Reversible Inhibition of Spreading of In Vitro Infection and Imbalance of Viral Protein Accumulation at Low pH in Viral Hemorrhagic Septicemia Rhabdovirus, a Salmonid Rhabdovirus

Abstract

The inhibition of viral hemorrhagic septicemia rhabdovirus (VHSV) in vitro infection by pHs of <7 (low pH) has been previously reported. Nevertheless, the details of the mechanism underlying this effect remain obscure. We present evidence showing that low-pH inhibition occurs during a viral postadsorption step. Thus, while VHSV bound, replicated within single cells, and presented its G protein on the membranes of infected cells at both low and physiological pHs, both cell-to-cell spreading of infection (as estimated by the appearance of foci of infected cells) and fusion (as estimated by a syncytium assay) were inhibited by this low pH. The decreased VHSV titers and the inhibition of both cell-to-cell spreading of infection and fusion could be reversed by adjusting the pH to 7.5 at any time during infection. This effect should be taken into account to avoid false negatives in the diagnosis of VHSV by cell culture. On the other hand, the cell-to-cell spreading of infection at pH 7.5 could be stopped at any time by reducing the pH to 6.5. Since at low pH there were changes in the protein G conformation and smaller and imbalanced amounts of N with respect to M1, M2, and G viral proteins, alterations of the assembly and/or budding of VHSV are most probably involved in the absence of newly released infective virions.In many enveloped viruses, after the virus particle is internalized by a receptor-mediated endocytosis mechanism (6), viral and cell membrane fusions are triggered by the decrease of the endosomal pH (30). Conformational changes are induced by the low pH in the viral glycoproteins (25, 51) to cause fusion. Thus, agents that inhibit the lowering of the pH in the endosomes, such as NH 4 ϩ , also inhibit viral fusion and infectivity (11,44,49). However, it has been shown that a low-pH environment outside the host cells can also inhibit the in vitro viral replication and/or fusion of viruses such as vesicular stomatitis virus (VSV) (32), rabies virus (RV) (21,22,26,35), viral hemorrhagic septicemia rhabdovirus (VHSV) (10, 50, 52), influenza virus (5), poliovirus (18), or retrovirus (4,42,56).It was proposed earlier that the inhibition of VSV infectivity caused by low pH was due not to inhibition of viral RNA or protein synthesis (18) but to some yet-unidentified alterations in the nucleocapsid replication complex (32). Since then, there have been no further references about the mechanisms causing low-pH inhibition of VSV or any other rhabdoviruses, such as RV or VHSV. However, the study of this aspect of rhabdovirus infection might have important implications for the inactivation of virus in biological fluids (29) and for laboratories responsible for proper surveillance of these diseases, which should correctly diagnose possible false negatives.To further study this phenomenon, we choose VHSV (a salmonid rhabdovirus) as a model among rhabdoviruses, because VHSV causes cytopathic effects of established fish cell lines only at pH 7.0 or higher (10, 50, 52), and this might cause problems in cell culture detectio...

Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus

“…In vitro, those antibody-armed NK-like effector cells could have attached to VHSV-infected target cells. Since it has been demonstrated that VHSV-infected cells incorporate G protein into their cell membrane [54], this attachment could result in ADCC. Therefore the question of ADCC should be a subject of further investigations.…”

Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

“…In all these cases, the change in conformation at physiological pH at position 140 or 433 (as estimated by binding of MAb C10) and 235 (as estimated by binding of MAb 2F1A12) did not abolished fusion. The increase in the binding of MAbs C10 and 2F1A12 to wild-type pG at low pH (31) might indicate that the conformation at low pH required for fusion is less strongly affected by these mutations. Figure 1 shows that the mutants with fusion activity at position P79 or L85 (around position 80) and position T135 (around position 140) and the mutants resistant to neutralization by MAb C10 (21) were mapped at positions around either amino acid 80 or 140, the two locations around which the number of amino acid changes in 22 isolates of VHSV was the greatest.…”

In VitroNeutralization of Viral Hemorrhagic Septicemia Virus by Plasma from Immunized Zebrafish

“…32 The use of tamponed water with high pH facilitated the reproducibility of the pH-sensitive infection with VHSV. 33 All the experimental protocols were performed with the approval of the Ethics Committee at INIA.…”