Instantaneous Inactivation of Herpes Simplex Virus by Silicon Nitride Bioceramics

Abstract

Hydrolytic reactions taking place at the surface of a silicon nitride (Si₃N₄) bioceramic were found to induce instantaneous inactivation of Human herpesvirus 1 (HHV-1, also known as Herpes simplex virus 1 or HSV-1). Si₃N₄ is a non-oxide ceramic compound with strong antibacterial and antiviral properties that has been proven safe for human cells. HSV-1 is a double-stranded DNA virus that infects a variety of host tissues through a lytic and latent cycle. Real-time reverse transcription (RT)-polymerase chain reaction (PCR) tests of HSV-1 DNA after instantaneous contact with Si₃N₄ showed that ammonia and its nitrogen radical byproducts, produced upon Si₃N₄ hydrolysis, directly reacted with viral proteins and fragmented the virus DNA, irreversibly damaging its structure. A comparison carried out upon testing HSV-1 against ZrO₂ particles under identical experimental conditions showed a significantly weaker (but not null) antiviral effect, which was attributed to oxygen radical influence. The results of this study extend the effectiveness of Si₃N₄'s antiviral properties beyond their previously proven efficacy against a large variety of single-stranded enveloped and non-enveloped RNA viruses. Possible applications include the development of antiviral creams or gels and oral rinses to exploit an extremely efficient, localized, and instantaneous viral reduction by means of a safe and more effective alternative to conventional antiviral creams. Upon incorporating a minor fraction of micrometric Si₃N₄ particles into polymeric matrices, antiherpetic devices could be fabricated, which would effectively impede viral reactivation and enable high local effectiveness for extended periods of time.

Keywords: Herpes simplex virus; Raman spectroscopy; human herpesvirus; instantaneous inactivation; reverse transcription polymerase chain reaction; silicon nitride; surface hydrolysis.

Figure 1

Results of immunochemical testing on Vero cells infected with HSV-1 virus after 1 min (a) or 10 min (b) exposure to Si₃N₄ or ZrO₂ powders. All data are plotted (with statistical validation) in terms of PFU counts (cf. labels in the inset). Sham refers to a virus sample unexposed to ceramic particles. In (c) and (d), virus reduction percentage rates are plotted for virions exposed to ceramic particles for 1 and 10 min, respectively.

Figure 2

Results of RT-PCR tests to evaluate viral DNA fragmentation upon exposure to Si₃N₄ or ZrO₂ ceramic powders for 1 and 10 min (in (a) and (b), respectively); a comparison of supernatants and powders in comparison with virions simply suspended in water is given using evaluations of viral DNA. Red inset labels in (a,b) comply with ANOVA variance statistics.

Figure 3

Raman spectra in the wavenumber interval 600~1800 cm⁻¹ of (a) the pristine HSV-1 and of (b) and (c) of the same virus after exposure for 1 min in aqueous suspension to Si₃N₄ and ZrO₂ particles, respectively. The spectra were normalized to their maximum signal and deconvoluted into Lorentzian-Gaussian sub-band components. Four zones are emphasized in (a), and labels show maximum wavenumbers for selected bands (the inset blue, green, and orange wavenumbers are in cm⁻¹ units). Met, Tyr, Trp, and Phe are abbreviations for methionine, tyrosine, tryptophan, and phenylalanine, respectively. Additional vibrations related to structural modifications induced by exposure to ceramic powders are emphasized with pink asterisks, as mentioned in the text.

Figure 4

Schematic drafts of (a) methionine (Met) and (b) cysteine (Cys) trans and gauche rotamers with related vibrational stretching modes. In (c), (d), and (e), highly resolved Zone I of the HSV-1 spectrum before and after 1 min exposure in aqueous suspensions of Si₃N₄ and ZrO₂ micrometric powders, respectively; spectra are deconvoluted into a sequence of Lorentzian-Gaussian sub-band components (the inset wavenumbers are in cm⁻¹ units).

Figure 5

Schematic drafts of (a) zwitterionic, (b) partly hydrated, and (c) fully hydrated tyrosine (Tyr) molecules. In (d), (e), and (f), a highly resolved Zone II of the HSV-1 spectrum indicates the virus before and after 1 min exposure in aqueous suspension to Si₃N₄ and ZrO₂ micrometric powder, respectively; spectra are deconvoluted into a sequence of Lorentzian-Gaussian sub-band components (the inset wavenumbers are in cm⁻¹ units; values of Raman ratios R_Tyr = I₈₅₁/I₈₂₃ are given inset). Additional vibrations related to structural modification upon exposure to ceramic powders are emphasized with asterisks as discussed in the text.

Figure 6

Schematic drafts of (a) protonated and partly deprotonated tryptophan (Trp) molecules. In (b), (c), and (d), highly resolved spectra of the HSV-1 virus (in the wavenumber interval 1280~1420 cm⁻¹) before and after 1 min exposure in aqueous suspension to Si₃N₄ and ZrO₂ micrometric powders, respectively, are deconvoluted into a sequence of Lorentzian-Gaussian sub-band components (the inset wavenumbers shown in green, blue, and royal blue are in cm⁻¹ units; inset values are of Raman ratios R_Trp = I₁₃₆₄/I₁₃₃₉). Additional vibrations related to structural modification upon exposure to ceramic powders are emphasized with asterisks as discussed in the text.

Figure 7

(a) Schematic draft of linked DNA nucleobases with wavenumbers of their respective ring vibrational fingerprints (cf. labels); in (b), comparison between relative intensities of fingerprint signals for phosphate-deoxyribose backbone, adenine, cytosine, thymine, and guanine (cf. labels in (a)) in the spectrum of unexposed HSV-1 virions (taken as 100%) and spectra of HSV-1 virions after 1 min exposure in aqueous suspension to Si₃N₄ and ZrO₂ micrometric powders (cf. intensity reductions indicated in colored inset labels).

Figure 8

(a) Schematic drafts of Amide I vibrations and secondary structures of proteins with their relative wavenumber intervals. In (b), (c), and (d), highly resolved spectra of the HSV-1 virus in Zone IV (1600~1750 cm⁻¹) before and after 1 min exposure to Si₃N₄ and ZrO₂ micrometric powders in aqueous suspension, respectively (cf. labels in inset); spectra are deconvoluted into a sequence of Lorentzian-Gaussian sub-band components (the inset wavenumbers are in cm⁻¹ units; abbreviations βs, αh, rc, βt₁, and βt₂ refer to β-sheet, α-helix, random coil, and Type I and Type II β-turn rotamers, respectively). Inset labels also give the relative fractions of different secondary structures. Additional vibrations related to structural modification upon exposure to ceramic powders are emphasized with asterisks as discussed in the text.

Figure 9

Schematic drafts of: (a) the proposed interaction between methionine residues contained in the envelope glycoproteins of HSV-1 virions and deprotonated silanols formed in the highly alkaline environment at the Si₃N₄ solid surface, and (b) the interaction between HSV-1 solid surface interaction upon Si₃N₄ hydrolysis. The most probable scenario behind the electrostatic attraction between Si₃N₄ surface and envelope proteins involves deprotonated silanol groups at the surface of Si₃N₄ strongly attracting the C-COOH terminus of methionine and cysteine residues. A schematic diagram of the chemical reactions leading to DNA backbone cleavage (c) and a draft of damaged HSV-1 undergoing a structural order → disorder transition due to the presence of ammonia (d); backbone damage starts with deprotonation at the 2′-hydroxyl group by NH₃, proceeds with destabilization of the ribose ring chain with the formation of a transient pentaphosphate, and ends with bond cleavage upon interaction with acidic NH₄⁺ ions. (e) The destabilization process of the α-helix structure as triggered by the disruption of its hydrogen bonds in a highly alkaline environment; and (f) the draft of the protein structural modifications from α-helix to β-sheet, ultimately leading to the loss of glycosylation sites in envelope glycoproteins.