Differential Nervous Necrosis Virus (NNV) Replication in Five Putative Susceptible Cell Lines

Viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is one of the most threatening viral diseases affecting marine fish worldwide. In vitro propagation of NNV strains is essential for the design of effective control measures. In the present study we analysed both the susceptibility and the permissiveness of five fish cell lines (E-11, GF-1, SAF-1, DLB-1, and SaB-1) to three NNV strains (one RGNNV, one SJNNV, and one reassortant RGNNV/SJNNV). E-11 and DLB-1 were demonstrated to be highly susceptible to NNV strains, with average adsorption efficiency (AE) values higher than 90%. SAF-1 also showed high susceptibility (AE 88%), whereas GF-1 can be regarded as moderately susceptible (AE around 50%). On the contrary, SaB-1 can be considered a poorly susceptible cell line (AE values below 20%). E-11 and GF-1 cell lines provided the highest production rates for RGNNV and RG/SJ (around 103) and both cell lines can be regarded as fully permissive for these viral types. However, the SJNNV production rate in GF-1 was only 17.8 and therefore this cell line should be considered semi-permissive for this genotype. In SAF-1 cells, moderate viral replication was recorded but differences in intracellular and extracellular production suggest that viral progeny was not efficiently released. In DLB-1 and SaB-1 the final viral titres obtained in E-11 were lower than those of the inoculum. However, RNA1 synthesis values seem to indicate that RGNNV replication in DLB-1 and SAF-1 could have been underestimated, probably due to a poor adaptation of the virus grown in these cell lines to E-11. Based on all these results, E-11 seems to be the most appropriate cell for in vitro culture of RGNNV, SJNNV, and reassortant strains.

Long-distance airborne dispersal of SARS-CoV-2 in COVID-19 wards

Evidence suggests that SARS-CoV-2, as well as other coronaviruses, can be dispersed and potentially transmitted by aerosols directly or via ventilation systems. We therefore investigated ventilation openings in one COVID-19 ward and central ducts that expel indoor air from three COVID-19 wards at Uppsala University Hospital, Sweden, during April and May 2020. Swab samples were taken from individual ceiling ventilation openings and surfaces in central ducts. Samples were subsequently subjected to rRT-PCR targeting the N and E genes of SARS-CoV-2. Central ventilation HEPA filters, located several stories above the wards, were removed and portions analyzed in the same manner. In two subsequent samplings, SARS-CoV-2 N and E genes were detected in seven and four out of 19 room vents, respectively. Central ventilation HEPA exhaust filters from the ward were found positive for both genes in three samples. Corresponding filters from two other, adjacent COVID-19 wards were also found positive. Infective ability of the samples was assessed by inoculation of susceptible cell cultures but could not be determined in these experiments. Detection of SARS-CoV-2 in central ventilation systems, distant from patient areas, indicate that virus can be transported long distances and that droplet transmission alone cannot reasonably explain this, especially considering the relatively low air change rates in these wards. Airborne transmission of SARS-CoV-2 must be taken into consideration for preventive measures.

Long-distance airborne dispersal of SARS-CoV-2 in COVID-19 wards

Evidence suggests that SARS-CoV-2, as well as other coronaviruses, can be dispersed and potentially transmitted by aerosols directly or via ventilation systems. We therefore investigated ventilation openings in one COVID-19 ward and central ducts that expel indoor air from three COVID-19 wards at Uppsala University Hospital, Sweden, during April and May 2020. Swab samples were taken from individual ceiling ventilation openings and surfaces in central ducts. Samples were subsequently subjected to rRT-PCR targeting the N and E genes of SARS-CoV-2. Central ventilation HEPA filters, located several stories above the wards, were removed and portions analyzed in the same manner. In two subsequent samplings, SARS-CoV-2 N and E genes were detected in seven and four out of 19 room vents, respectively. Central ventilation HEPA exhaust filters from the ward were found positive for both genes in three samples. Corresponding filters from two other, adjacent COVID-19 wards were also found positive. Infective ability of the samples was assessed by inoculation of susceptible cell cultures but could not be determined in these experiments. Detection of SARS-CoV-2 in central ventilation systems, distant from patient areas, indicate that virus can be transported long distances and that droplet transmission alone cannot reasonably explain this, especially considering the relatively low air change rates in these wards. Airborne transmission of SARS-CoV-2 must be taken into consideration for preventive measures.

Contributions of the four essential entry glycoproteins to HSV-1 tropism and the selection of entry routes

ABSTRACTHerpes Simplex viruses (HSV-1 and HSV-2) encode up to 15 glycosylated and unglycosylated envelope proteins. Four of these, gB, gH, gL, and gD, are essential for entry and mediate cell-cell fusion when co-expressed in uninfected, receptor-bearing cells. However, their contributions to HSV-1 tropism and the selection of entry routes are unclear. To begin addressing this, we previously pseudotyped VSV lacking its native glycoprotein, G, with HSV-1 glycoproteins gB, gH, gL, and gD. This novel VSVΔG-BHLD pseudotype recapitulated several aspects of HSV-1 entry: it could enter murine C10 cells, required gB, gH, gL, gD, and a cellular receptor for entry, and was sensitive to neutralization by gB and gH/gL antibodies. Here, we screened six additional HSV-1-susceptible cell lines and found that only two, C10 and CHO-HVEM cells, reproducibly supported a receptor-dependent entry by VSVΔG-BHLD. We then compared VSVΔG-BHLD and HSV-1 entry routes into these two cell lines using a combination of chemical and genetic inhibitors of cellular uptake pathways. We discovered that the VSVΔG-BHLD pseudotype not only has a narrower tropism but also uses entry pathways different from those used by HSV-1. We conclude that while the four essential HSV-1 entry glycoproteins enable entry in certain contexts, they are insufficient for entry into any HSV-1-susceptible cell nor do they specify native HSV-1 entry routes. We hypothesize that the HSV-1 envelope proteins outside the essential four (so-called “non-essential”) contribute towards the tropism and the selection of native HSV-1 entry routes. Our work draws attention to the need for systematic investigation of the HSV-1 entry mechanisms and the roles of the envelope proteins that were long considered non-essential in the selection of target cells, routes of entry, and pathogenesis.AUTHOR SUMMARYDifferent viruses enter cells by diverse routes, but how that choice is made is not always clear. Understanding the mechanisms behind these choices is vital for finding strategies to prevent viral infections. In enveloped viruses, viral proteins embedded in the envelope accomplish this task. While most enveloped viruses encode one or two envelope proteins, Herpes Simplex viruses (HSV) encode up to 15. Four of these are deemed essential for entry (gB, gH, gL, and gD) whereas the rest have been termed non-essential. While these four proteins are essential, their contributions to HSV-1 cellular tropism and entry pathways have not been fully elucidated. Here, we generated virions that have only the four essential HSV-1 glycoproteins on their surface. We show that the VSVΔG-BHLD pseudotype has a narrower tropism than HSV-1 and uses different entry pathways. Thus, the four essential HSV-1 entry glycoproteins alone do not define HSV-1 tropism or specify native entry routes. We hypothesize that the HSV-1 envelope proteins outside the essential four may contribute towards tropism and entry route selection. Our work emphasizes the need to investigate the roles of the so-called non-essential envelope proteins in HSV entry. This is important because HSV enters natural target cells, epithelial cells and neurons, by different, poorly defined routes. Mechanistic understanding of HSV entry is essential for understanding its pathogenesis and developing new strategies to prevent HSV entry and spread.

COMPARATIVE STUDIES OF FELINE VIRAL RHINOTRACHEITIS VIRUS FOR ITS REPLICATION PROPERTIES IN DIFFERENT CELL CULTURES

The results of comparative studies of feline viral rhinotracheitis virus for its culture properties in primary and continuous cell cultures of feline origin (FK, FK (subculture), CrFK, FS, CC-81, FC/Tg) are presented. It was found that viral rhinotracheitis virus replication, irrespective of the route of infection and the culture technique, was consistent and practically equal in susceptible cell cultures. The most pronounced cytopathic effect (more than 75% monolayer degeneration) was observed in all types of cell cultures in 48–72 hours of cultivation. However, the accumulation of feline viral rhinotracheitis virus Grand strain was highest when preliminary adsorption occurred within the specified period of time, monolayer cell cultures were infected with the virus at a dose of 5.5 lg TCID50/ml and roller bottle cultivated, and the рН of the medium was maintained at 7.0–7.4. Single freezing of the virus at a temperature of minus 60 degrees Celsius upon the completion of the cultivation cycle (during 60–72 hours) and its thawing were found to significantly increase the virus titre by 0.5 lg TCID50/ml.

Heparan Sulfate Is an Attachment Factor for Foamy Virus Entry

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID50s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P< 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

Emergence of Linezolid-Resistant Mutants in a Susceptible-Cell Population of Methicillin-Resistant Staphylococcus aureus

ABSTRACTMethicillin-resistantStaphylococcus aureuswith a MIC of linezolid of 4 μg/ml, isolated from a patient who had undergone unsuccessful linezolid therapy, yielded linezolid-resistant mutants in blood agar at 48 h of incubation. The resistant clones showed a MIC of linezolid ranging from 8 to 64 μg/ml and accumulated the T2500A mutation(s) of the rRNA genes. Emergence of these resistant clones appears to be facilitated by a cryptic mutation or mutations associated with chloramphenicol resistance.