[Persistence of latex agglutination inhibition antibodies in rabbits following application of poliomyelitis-, Coxsackie-, Echo- and encephalomyocarditis viruses. Brief report].
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Katsuobushi is a traditional processed seafood product used in Japanese-style cooking, and when it is produced through fermentation by fungi, it is called karebushi. The fungi involved in katsuobushi fermentation are collectively referred to as katsuobushi molds. We previously discovered seven novel viruses from katsuobushi molds and determined their genome sequences. However, our previous explorations used only nine fungal strains available from culture collections, leaving the diversity of viruses infecting fungi involved in katsuobushi fermentation unclear. Therefore, in this study, we aimed to isolate fungi from commercially available karebushi and clarify the prevalence of viruses in the isolates. Karebushi produced by three manufacturers was obtained, and 30 fungal strains (including Aspergillus spp.) were isolated from each. Double-stranded RNA (dsRNA) fractions were prepared from the mycelia of the isolated strains. Electrophoresis suggested that a relatively high proportion of the isolates harbored dsRNA elements consistent with RNA virus infection (30-70% per manufacturer; 59% overall). Furthermore, dsRNA sequencing identified four novel viruses in isolates of Aspergillus chevalieri and Aspergillus montevidensis: a beny-like virus, a gammapartitivirus, a narnavirus, and a victorivirus, in addition to two previously reported viruses. Notably, this represents the first report of a beny-like virus in Aspergillus spp. This study provides insights into the diversity of viruses infecting fungi involved in katsuobushi fermentation.
Sixteen established cell lines of simian virus 40 (SV40)-transformed human amnion cells were examined for SV40 production. Many of these lines produced SV40 for extensive periods. Virus production had not ceased for 2 lines after 18 months, for 3 lines after 12 months, and for 3 lines at 3 months after recovery from "crisis". Three lines became virus-free in the first month, 1 line in the second month, 1 in the third month, and 1 in the fourth month, and 2 lines stopped virus production between 6 and 11 months after recovery. The virus titers were relatively low. Inclusion body-containing cells were infrequent. In contrast, in most cultures of SV40-transformed human fibroblasts rescued from crisis, no infectious virus was demonstrated, although exceptions have been reported. Virus was produced after heterokaryon formation of cells of the virus-free amnion lines with CV-1 cells in the presence of inactivated Sendai virus, as observed for SV40-transformed human fibroblasts. During the crisis period, some of the SV40-transformed amnion cells produced substantial amounts of virus. Titers decreased during the later periods of crisis. The most pronounced decrease in titers was in cultures from which established lines were recovered.
As arthropod-borne viruses continue to threaten populations globally, there is a pressing need for experimental systems that enable rapid antiviral discovery. Reverse-genetics platforms producing recombinant reporter orthoflaviviruses have been developed to address this gap. Here, we present two new recombinant tick-borne encephalitis viruses (TBEVrec) generated on a European-subtype Haselmühl Tiho1 isolate backbone. A reporter gene, either eGFP or Nluc, was inserted in the capsid-coding region of the genome downstream of the capsid RNA regulatory signal and separated from the complete viral polyprotein by a 2A self-cleaving peptide. TBEVrec was better rescued using the circular polymerase extension reaction (CPER) than with the infectious subgenomic amplicon (ISA) method. TBEVrec replicated efficiently in relevant human cell lines, with comparable replication to wild-type TBEV in a neuronal cell line and moderately reduced titers and RNA levels in immune-derived cell lines. Using either eGFP or Nluc, we illustrate how TBEVrec enabled high-content RNAi screening, highlighting Nucleolin and PRKD1 as potential TBEV host factors, and drug testing on a benchtop plate reader. Nanopore sequencing of the eGFP insert revealed that the reporter is excised without affecting flanking regions. Comparative analysis of eGFP and Nluc further shows that this instability is time- and cell type-dependent, and that Nluc is comparatively more stable. From these observations, we outline safeguards and design principles that are broadly applicable both to the rescue of existing constructs and to the design of future recombinant reporter virus platforms.
A review of virus thermal inactivation data published in the literature demonstrated variations in reported virus resistance. Examination of the methods used indicated that numerous studies were made by heat processing virus suspensions in test tubes. Duplication of some of the methods using milk suspensions of poliovirus 1 showed virus persistence after heating as a result of uneven temperature distribution inside the test tubes. Unless the containers (preferably sealed ampoules or capillary tubes) are completely submerged in the water bath and agitated vigorously, apparent virus persistence may be encountered.
Influenza virus type C could be propagated to high yield in primary chick embryo kidney cell culture (PCEK) provided that trypsin (2 microgram/ml) was used as a medium supplement. The virus could also be titrated by plaque assay using PCEK host cells and influenza C virus that had been plaque-purified in PCEK cells could then be serially passaged to high titer using the allantoic route of 10--11-day-old embryonated eggs.
Wild-type Marburg virus (MARV) can only be handled in biosafety level 4 facilities. By removing an essential gene from the virus genome, deficient virus particles can be generated that are only capable of replication if the missing gene product is provided in trans. As a result, these viruses are restricted to specific cell lines, making them safe to handle at lower biosafety levels. Here, we provide a detailed overview of how to generate MARV in which the VP30 gene has been replaced by a green fluorescent reporter gene, as well as how to use lentiviral transduction to create stable cell lines expressing MARV VP30. These cell lines can be used for the propagation and confinement of the resulting reporter virus.
Bedbugs of the species Cimex hemipterus (F) were collected on four separate occasions from the bedding in the huts of village dwellers in Senegal, West Africa. Hepatitis-B surface antigen (HBSAg) was detected in unengorged nymph and adult bedbugs in each of the first three collections. 3 of 28 such specimens were HBSAg(+) in the first collection and 3 of 17 specimens were positive in the second collection. In the third, 6 of 9 were HBSAg(+) when the bed occupant was known to be HBSAg(+). 2 of these 6 positive insects did not contain human serum proteins. Bedbugs in the fourth collection were captured and kept alive without a blood meal for 30 days. 3 of 89 of these samples were HBSAg(+). These are the highest field infection-rates of hepatitis-B virus reported in any insect species. The bedbug must be considered a potential vector of hepatitis-B virus.
In Primorye Territory, USSR, cucumber mosaic virus (CMV-I), arabis mosaic virus (AMV), raspberry, ringspot virus (RRSV), and tomato ringspot virus (TRSV) were identified on berry crops (currant, raspberry, honeysuckel). With respect to indicator plants and physico-chemical and serological properties, the isolates obtained do not differ from other isolates of these viruses, reported on berry crops in Europe and North America.
A virus of the RNA type, having no lipid envelope, acid-fast and stable to heat, was isolated from an aborted fetus and from the kidney of a piglet of a lower viability at birth. It was readily cultured, multiplying quickly in primary cell cultures of pig kidney and in the SPEV cell line, producing a characteristic cytopathic effect. It gave no hemagglutination with human, chicken, and swine erythrocytes. Serologically, it was identified as a swine enterovirus of serotype I, identical with the viruses described as the etiologic agents of SMEDI.
A disseminated herpes simplex virus (HSV) infection involving the central nervous system and accompanied by chrioretinitis in a a 3-week-old girl is described. The aetiologic diagnosis was established on the basis of vrius isolated from skin vesicles, and a significant rise in complement fixing antibodies to HSV type 1. The mode of transmission of the virus to the infant apparently was direct contact with an oral lesion in the mother, that was present at the time of delivery. The patient survived but became blind and microcephalic, with severe neurological sequelae. The virus isolated was identified as HSV type 1, which is an infrequent finding in herpetic chorioretinitis of the newborn.
Remdesivir (RDV) is a nucleotide analog prodrug approved for COVID-19 treatment that inhibits the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp; nsp12). Although RDV maintains activity against circulating variants of concern, ongoing evaluation of resistance-associated substitutions is critical for clinical care, particularly in settings of prolonged viral replication such as immunocompromised individuals. We assessed the phenotypic impact of nsp12 substitutions identified from in vitro resistance selection, RDV clinical reports, and global sequence surveillance. Using a recombinant infectious SARS-CoV-2 reporter virus, we compared susceptibility of these nsp12 substitutions to RDV and its parent nucleoside, GS-441524. After confirming concordant resistance profiles between RDV and GS-441524, we assessed RDV susceptibility in a complementary non-infectious replicon system. In both systems, single nsp12 substitutions remained fully susceptible to RDV within their respective assay variability limits. Of the double substitutions tested, S759A/V792I conferred the largest reduction in antiviral susceptibility (∼15-fold) but was associated with impaired replication kinetics. Given the strong concordance between the two assays, the replicon system also enabled phenotypic characterization of substitutions E802A, E802D, and P323L/E802D that could not be rescued as infectious virus. Analysis of >17 million SARS-CoV-2 genomes in GISAID showed that all tested nsp12 substitutions had low prevalence (≤0.1%), except P323L (98.8%). Collectively, these data reinforce the high genetic barrier to RDV resistance, as reduced susceptibility is typically accompanied by substantial reductions in replication. Our findings support the continued clinical utility of RDV and highlight the complementary value of SARS-CoV-2 infectious virus and replicon systems for antiviral resistance surveillance and phenotyping.
The current methods of diagnosing and treating herpes genitalis in clinics for sexually transmitted diseases in England and Wales are reported. Virus cultures were used as a diagnostic test in some or all patients in nearly two-thirds of clinics. Darkground examinations were performed on all or selected patients in most clinics, but fewer than half the clinics performed them on three or more separate occasions. In over 80% of clinics attempts were made to see some or all contacts of patients with herpes. The treatments prescribed varied, the commonest being saline washes, idoxuridine, and oral antibiotics. Serological tests were repeated after the disappearance of the lesions in 90% of clinics, while in 60% of clinics women with herpes genitalis were advised to have cytological examination at regular intervals in future. The importance of excluding syphilis in patients presenting with genital ulceration, the most appropriate treatments for herpes, and the possible association between the disease and cervical carcinoma are discussed.
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a bat-originated alphacoronavirus that causes devastating enteric disease in neonatal piglets and possesses significant potential for cross-species transmission. While the early stages of the coronavirus life cycle have been extensively characterized, the host factors indispensable for virion assembly and subsequent export remain largely enigmatic. Here, by performing a genome-wide CRISPR-Cas9 knockout screen using a recombinant icSADS-CoV-GFP reporter virus, we identified the small GTPase Rab10 as a critical host dependency factor for SADS-CoV infection. Viral life cycle analysis revealed that Rab10 is not required for viral attachment, entry, or initial genome replication, but is essential for the virion transport and non-lytic egress. Rab10 deficiency markedly reduced the extracellular release of viral RNA, viral proteins, and infectious progeny, as well as the secretion of SADS-CoV virus-like particles. Confocal imaging showed that Rab10 and viral protein-positive intracellular structures were associated with LMAN1, TGN46, and LAMP1 positive compartments. These findings support a model in which Rab10 coordinates a virus-containing vesicles trafficking pathway associated with ERGIC-TGN-lysosome compartments. Mechanistically, Rab10 facilitates the loading of the viral envelope (E) protein into transport vesicles derived from the ERGIC. Rab10 associates with the SADS-CoV E protein, and mapping analyses implicated the C-terminal PDZ-binding motif, particularly residue V75, in efficient Rab10 association and viral release. Collectively, our findings identify Rab10 as a host regulator of SADS-CoV non-lytic egress and highlight the E-Rab10 interaction and the vesicular trafficking machinery as a potential target for developing antiviral strategies.