Various experimental models have been developed to examine the antiviral properties of particular agents, with testing recommended to be taken in a stepwise approach. The European Committee of Standardization (CEN) recommends that the first phase involve an in vitro suspension test with enveloped viruses (representing the bulk of emerging infectious disease threats). Phase 2 involves similar conditions, but with non-enveloped viruses, while the third and final phase involves human hands in a simulation study. In addition to a virucidal hand test, variations on the latter step can include a quantitative non-porous surface test without mechanical action, a quantitative carrier test, and a so-called 4-field test involving surface disinfection with mechanical action (Fig. 1).
Similarly, the US Center for Disease Control recommends a standardised method simulating hand washing with the antiseptic formula to be tested to determine efficacy in reducing hand microflora . New test models have enabled the assessment of PVP-I formulations against highly infective and dangerous pathogens that may not be possible to test in vivo due to clinical risks and ethical considerations. Various commercially available formulations have achieved the standards of the latest European guidelines (EN 14476 and EN 1499), with efficacy demonstrated in both the Enveloped Virus Test Model (MVA) and Non-Enveloped Virus Test Model (MNV). The use of such model viruses can provide valuable data for informed decision-making during public health crises.
EN 14476 is a standardised inactivation assay that involves a virus suspension, an interfering substance (such as bovine serum albumin), and the substance to be tested . A virus control mixture is used to compare the effects of the antiviral product following a specified contact time (e.g. 15, 30 or 60 s), with virucidal activity calculated by determining the difference in logarithmic titre between the virus control and the test virus cultures.
The assessment of microbicidal efficacy can be challenging, due to the difficulties in direct observation and the sheer numbers of cells or particles involved. Consensus within the medical community has settled upon a minimum measure required to evaluate microbicidal efficacy, referred to as the log10 reduction factor. This is a mathematical term measured by titration at the endpoint and indicates the reduction in the number of living or viable microbes after treatments such as sanitisation, disinfection, or cleaning. European Standards (EN) stipulate a minimum level of ≥ 4 log10 reduction in titre for viruses and fungi, and a ≥ 5 log10 reduction for bacteria, representing reductions in the absolute number of microbes by 99.99% and 99.999%, respectively (Table 3).
The introduction and use of model viruses has significantly aided in the investigation of new anti-virucidal agents, particularly during times of pressing need. For example, in December 2013, the Ebola virus was first discovered in Guinea, and rapidly became one of the most complex epidemics in recent history. Due to its high biosecurity level, research into vaccines and containment measures for the virus was highly limited. Although yet to be confirmed as a surrogate for Ebola virus, the modified vaccinia virus (MVA) was introduced in 2014 with a reference claim against “enveloped viruses for hygienic hand rub and hand wash” . Such models allow for reasonable progress to be made in comparing antiviral agents in certain settings. Similarly, Middle East Respiratory Syndrome (MERS) was first discovered in 2012, with the virus now having infected more than 1300 victims in 26 countries, resulting in more than 480 deaths. Transmission is known to frequently occur in healthcare settings, highlighting the need for suitable models to test containment measures. The modified vaccinia virus, Ankara, has been used as a test model for MERS, with similar structural features and cultivation measures .
The influenza virus has been responsible for some of the most significant epidemics in the modern world, with annual outbreaks resulting in approximately 3–5 million cases of severe illness and between 250,000 and 500,000 deaths per year . An influenza study using plaque inhibition assays showed that a 1.56-mg/ml PVP-I treatment can inhibit infections in MDCK cells by human (eight strains) and avian (five strains) influenza A viruses, including H1N1, H3N2, H5N3 and H9N2, from 23 to 98%. Receptor binding analysis revealed that haemagglutinin inhibition was the likely cause of the PVP-I virucidal activity, rather than the inhibition of host-specific sialic acid receptors. The finding also demonstrates two specific mechanisms of reduction of viral growth, namely, PVP-I blockade of viral attachment to the host cell receptors and the inhibition of viral release from infected cells .
PVP-I formulations are also known to have broad antiviral properties. These effects are mechanistically similar in principle to iodine’s antibacterial activity. For example, the virucidal mechanisms of action of PVP-I have been determined to involve the inhibition of essential viral enzymes such as neuraminidase. The inactivation of this enzyme blocks viral release from the host cell, preventing further spread of the virus to uninfected cells. In addition, PVP-I also inhibits viral haemagglutinin, resulting in the blockade of attachment to host cell receptors. By simultaneously targeting both critical aspects of the viral machinery needed for replication, PVP-I reduces the likelihood of resistance emerging through sudden mutation.
Under such guidelines, PVP-I formulations have been shown to elicit viral inactivation of > 99.99% in test systems using a modified vaccinia virus . Virucidal efficacy has in some cases been determined to occur within 15 s of contact. Following a hand simulation study with the murine norovirus, it was found that hand washing with PVP-I was more effective than chlorhexidine and soft soap, a gold standard recommended by the WHO. PVP-I was also shown to be more virucidal against both enterovirus and coxsackievirus when compared to other disinfectants.
The need to develop potent antiviral formulations suitable for widespread use has been brought to prominence by the emergence of rapid viral outbreaks over the past decade, many of which have been coronaviruses. The Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a single-stranded RNA virus first identified during an outbreak in 2012 that eventually spread to 21 countries worldwide, triggering mass media coverage . To date, the virus remains categorised as a high biosafety risk, with containment remaining the primary measure to combat outbreaks, as no vaccines or specific antiviral treatments have yet been developed. However, randomised controlled clinical trials have shown that PVP-I and alcohol-based hand rubs are more effective than soap-based hand washes for hand hygiene in the presence of such transmissible viruses .
In a study evaluating mouthwash, surgical scrubs, and skin cleanser formulations of PVP-I for antiviral activity against the MERS coronavirus, it was shown that the viral titre could be reduced by a factor of C4 log10, corresponding to a c.99.99% inactivation level . This remarkable level of potency was achieved within 15 s of application of each PVP-I formulation, which included a 7.5% PVP-I surgical scrub, a 1% PVP-I gargle/mouthwash and a 4% PVP-I skin cleanser formulation under the brand name Betadine (Mundipharma, Limburg, Germany). The findings indicate that PVP-I-based hand hygiene products can be used to decontaminate virally-infected skin, while PVP-I mouthwash can reduce viral load in the oral cavity and the oropharynx, potentially aiding in the support of hygiene measures needed to reduce the severity of future MERS outbreaks.
An earlier cooperative study presenting results in comparison with other antiseptics has shown how PVP-I impacts the infectivity of some of the most significant human pathogenic viruses, including polio-, HIV-1, adeno-, rota-, mumps, rhino-, coxsackie-, rubella, herpes-, measles, and influenza viruses. Mumps and adeno-viruses have been decimated in test settings by a more than 3-log reduction within 60 s of exposure to PVP-I concentrations higher than 0.5% . Influenza virus was inactivated by a more than 5-log reduction following 15 s at the same dose levels, while HIV was reduced by a more than 4.5-log reduction after a 30-s exposure to doses higher than 0.05%. However, coxsackievirus and poliovirus type 1 were observed to be not as sensitive to PVP-I inactivation, with both viruses requiring doses higher than 0.125% for inactivation, as was the case for rhinovirus. The authors concluded that PVP-I preparations were effective against measles, mumps, herpes, HIV, influenza, and rota-viruses, while rubella, polio-, adeno-, and rhino-viruses were only sensitive to higher doses. The fact that both virus types could be either sensitive or resistant regardless of whether they were enveloped or non-enveloped suggests that mechanisms specific to certain viral types are likely to some extent to influence iodine sensitivity. Overall, the findings are of particular relevance given that an overwhelming proportion of sore throat cases are thought to be of viral origin, and there appears to remain an overprescribing of antibiotics in such cases.