Vertical mother-to-infant transmission of herpes simplex virus 2 is correlated with tropism due to mutations in viral UL13.

Although neonates are commonly exposed to vaginal herpes simplex virus (HSV)-2, neonatal herpes is rare. Therefore, we analyzed paired infant and maternal HSV-2 isolates from two cases of mother-to-infant transmission to identify viral factors contributing to vertical transmission. Sixteen infant isolates with neonatal herpes and 27 genital isolates in their third trimester were included. The infant isolates were significantly more temperature-independent than the maternal isolates. Sequence comparison revealed viral UL13 protein kinase (UL13-PK) mutation in the infant isolates in both cases. In the expanded cohort, infant isolates (5/18) had significantly more UL13-PK mutations than genital isolates (1/29). Isolates within 8 days post-birth (3/4) had a significantly higher frequency of UL13-PK mutation than those after 9 days (2/14), suggesting a close association between UL13-PK mutations and vertical transmission. Elongation factor 1-delta was identified as a target of UL13-PK by proteomic analysis of UL13-PK-positive and -negative HepG2 cells. The mixed infant isolates with the intact and mutated UL13-PK conferred altered cell tropism, temperature independence adapting to fetal temperature, and better growth properties in Vero and hepatoblastoma HepG2 cells than in HSV-2 with intact and mutated UL13-PK alone, indicating that viral UL13-PK mutation is essential for vertical HSV-2 transmission.

Preemptive Intravenous Human Immunoglobulin G Suppresses BK Polyomavirus Replication and Spread of Infection In Vitro.

BK polyomavirus (BKPyV) infection causes various diseases in immunocompromised patients. Cells from human lung and kidney were infected with BKPyV and treated with commercially available intravenous immunoglobulin G (IVIG). Its effects on BKPyV replication and spread of infection were investigated, focusing on administration timing. IVIG treatment 3 hours after infection suppressed BKPyV replication assessed by real-time PCR and expression of the viral capsid protein 1 and large T-antigen. IVIG effectively reduced the number of BKPyV-infected cells 2 weeks after infection in an antibody titer-dependent manner. Virus release in the culture supernatants was not influenced by IVIG treatment 6-80 hours and 3-9 days after infection. Collectively, IVIG did not affect viral release from infected cells but inhibited the spread of infection by neutralizing the released virus and blocking the new infected cell formation, indicating greater efficacy in early localized infection. BKPyV replication resumed in IVIG-treated cultures at 7 days after IVIG removal. Early prophylactic administration of IVIG is expected to reduce the growth and spread of BKPyV infection, resulting in the reduction of infected cell lesions and prevention of BKPyV-associated diseases.

Fosphenytoin Alleviates Herpes Simplex Virus Infection-Induced Provoked and Spontaneous Pain-Like Behaviors in Mice.

In this study, we investigated the effects of fosphenytoin (fPHT), a water-soluble prodrug of phenytoin, on the pain responses of a mouse herpes zoster (HZ) pain model. Transdermal herpes simplex virus type 1 (HSV-1) inoculation induced mechanical allodynia and hyperalgesia of the hind paw and spontaneous pain-like behaviors, such as licking the affected skin. Intravenous injection of fPHT (15 and 30 mg/kg) alleviated HSV-1-induced provoked pain (allodynia and hyperalgesia). The suppressive effects of fPHT on provoked pain were weaker than those of diclofenac and pregabalin which were used as positive controls. fPHT, diclofenac, and pregabalin significantly suppressed HSV-1-induced spontaneous pain-like behaviors. Among them, high-dose fPHT (30 mg/kg) showed the strongest suppression. Intravenous fPHT may become a viable option for an acute HZ pain, especially for spontaneous pain.

Successful treatment with famciclovir for varicella zoster virus infection resistant to acyclovir.

Varicella zoster virus (VZV) reactivates more frequently in immunocompromised patients than immunocompetent subjects and is a significant cause of morbidity and mortality. Acyclovir is frequently used for treatment against VZV reactivation. However, long-term use of acyclovir can result in the emergence of VZV strain resistant to acyclovir. Here, we report a 67-year-old man with adult T-cell leukemia who suffered from herpes zoster with acyclovir-resistant VZV after long-term prophylaxis. The isolated viruses from his skin lesions were a mixture of acyclovir-resistant and acyclovir-susceptible strains. Sequence analysis showed the presence of thymidine kinase (TK) mutations in the resistant clones. Interestingly, oral administration of famciclovir, a prodrug form of penciclovir, resulted in resolution of his herpes zoster, although most acyclovir-resistant strains of VZV were reported to be resistant to penciclovir. This implied that a certain amount of susceptible VZV with wild-type viral TK gene was present in vivo, and that famciclovir could be phosphorylated intracellularly by the intact viral kinases. As famciclovir is more potent and longer-acting than acyclovir, the susceptible strains might have suppressed the generation and proliferation of the resistant in vivo. Even when VZV is developing resistance to acyclovir, famciclovir might be effective at least in the early resistant phase.

Improvement of refractory acyclovir-resistant herpes simplex virus type 1 infection by continuous acyclovir administration.

Resistant herpes simplex virus type 1 (HSV-1) infection is sometimes fatal for immunocompromised patients. Here, we report 10-year-old girl receiving hematopoietic stem cell transplantation developed refractory HSV-1 infection, which was persisted to intermittent acyclovir (ACV) or foscarnet (FOS) administrations but was improved by continuous ACV administration. The isolates from the lesion were identified with low susceptibilities to ACV and FOS by plaque reduction assay due to DNA pol gene mutation. Continuous ACV administration overcomes the efficacy of intermittent administration and could be the best option to treat severe HSV-1 infectious patients.

Antiviral Drugs Against Alphaherpesvirus.

The discovery of acyclovir and penciclovir has led to the development of a successful systemic therapy for treating herpes simplex virus infection and varicella-zoster virus infection, and the orally available prodrugs, valacyclovir and famciclovir, have improved antiviral treatment compliance. Acyclovir and penciclovir are phosphorylated by viral thymidine kinase and are incorporated into the DNA chain by viral DNA polymerase, resulting in chain termination. Helicase-primase plays an initial step in DNA synthesis to separate the double strand into two single strands (replication fork) and is a new target of antiviral therapy. The helicase-primase inhibitors (HPIs) pritelivir and amenamevir have novel mechanisms of action, drug resistance properties, pharmacokinetic characteristics, and clinical efficacy for treating genital herpes. The clinical study of amenamevir in herpes zoster has been completed, and amenamevir has been submitted for approval for treating herpes zoster in Japan. The clinical use of HPIs will be the beginning of a new era of anti-herpes therapy.

Acyclovir-resistant herpes simplex virus 1 infection early after allogeneic hematopoietic stem cell transplantation with T-cell depletion.

We previously reported that oral low-dose acyclovir (200 mg/day) for the prevention of herpes simplex virus (HSV) infections after allogenic hematopoietic stem cell transplantation (HSCT) is effective without the emergence of acyclovir-resistant HSV infections. However, HSV infections are of significant concern because the number of allogeneic HSCT with T-cell depletion, which is a risk factor of the emergence of drug-resistant HSV infections, has been increasing. We experienced a 25-year-old female who received allogenic HSCT from an unrelated donor with 1-antigen mismatch using anti-thymocyte globulin. Despite acyclovir prophylaxis (200 mg/day), she developed the right palatal ulcer that was positive for HSV-1 specific antigen by fluorescent antibody on day 20 and developed new hypoglossal and tongue ulcers on day 33. Replacement of acyclovir with foscarnet improved her ulcers. We isolated 2 acyclovir-resistant and foscarnet-sensitive strains from the right palatal and hypoglossal ulcers, which had the same frame shift mutation in the thymidine kinase genes. The rate of proliferation of the isolate from the hypoglossal ulcer was faster than that from the right palatal ulcer in the plaque reduction assay. HSV strains that acquired acyclovir-resistant mutations at the right palatal ulcer with larger plaque might spread to the hypoglossal ulcer as the secondary site of infection because of better growth property. Second-line antiviral agents should be considered when we suspect treatment failure of HSV infection, especially in HSCT with T-cell depletion. Further studies are required whether low-dose acyclovir prophylaxis leads to the emergence of virological resistance.

Functional differences between antiviral activities of sulfonated and intact intravenous immunoglobulin preparations toward varicella-zoster virus and cytomegalovirus.

Intravenous immunoglobulin (IVIG) is used to treat severe viral infection, especially varicella-zoster virus (VZV) and cytomegalovirus (CMV) infections. The neutralization antibody titers of eleven IVIG preparations from four companies were examined using VZV and CMV with and without complement. The neutralizing antibody titers of intact IgG preparations were three to six times higher after addition of complement. The effectiveness of the sulfonated IgG preparation was not enhanced by complement, but desulfonated IgG regained enhanced neutralization activity with complement. Antibody-dependent cellular cytotoxicity (ADCC) toward VZV-infected cells was observed with both intact and sulfonated IVIG and guinea pig splenocytes, but ADCC toward CMV-infected cells was not, although NK cell activity toward cells infected with VZV or CMV was detected by splenocytes. Sulfonated IVIG had no complement-activated neutralization of VZV and CMV but retained ADCC toward VZV with less activity after dilution than with intact IVIG. Because sulfonated IVIG is converted to the intact form after intravenous administration, it would show complement-enhanced neutralization and ADCC activity similar to that of intact IVIG in vivo. In this study we showed the effects of intact and sulfonated IgG on the functional activity of IgG against VZV and CMV.

Virus specific cell-mediated immunity may play a role in controlling reactivated human herpesvirus 6B in patients under measles induced immunosuppression.

For a better understanding of the cellular immune responses to reactivated HHV 6B the lymphoproliferative response to human herpesvirus 6B (HHV 6B) antigen was measured in three consecutive specimens obtained biweekly from 22 young children and infants suffering from acute measles, and in 19 influenza patients and nine healthy control subjects. HHV 6B DNA in peripheral blood mononuclear cells (PBMCs) was detected in 18 of 22 subjects with measles, but not in the influenza patients or the healthy population. A novel reactivation profile of HHV 6B was found in patients with measles in the milder form of immunosuppression than in patients with organ transplantation. HHV 6B specific lymphoproliferation activities increased correspondingly with reactivation of HHV 6B assessed by detecting HHV 6B DNA in PBMCs in patients with measles, but no significant change in either the antibody response to HHV 6B or DNAemia occurred in serial specimens obtained either from patients with influenza or healthy subjects. This novel form of HHV 6B reactivation without antibody response was observed in patients with measles. The dynamic fluctuations in lymphoproliferative responses in measles may represent the balance between HHV 6B reactivation and its suppression by the host immune system.

Characterization of susceptibility variants of influenza virus grown in the presence of T-705.

T-705 (favipiravir) is a potent inhibitor of RNA polymerases of influenza viruses. Susceptibility variants were isolated during passages in the presence of T-705. Nine variants with 0.4 to 2.1 times the 50% inhibitory concentration for plaque formation of the parent A/PR/8/34 (H1N1) strain had amino acid variations in the PB1, PB2, and PA genes of the RNA polymerase complex. However, the variation patterns in the RNA polymerase complex indicated that T-705 does not work as a mutagen, and resistant mutants were not isolated, possibly because a mutation leading to resistance would be lethal to the RNA polymerase function.

Novel deletion in glycoprotein G forms a cluster and causes epidemiologic spread of herpes simplex virus type 2 infection.

The herpes simplex virus type 2 (HSV-2) glycoprotein G (gG-2) gene of 106 clinical isolates was analyzed and six isolates were identified with 63 nucleotides comprising 21 amino acids (aa) deleted in the immunodominant region. Compared with strain HG52, variations in the gG-2 gene were found at 276 and 27 sites in nucleotide and aa sequences, respectively, in the 106 strains. Significant variations in both nucleotides and aa were accumulated in the immunodominant region rather than in the other regions (P < 0.001), indicating that the immunodominant region might be indispensable in vivo and a hot spot for variation. The frequency of 21 aa-deleted strains (HSVΔ21/gG-2) among clinical isolates was 5%, indicating the advantage of this deletion of gG-2 for epidemiological expansion. Phylogenetic analysis of the 106 strains indicated that the HSVΔ21/gG-2 strains formed a cluster among the various variations but that their genomes showed different endonuclease digestion patterns. The antibody titers to total HSV antigens of patients infected with wild HSV-2 and HSVΔ21/gG-2 were similar, but patients with HSVΔ21/gG-2 had a lower antibody titer to gG-2 than those with wild HSV-2 (P < 0.001). HSVΔ21/gG-2 might be less immnunogenic and reduce antibody production to gG-2, while its pathogenicity in humans was not distinguished in its clinical manifestations. Thus, infection with HSVΔ21/gG-2 caused genital lesions similar to wild HSV-2 infection, but evaded the immune response to gG-2 to allow epidemiological spread, indicating the importance of this deletion in the immunodominant region of gG-2 in the pathogenesis and transmission of genital herpes.

Rapid detection of human cytomegalovirus UL97 and UL54 mutations for antiviral resistance in clinical specimens.

Drug-resistant cytomegalovirus appears during prolonged anti-cytomegalovirus therapy. Assays for human cytomegalovirus viral protein kinase (UL97) and viral DNA polymerase (UL54) gene mutations conferring drug resistance have been used rather than susceptibility assays to assess clinical specimens. In this study a sensitive system for genotype assay of UL97 and UL54 in clinical specimens with as few as six copies/µg of DNA was developed.

Neutralizing anti-gH antibody of Varicella-zoster virus modulates distribution of gH and induces gene regulation, mimicking latency.

The anti-glycoprotein H (gH) monoclonal antibody (anti-gH-MAb) that neutralizes varicella-zoster virus (VZV) inhibited cell-to-cell infection, resulting in a single infected cell without apoptosis or necrosis, and the number of infectious cells in cultures treated with anti-gH-MAb declined to undetectable levels in 7 to 10 days. Anti-gH-MAb modulated the wide cytoplasmic distribution of gH colocalized with glycoprotein E (gE) to the cytoplasmic compartment with endoplasmic reticulum (ER) and Golgi markers near the nucleus, while gE retained its cytoplasmic distribution. Thus, the disintegrated distribution of gH and gE caused the loss of cellular infectivity. After 4 weeks of treatment with anti-gH-MAb, no infectious virus was recovered, even after cultivation without anti-gH-MAb for another 8 weeks or various other treatments. Cells were infected with Oka varicella vaccine expressing hepatitis B surface antigen (ROka) and treated with anti-gH-MAb for 4 weeks, and ROka was recovered from the quiescently infected cells by superinfection with the parent Oka vaccine. Among the genes 21, 29, 62, 63, and 66, transcripts of gene 63 were the most frequently detected, and products from the genes 63 and 62, but not gE, were detected mainly in the cytoplasm of quiescently infected cells, in contrast to their nuclear localization in lytically infected cells. The patterns of transcripts and products from the quiescently infected cells were similar to those of latent VZV in human ganglia. Thus, anti-gH-MAb treatment resulted in the antigenic modulation and dormancy of infectivity of VZV. Antigenic modulation by anti-gH-MAb illuminates a new aspect in pathogenesis in VZV infection and the gene regulation of VZV during latency in human ganglia.

Microemulsion-based oxyresveratrol for topical treatment of herpes simplex virus (HSV) infection: physicochemical properties and efficacy in cutaneous HSV-1 infection in mice.

The physicochemical properties of the optimized microemulsion and the permeating ability of oxyresveratrol in microemulsion were evaluated, and the efficacy of oxyresveratrol microemulsion in cutaneous herpes simplex virus type 1 (HSV-1) infection in mice was examined. The optimized microemulsion was composed of 10% w/w of isopropyl myristate, 35% w/w of Tween 80, 35% w/w of isopropyl alcohol, and 20% w/w of water. The mean particle diameter was 9.67 ± 0.58 nm, and the solubility of oxyresveratrol in the microemulsion was 196.34 ± 0.80 mg/ml. After accelerated and long-term stability testing, the microemulsion base and oxyresveratrol-loaded microemulsion were stable. The cumulative amount of oxyresveratrol permeating through shed snake skin from microemulsion at 6 h was 93.04 times compared to that of oxyresveratrol from Vaseline, determined at 20% w/w concentration. In cutaneous HSV-1 infection in mice, oxyresveratrol microemulsion at 20%, 25%, and 30% w/w, topically applied five times daily for 7 days after infection, was significantly effective in delaying the development of skin lesions and protecting from death (p < 0.05) compared with the untreated control. Oxyresveratrol microemulsion at 25% and 30% w/w was significantly more effective than that of 30% w/w of oxyresveratrol in Vaseline (p < 0.05) and was as effective as 5% w/w of acyclovir cream, topically applied five times daily (p > 0.05). These results demonstrated that topical oxyresveratrol microemulsion at 20-30% w/w was suitable for cutaneous HSV-1 mouse infection.

[Mechanism of action of antiviral drugs].

Antiviral therapy is now one of routine practices and as common as chemotherapy against bacterial infection. Therefore it is important for the clinicians to understand the differences between bacterial and viral infections in order to use antiviral drugs properly. This review focuses the difference of the mechanism of action of antiviral drugs and antibiotics and the importance of host immune status to recover from microbial infection.

Antiviral therapy for COVID-19: Derivation of optimal strategy based on past antiviral and favipiravir experiences.

Favipiravir, a broad-spectrum RNA-dependent RNA polymerase inhibitor, inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at significantly lower concentrations than the plasma trough levels achieved by the dosage adopted for influenza treatment and exhibits efficacy against coronavirus disease 2019 (COVID-19) pneumonia. Although high doses of favipiravir are required due to the molecule being a purine analog, its conversion into the active form in infected cells with active viral RNA synthesis enhances the antiviral specificity and selectivity as a chain terminator with lethal mutagenesis. Another characteristic feature is the lack of generation of favipiravir-resistant virus. COVID-19 pneumonia is caused by strong cell-mediated immunity against virus-infected cells, and the inflammatory response induced by adaptive immunity continues to peak for 3 to 5 days despite antiviral treatment. This has also been observed in herpes zoster (HZ) and cytomegalovirus (CMV) pneumonia. Inflammation due to an immune response may mask the effectiveness of favipiravir against COVID-19 pneumonia. Favipiravir significantly shortened the recovery time in patients with mild COVID-19 pneumonia by 3 days with the start of treatment by the 5th day of symptom onset. Since both CMV and COVID-19 pneumonia are caused by adaptive immunity and prevention of cytomegalovirus pneumonia is the standard treatment due to difficulties in treating refractory CMV pneumonia, COVID-19 pneumonia should be prevented with early treatment as well. In the present study, we have comprehensively reviewed the optimal antiviral therapy for COVID-19 based on clinical trials of favipiravir for the treatment of COVID-19 pneumonia and the concurrently established therapies for other viral infections, particularly HZ and CMV pneumonia. Optimally, antivirals should be administered immediately after COVID-19 diagnosis, similar to that after influenza diagnosis, to prevent COVID-19 pneumonia and complications resulting from microangiopathy.

Everolimus reduces BK polyomavirus infection by suppressing its replication and spread of infection.

BK polyomavirus-associated nephropathy is one of serious complications in transplant recipients. Everolimus-a mammalian target of rapamycin inhibitor-has been shown to reduce the incidence of BK polyomavirus infection in transplant recipients. In this study, the effects of everolimus were examined on viral replication and the spread of infection in BK polyomavirus-infected cultures. BK polyomavirus replicated in renal and pulmonary cells, contrary to that in hepatocytes, and spread as diffusely scattered patterns of infected cells, unlike plaque formation through the cell-to-cell mode. BK polyomavirus is stable to heat up to 65 °C with a particle per infectivity ratio of 5000, and the replication cycle was for approximately 34 h. Everolimus administration remarkably reduced the viral replication to 20% in cells treated with 0.1-10 ng/mL, the concentration at which everolimus reached the serum of transplant recipients. In addition, it reduced the amount of viral capsid protein 1 at 5 ng/mL without reducing the ratio of viral capsid protein 1 versus ß-actin, and it also retained the pattern of viral capsid protein 1 localization in the nuclei. Everolimus suppressed the number of infected cells to 32.8% during a 14-day treatment, indicating the reduction of BK polyomavirus-infected cell mass to 18.8% of untreated cultures by modifying cellular functions. The reduction in the total number of BK polyomavirus infected cells by everolimus indicates that everolimus alleviates BK polyomavirus infection, including nephropathy in transplant recipients.

Antibody to varicella-zoster virus immediate-early protein 62 augments allodynia in zoster via brain-derived neurotrophic factor.

Varicella-zoster virus (VZV) expresses immediate-early protein 62 (IE62), and zoster is associated with neuropathic pain. Brain-derived neurotrophic factor (BDNF) is involved in the neuronal mechanism underlying pain hypersensitivity. Zoster is associated with prodrome and the robust production of booster antibody to VZV. We hypothesized that the intrathecal production of antibody to IE62 cross-reacting with BDNF and the nerve injury by skin lesions may augment allodynia in zoster by enhancing BDNF activity. One of three monoclonal antibodies against the 268-556 peptide of IE62 recognized BDNF. Immunological cross-reactivity between IE62 and BDNF and the effects of anti-IE62 monoclonal antibody (anti-IE62 MAb) cross-reactivity with BDNF on BDNF activity in cultured neurons were examined. Anti-IE62 MAb and anti-BDNF MAbs recognized the 414-429 peptide of IE62 and the BDNF dimer. Anti-IE62 MAb significantly augmented BDNF-related transcription in neurons and the morphological development of spinal dorsal horn neurons. Sera from patients recognized IE62 and BDNF and enhanced BDNF activity in neurons. The effect of anti-IE62 antibody on mechanical allodynia was characterized by the threshold of allodynia using von Frey filaments in a spinal nerve injury (SNI) in mice. The administration of anti-IE62 MAb to or immunization with cross-reacting IE62 protein to mice significantly enhanced mechanical allodynia on the side with SNI but not on the uninjured side. Anti-IE62 antibody augmented BDNF activity in neurons and allodynia in mice with SNI. The intrathecal production of anti-IE62 antibody augmenting BDNF activity and peripheral nerve injury by zoster may participate in the pathogenesis of allodynia in zoster.

Efficacy of Brazilian Propolis against Herpes Simplex Virus Type 1 Infection in Mice and Their Modes of Antiherpetic Efficacies.

Ethanol extracts (AF-06, 07, and 08, 10 mg/kg) of Brazilian propolis were administered orally to cutaneously herpes simplex virus type 1 (HSV-1)-infected mice three times daily on days 0 to 6 after infection to evaluate their efficacies against HSV-1 infection and significantly limited development of herpetic skin lesions. AF-07 and 08 significantly reduced virus titers in brain and/or skin on day 4 without toxicity, but AF-08 had no anti-HSV-1 activity in vitro. AF-06 and 08 significantly enhanced delayed-type hypersensitivity (DTH) to inactivated HSV-1 antigen in infected mice. Oral AF-08-administration significantly augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice, while direct exposure of splenocytes of infected mice to AF-06 significantly elevated IFN-γ production in vitro. Thus, AF-08 might have components that are active in vivo even after oral administration and those of AF-06 might be active only in vitro. Because DTH is a major host defense for intradermal HSV-1 infection, augmentation of DTH response by AF-06 or 08, directly or indirectly, respectively, may contribute to their efficacies against HSV-1 infection. In addition, AF-06 and 07 possibly contain anti-HSV-1 components contributing to their efficacies. Such biological activities of Brazilian propolis may be useful to analyze its pharmacological actions.

[Varicella vaccine potency and stability during transport and delivery].

Oka varicella vaccine was developed to confer active immunity to varicella-zoster virus (VZV) in immunocompromized and immunocompetent children. It is now used to prevent varicella in about 20 million people worldwide. Although VZV infectivion is relatively unstable compared to other viruses, cell-free virus is stabilized and lyophilized vaccine has been developed. Virus titers were evaluated in vaccine distributed to six clinics in 5 years. Yearly mean virus titers at the vaccine producer were 42,000-67,000 plaque-forming units per dose, corresponding to Oka varicella vaccine (Zostavax) used to prevent zoster and postherpetic neuralgia by Oxman et al. Virus titer was found to be stable during delivery to clinics. Virus titers of varicella vaccine were equivalent to Zostavax and vaccine delivered to clinics had enough virus titer to confer active immunity to VZV in this study.