylogenetic relationships indicate that these two dsRNAs together make up the genome of a novel Partitivirus. This virus was found to be closely related to the Pepper cryptic virus 1 and Raphanus sativus cryptic virus. It is suggested that this novel virus putatively named Citrullus lanatus cryptic virus be considered as a new member of the family Partitiviridae.     

Quantitation of antibodies to varicella-zoster virus by immune adherence hemagglutination.

Immune adherence hemagglutination was compared with the complement fixation test as a means of measuring antibodies to varicella-zoster virus. Analysis of acute- and convalescent-phase sera from patients infected with varicella-zoster or with herpes simplex virus showed the immune adherence hemagglutination test to be more sensitive than the complement fixation test, and greater cross-reactivity between the two viruses appeared to be associated with the increased sensitivity. The two assay methods were used to measure antibodies to varicella-zoster virus in 265 sera obtained from patients of different ages as well as sera from 26 patients with leukemia. There were 35 cases where antibodies were detected by immune adherence hemagglutination but not by complement fixation, whereas in five cases the converse was found. Our findings support the contention that immune adherence hemagglutination is the method of choice for detecting antibodies to varicella-zoster virus.     

Hepatitis A virus hemagglutination and a test for hemagglutination inhibition antibodies.

Like enteroviruses, hepatitis A virus (HAV) hemagglutinated various species of erythrocytes under similar conditions. HAV-specific antibodies in both acute- and convalescent-phase sera were found to inhibit hemagglutination. The HAV hemagglutination inhibition test can be used for diagnosis, epidemiological surveillance, and vaccine assessment.     

Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae.

Bacterial attachment is believed to be an early step in gram-negative nosocomial pneumonia. The frequency of fimbria-associated adhesins among respiratory pathogens has not been studied in detail. In this study isolates belonging to the family Enterobacteriaceae, prospectively obtained from intensive care unit patients who were suspected of having nosocomial pneumonia, were examined for fimbria-associated adhesins. Type 3, P, type 1, and other fimbrial phenotypes were identified by specific hemagglutination and electron microscopy. The Klebsiella type 3 fimbrial phenotype was further characterized by using a monoclonal antibody. Also, both type 3 and Escherichia coli P fimbrial genotypes were detected by using DNA colony blot assays. The frequencies of genera or species isolated were as follows: Enterobacter (38.6%), Klebsiella (26.8%), Serratia (17.7%), E. coli (13%), and Proteus (5.2%). Isolates of Klebsiella oxytoca, K. pneumoniae, and Enterobacter cloacae most commonly possessed the type 3 fimbrial phenotype and genotype. The phenotype and genotype for E. coli P fimbriae (46.2 and 50%, respectively), a known pathogenic determinant in the urinary tract, were detected more frequently than expected. In addition, a previously unspecified hemagglutinin that was specific for porcine erythrocytes was almost uniformly expressed among isolates of Enterobacter aerogenes. Finally, the expression of the type 1 fimbrial phenotype was widely detected among the isolates tested but notably absent among K. oxytoca and Proteus mirabilis isolates. The frequency of the various fimbrial types identified suggests a role for these bacterial organelles in adherence to respiratory epithelia.     

Evolutionary relationships of virus species belonging to a distinct lineage within the Ampelovirus genus

A study of the evolutionary relationships of GLRaV-4,-5,-6 and -9, and two new Ampelovirus isolates (GLRaV-Pr and -De) related to grapevine leafroll disease was conducted based on molecular variability, positive selection analysis and maximum likelihood phylogenetic reconstructions. Sequences corresponding to the N-terminal HSP70h and full CP encoding genes were determined for these viruses and datasets including homologous genomic regions from different members of the Closteroviridae were analyzed. GLRaV-Pr and -De were further characterised as distinct from the other closely related species after determination of a large genomic region (4319-4358 nts). ML phylogenetic topologies for both genes established the closer phylogenetic relationships of GLRaV-4,-5,-6,-9,-Pr and -De in regard to the other ampeloviruses, revealing very low inter-species evolutionary distances for this multitudinous lineage. The HSP70h segment phylogeny and bootstrap analysis enabled the identification of species within this lineage and provides a useful taxonomic tool for the rapid demarcation of these viruses. Estimations of d(N)/d(S) using the CP and HSP70h datasets revealed that, within the Closteroviridae, these viruses are subjected to the strongest constraints against amino acid substitutions. These estimations demonstrated a distinct evolutionary trait for this lineage probably related to its particular ecological niche that involves successful adaptation to the host, transmission through vegetative propagation and lack of vectors with high transmission efficiency.     

Detection of antibodies to Epstein-Barr virus capsid antigen by immune adherence hemagglutination.

The immune adherence hemagglutination assay was found to be as sensitive and specific as the indirect immunofluorescence technique for titration of antibodies to Epstein-Barr virus capsid antigen. Satisfactory virus capsid antigen-specific and negative control antigens for the immune adherence hemagglutination assay were prepared from cell extracts of the Epstein-Barr virus producer P3HR-1 and the Epstein-Barr virus genome-negative BJAB lymphoblastoid cell lines, respectively. As the immune adherence hemagglutination assay can be used to titrate antibodies to both the heterophil antigen of the Paul-Bunnell type and to virus capsid antigen, it offers a promising alternative to the immunofluorescence methods in the serodiagnosis of Epstein-Barr virus infections which can be performed by most diagnostic laboratories.     

Specificity of the passive hemagglutination test for antibody to rubella virus and the passive hemagglutination response after vaccination.

Nonspecific reactions by the passive hemagglutination test for rubella viral antibody occurred with 1.5% of sera tested. Rises in passive hemagglutination titers (greater than or equal to 4X) occurred with some serum samples taken 14 days after vaccination.     

Insights into new bacteriophages of Lactococcus garvieae belonging to the family Podoviridae

Lactococcus garvieae is an emerging pathogen responsible for lactococcosis, a serious disease in trout aquaculture. The identification of new bacteriophages against L. garvieae strains may be an effective way to fight this disease and to study the pathogen’s biology. Three L. garvieae phages, termed WP-1, WWP-2 and SP-2, were isolated from different environments, and their morphological features, genome restriction profiles and structural protein patterns were studied. Random cloning of HindIII-cut fragments was performed, and the fragments were partially sequenced for each phage. Although slight differences were observed by transmission electron microscopy, all of the phages had hexagonal heads and short non-contractile tails and were classified as members of the family Podoviridae. Restriction digestion analysis of the nucleic acids of the different phages revealed that the HindIII and AseI digests produced similar DNA fragment patterns. Additionally, SDS-PAGE analysis indicated that the isolated phages have similar structural proteins. The sequence BLAST results did not show any significant similarity with other previously identified phages. To the best of our knowledge, this study provides the first molecular characterization of L. garvieae phages.     

Bluetongue virus hemagglutination and its inhibition by specific sera

Summary Bluetongue-virus (BTV) was found to agglutinate a variety of erythrocytes including sheep-, chicken-, guinea pig- and mouse-erythrocytes. Hemagglutination was inhibited specifically with type specific serum. A temperature dependence was only found for chicken erythrocytes, which showed a hemagglutination optimum at 37° C. The hemagglutination was lost upon treatment of the virus with 0.4 per cent trypsin as well as after treatment with 0.01M KJO₄. Heating of the virus preparation to 56° C resulted in the loss of the HA-activity. Gelchromatographic studies indicated that the hemagglutinating capacity is associated with the complete virion. Whereas virulent strains of BTV hemagglutinate a number of different erythrocytes the avirulent type tested produced only a slight hemaglutination with sheep red blood cells. However, specific antiserum produced with the avirulent strain yielded strong hemagglutination inhibition (HI) with the corresponding virulent strain. Treatment of sera prior to their use in the HI proved necessary to remove nonspecific inhibitors. The efficiency of KJO₄ in removing nonspecific inhibitors indicates that carbohydrates represent the major group of nonspecific inhibitors. The data represented recommend the hemagglutination inhibition test as a new method to identify the various BTV serotypes.With 1 Figure     

Studies on influenza virus hemagglutination inhibition by anti-host serum

Summary Hemagglutination inhibition activity of anti-host serum was enhanced by antiglobulin in an indirect HI test. The resulting influenza virus-anti-host-antiglobulin complexes were irreversible, whereas the virus-anti-host complexes were reversible. The dissociability of the virus antibody complexes was examined with antibody redistribution method.     

Mechanism of translation initiation by Dicistroviridae IGR IRESs

The Dicistroviridae is a growing virus family characterized by a dicistronic genome, wherein each open reading frame (ORF) is translated from an independent internal ribosome entry site (IRES). The 5′ IRES that translates the first open reading frame (ORF1) is similar to the picornaviral IRESs. However the second IRES, referred to as the intergenic region (IGR) IRES, - translates ORF2 by and uses an unusual mechanism of initiating protein synthesis. It folds into a compact RNA structure that can bind directly to 40S ribosomal subunits and form 80S complexes to initiate translation in the absence of any initiation factors. Despite its unusual mechanism, the IGR IRES has proven to be an elegant model for elucidating initiation mechanisms employed by IRESs, as well as making it a powerful research tool with diverse applications.     

Rat alpha 1 macroglobulin inhibits hemagglutination by influenza C virus

Purified alpha 1-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed.     

Watermelon bud necrosis tospovirus is a distinct virus species belonging to serogroup IV

Summary.  The nucleocapsid protein gene of a tospovirus infecting watermelon in India was cloned and sequenced. Sequence analyses showed that the gene was most closely related to those of watermelon silver mottle tospovirus (WSMV) from Taiwan and peanut bud necrosis tospovirus (PBNV) from India, the two definitive species of serogroup IV. Amino acid sequence similarity was 84% and 82% with WSMV and PBNV, respectively. On the basis of the sequence divergence and the previously determined host range differences, the watermelon tospovirus, designated as watermelon bud necrosis tospovirus, should be considered as a distinct species belonging to serogroup IV. Received October 3, 1997 Accepted March 2, 1998     

First description of Grapevine leafroll-associated virus 5 in Argentina and partial genome sequence

An accession of Vitis vinifera cv. Red Globe from Argentina, was found to be infected with Grapevine leafroll-associated virus-5 by ELISA. It was partially sequenced, and three ORFs, corresponding to HSP70h, HSP90h, and CP, were found. This isolate shares a high aminoacid identity with the previously reported sequence of the virus, and identities between 80% and 90% with previously reported GLRaV-9 and GLRaV-4 isolates. The analysis of the sequence supports the clustering together with GLRaV-4 and GLRV-9 inside the Ampelovirus genus.     

Detection of early hemagglutination inhibitory antibodies to rubella virus by pretreatment of sera with streptococcal cells

A technique that is based on absorption of sera with streptococcal cells and hemagglutination inhibition (HI) was evaluated for its feasibility for serologic diagnosis of recent rubella. The mixture of AR1 and AW43 cells removes IgG and IgA from the kaolin-treated sera, leaving IgM and a trace of IgA, probably oligomeric IgA. Consequently, after the absorption with streptococci, the HI antibodies are detectable exclusively in the early sera of patients with rubella. The streptococci (AR1 and AW43) have several advantages as the absorbent over the staphylococcus (Cowan I) that has been used routinely.