Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

IMPORTANCE: Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Macacine alphaherpesvirus 1 (B Virus) Infection in Humans, Japan, 2019.

Two human patients with Macacine alphaherpesvirus 1 infection were identified in Japan in 2019. Both patients had worked at the same company, which had a macaque facility. The rhesus-genotype B virus genome was detected in cerebrospinal fluid samples from both patients.

Pathological and virological findings of type I interferon receptor knockout mice upon experimental infection with Heartland virus.

Heartland virus (HRTV) causes generalized symptoms, severe shock, and multiple organ failure. We previously reported that interferon-α/ß receptor knockout (IFNAR-/-) mice infected intraperitoneally with 1 × 107 tissue culture-infective dose (TCID50) of HRTV died, while those subcutaneously infected with the same dose of HRTV did not. The pathophysiology of IFNAR-/- mice infected with HRTV and the mechanism underlying the difference in disease severity, which depends on HRTV infection route, were analyzed in this study. The liver, spleen, mesenteric and axillary lymph nodes, and gastrointestinal tract of intraperitoneally (I.P.) infected mice had pathological changes; however, subcutaneously (S.C.) infected mice only had pathological changes in the axillary lymph node and gastrointestinal tract. HRTV RNA levels in the mesenteric lymph node, lung, liver, spleen, kidney, stomach, intestine, and blood were significantly higher in I.P. infected mice than those in S.C. infected mice. Chemokine ligand-1 (CXCL-1), tumor necrosis factor (TNF)-α, interleukin (IL)-12, interferon (IFN)-γ, and IL-10 levels in plasma of I.P. infected mice were higher than those of S.C. infected mice. These results indicated that high levels of viral RNA and the induction of inflammatory responses in HRTV-infected IFNAR-/- mice may be associated with disease severity.

Susceptibility of Type I Interferon Receptor Knock-Out Mice to Heartland Bandavirus (HRTV) Infection and Efficacy of Favipiravir and Ribavirin in the Treatment of the Mice Infected with HRTV.

Heartland bandavirus (HRTV) is an emerging tick-borne virus that is distributed in the United States and that causes febrile illness with thrombocytopenia and leukocytopenia. It is genetically close to Dabie bandavirus, which is well known as severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV). The mortality rate of human HRTV infection is approximately 10%; however, neither approved anti-HRTV agents nor vaccines exist. An appropriate animal model should be developed to evaluate the efficacy of antiviral agents and vaccines against HRTV. The susceptibility of IFNAR-/- mice with HRTV infection was evaluated using subcutaneous, intraperitoneal, and retro-orbital inoculation routes. IFNAR-/- mice intraperitoneally infected with HRTV showed the most severe clinical signs, and the 50% lethal dose was 3.2 × 106 TCID50. Furthermore, to evaluate the utility of a novel lethal IFNAR-/- mice model, IFNAR-/- mice were orally administered favipiravir, ribavirin, or a solvent for 5 days immediately after a lethal dose of HRTV inoculation. The survival rates of the favipiravir-, ribavirin-, and solvent-administered mice were 100, 33, and 0%, respectively. The changes in bodyweights and HRTV RNA loads in the blood of favipiravir-treated IFNAR-/- mice were the lowest among the three groups, which suggests that favipiravir is a promising drug candidate for the treatment of patients with HRTV infection.

Longitudinal Trends of Prevalence of Neutralizing Antibody against Human Cytomegalovirus over the Past 30 Years in Japanese Women.

Neutralizing antibodies (NAbs) to human cytomegalovirus (HCMV) are associated with the risk of transplacental HCMV infection of the fetus in pregnant women. The IgG-positivity rate to HCMV determined by enzyme immunoassay (EIA) or indirect immunofluorescence assay has decreased from approximately 100% to 70% over the past 30 years in Japan. We tested serum samples from 630 Japanese women aged 20-49 years whose blood samples were obtained between 1980 and 2015. IgG titer was measured using an EIA-based assay. HCMV-NAb titer was measured using a neutralization test assay with an HCMV isolate on human retinal epithelial cells. Longitudinal transitions in HCMV-NAb prevalence were clarified. The prevalence of HCMV-EIA-IgG, and HCMV-NAb at a titer of 16-fold, and HCMV-NAb at a titer of 100-fold, changed from 96.7% to 78.9%, 93.3% to 85.6%, and 35.5% to 41.1%, respectively, between 1980-1990 and 2010-2015. Prevalence of HCMV-NAb at a titer of 16-fold decreased by 7.7%, whereas that at a titer of 100-fold increased by 5.6%. A high titer of HCMV-NAb in pregnant women is expected to reduce the risk of intrauterine HCMV transmission from the mother to the fetus. The association between the risk of congenital HCMV infection and the prevalence of HCMV-NAb remains to be addressed.

Virulence of Herpes Simplex Virus 1 Harboring a UAG Stop Codon between the First and Second Initiation Codon in the Thymidine Kinase Gene.

Herpes simplex virus 1 (HSV-1)-TK (8UAG) expresses a truncated thymidine kinase (TK) translated from the second initiation codon due to a stop codon (UAG) at the 8th position (counted from the first initiation codon). Here, we showed that the sensitivity of HSV-1-TK (8UAG) to acyclovir (ACV) is similar to that of the control HSV-1 wild-type (WT), which expresses an intact TK protein. However, HSV-1-TK (44UAG), which expresses a truncated TK due to a UAG codon at position 44, showed lower sensitivity to ACV. A mouse infection model was used to compare the virulence of HSV-1-TK (8UAG) and HSV-1-TK (44UAG) to that of HSV-1 WT. The 50% lethal dose (LD50) for HSV-1-TK (44UAG) was 7.8-fold higher than that for HSV-1-TK (8UAG), whereas the LD50 for HSV-1-TK (8UAG) was the same as that for the parental HSV-1 WT. There were no statistically significant differences among HSV-1-TK (44UAG), HSV-1-TK (8UAG), and HSV-1 WT with respect to replication capacity and viral TK mRNA expression in the mouse brain. Thus, the virulence of HSV-1 expressing the truncated viral TK translated from the second initiation codon might depend on the position of the UAG stop codon.

Development of an RT-LAMP Assay for the Rapid Detection of SFTS Virus.

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >103.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease.

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 103 TCID50 and 105 TCID50. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.

Association of human cytomegalovirus (HCMV) neutralizing antibodies with antibodies to the HCMV glycoprotein complexes.

BACKGROUND: Human cytomegalovirus (HCMV) causes asymptomatic infections, but also causes congenital infections when women were infected with HCMV during pregnancy, and life-threatening diseases in immunocompromised patients. To better understand the mechanism of the neutralization activity against HCMV, the association of HCMV NT antibody titers was assessed with the antibody titers against each glycoprotein complex (gc) of HCMV. METHODS: Sera collected from 78 healthy adult volunteers were used. HCMV Merlin strain and HCMV clinical isolate strain 1612 were used in the NT assay with the plaque reduction assay, in which both the MRC-5 fibroblasts cells and the RPE-1 epithelial cells were used. Glycoprotein complex of gB, gH/gL complexes (gH/gL/gO and gH/gL/UL128-131A [PC]) and gM/gN were selected as target glycoproteins. 293FT cells expressed with gB, gM/gN, gH/gL/gO, or PC, were prepared and used for the measurement of the antibody titers against each gc in an indirect immunofluorescence assay (IIFA). The correlation between the IIFA titers to each gc and the HCMV-NT titers was evaluated. RESULTS: There were no significant correlations between gB-specific IIFA titers and the HCMV-NT titers in epithelial cells or between gM/gN complex-specific IIFA titers and the HCMV-NT titers. On the other hand, there was a statistically significant positive correlation between the IIFA titers to gH/gL complexes and HCMV-NT titers. CONCLUSIONS: The data suggest that the gH/gL complexes might be the major target to induce NT activity against HCMV.

New Mechanism of Acyclovir Resistance in Herpes Simplex Virus 1, Which Has a UAG Stop Codon between the First and Second AUG Initiation Codons.

Morphological changes in the structure of the herpes simplex virus 1 (HSV-1) viral thymidine kinase (vTK) polypeptide usually lead to conferring acyclovir (ACV) resistance. HSV-1 I4-2, in which a UAG stop codon is present at the 8th position between the 1st initiation AUG codon (1st position) and the 2nd initiation AUG codon (46th position) of the HSV-1 vTK gene, showed sensitivity to ACV. In contrast, HSV-1 KG111, in which a UAG stop codon was artificially inserted at the 44th position, showed resistance to ACV at 39˚C. The mechanism underlying the difference in the sensitivity profiles was elucidated. The virus recombinants HSV-1-TK(8UAG) and HSV-1-TK(44UAG) containing a UAG stop codon at the 8th and 44th positions counted from the 1st initiation codon, respectively, were generated and tested for susceptibility to antiviral compounds. HSV-1-TK(8UAG) and HSV-1-TK(44UAG) were sensitive and resistant to ACV and BVdU at 37˚C, respectively. The expression level of the truncated vTK translated from the 2nd initiation codon in Vero cells infected with HSV-1-TK(44UAG) was clearly less than that with HSV-1-TK(8UAG) in a temperature-dependent manner. The differences in the antiviral sensitivity profiles were due to the position of the UAG stop codon between the 1st and the 2nd initiation codons.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

Conditional expression of a dominant-negative form of Epstein-Barr virus (EBV) nuclear antigen EBNALP inhibits EBV-positive lymphoblastoid cell growth.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that transforms primary B lymphocytes, yielding lymphoblastoid cell lines (LCLs). EBV-encoded nuclear antigen 2 (EBNA2) and EBV-encoded nuclear antigen leader protein (EBNALP) are the first viral products expressed after EBV infection of primary B lymphocytes and are essential for EBV-induced B-lymphocyte growth transformation. EBNA2 functions as a transcriptional activator of viral and cellular genes, with EBNALP as a coactivator for EBNA2-mediated transcriptional activation. We previously reported that mutant EBNALP with a C-terminal 10-amino-acid truncation loses the ability to coactivate, and has a dominant-negative effect on wild-type-EBNALP-mediated coactivation. However, the functional relevance of EBNALP in maintenance of LCL cell growth has not been investigated. To address this, we have constructed LCL-derived cell clones in which this dominant-negative form of EBNALP (DNLP) is conditionally expressed by the Cre-loxP system. We used these cells to evaluate the effect of DNLP expression on EBV-induced cell proliferation. After drug treatment, the DNLP-expressing LCL clones showed reduced cell proliferation and viability. These results indicate that EBNALP is critical for maintaining LCL growth and EBV-induced cell proliferation.

Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice.

Middle East respiratory syndrome-coronavirus (MERS-CoV) is an emerging virus that causes severe disease with fatal outcomes; however, there are currently no approved vaccines or specific treatments against MERS-CoV. Here, we developed a novel bivalent vaccine against MERS-CoV and rabies virus (RV) using the replication-incompetent P-gene-deficient RV (RVΔP), which has been previously established as a promising and safe viral vector. MERS-CoV spike glycoprotein comprises S1 and S2 subunits, with the S1 subunit being a primary target of neutralizing antibodies. Recombinant RVΔP, which expresses S1 fused with transmembrane and cytoplasmic domains together with 14 amino acids from the ectodomains of the RV-glycoprotein (RV-G), was developed using a reverse genetics method and named RVΔP-MERS/S1. Following generation of RVΔP-MERS/S1 and RVΔP, our analysis revealed that they shared similar growth properties, with the expression of S1 in RVΔP-MERS/S1-infected cells confirmed by immunofluorescence and western blot, and the immunogenicity and pathogenicity evaluated using mouse infection experiments. We observed no rabies-associated signs or symptoms in mice inoculated with RVΔP-MERS/S1. Moreover, virus-specific neutralizing antibodies against both MERS-CoV and RV were induced in mice inoculated intraperitoneally with RVΔP-MERS/S1. These findings indicate that RVΔP-MERS/S1 is a promising and safe bivalent-vaccine candidate against both MERS-CoV and RV.

Differences in the Likelihood of Acyclovir Resistance-Associated Mutations in the Thymidine Kinase Genes of Herpes Simplex Virus 1 and Varicella-Zoster Virus.

Acyclovir (ACV) resistance-associated mutations in two recombinant herpes simplex virus 1 (HSV-1) clones were compared. Recombinant HSV-1 lacking its thymidine kinase (TK) and expressing varicella-zoster virus (VZV) TK ectopically had no mutations in the VZV TK gene. In contrast, recombinant HSV-1 expressing HSV-1 TK ectopically harbored mutations in the HSV-1 TK gene. These results suggest that the relatively low frequency of ACV-resistant VZV is a consequence of the characteristics of the TK gene.

Acyclovir Sensitivity and Neurovirulence of Herpes Simplex Virus Type 1 with Amino Acid Substitutions in the Viral Thymidine Kinase Gene, Which Were Detected in the Patients with Intractable Herpes Simplex Encephalitis Previously Reported.

Several cases of herpes simplex encephalitis (HSE) caused by acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) have been reported. Amino acid substitutions of R41H, Q125H, and A156V in the viral thymidine kinase (vTK) gene have been reported to confer ACV resistance. Recombinant HSV-1 clones, containing each amino acid substitution in the vTK gene, were generated using the bacterial artificial chromosome system. A recombinant HSV-1 with the Q125H substitution showed ACV resistance while the R41H or A156V substitutions were ACV-sensitive. Furthermore, the Q125H recombinant HSV-1 was less virulent than the repaired virus, but it maintained neurovirulence in mice at relatively high levels. Substitution of Q125H, which was detected in the neonatal HSE patient, conferred ACV resistance, but the substitutions of R41H and A156V, which were detected in immunocompetent adult HSE patients, did not. This suggests that HSE caused by ACV-resistant HSV-1 might be a very rare event to occur during the course of ACV treatment in immunocompetent patients. Showing resistance to ACV treatment does not always indicate emergence of ACV-resistant HSV-1 in HSE patients.

A Novel System for Constructing a Recombinant Highly-Attenuated Vaccinia Virus Strain (LC16m8) Expressing Foreign Genes and Its Application for the Generation of LC16m8-Based Vaccines against Herpes Simplex Virus 2.

A novel system was developed for generating highly attenuated vaccinia virus LC16m8 (m8, third-generation smallpox vaccine) that expresses foreign genes. The innovations in this system are its excisable selection marker, specificity of the integration site of a gene of interest, and easy identification of clones with a fluorescent signal. Using this system, recombinant m8s, which expressed herpes simplex virus 2 (HSV-2) glycoprotein B (gB)-, gD-, or both gB and gD (gB + gD), were generated, and their efficacy was evaluated. First, the induction of a specific IgG against these HSV-2 glycoproteins in mice infected with one of these recombinant m8s was confirmed by an immunofluorescent assay. Next, mice preinfected with one of the recombinant m8s were infected with HSV-2 at a lethal dose to examine the vaccine efficacy. The fatality rate among the mice preinfected with either the recombinant gB + gD- or gD-expressing m8 significantly decreased in comparison with the control. The survival rate in male and female mice preinfected with either the recombinant gB + gD- or gD-expressing m8 increased to 100% and 60%, respectively, while most of the control mice died. In summary, this new system may be applicable to creation of a novel m8-based vaccine.

Construction and characterization of bacterial artificial chromosomes harboring the full-length genome of a highly attenuated vaccinia virus LC16m8.

LC16m8 (m8), a highly attenuated vaccinia virus (VAC) strain, was developed as a smallpox vaccine, and its safety and immunogenicity have been confirmed. Here, we aimed to develop a system that recovers infectious m8 from a bacterial artificial chromosome (BAC) that retains the full-length viral genomic DNA (m8-BAC system). The infectious virus was successfully recovered from a VAC-BAC plasmid, named pLC16m8-BAC. Furthermore, the bacterial replicon-free virus was generated by intramolecular homologous recombination and was successfully recovered from a modified VAC-BAC plasmid, named pLC16m8.8S-BAC. Also, the growth of the recovered virus was indistinguishable from that of authentic m8. The full genome sequence of the plasmid, which harbors identical inverted terminal repeats (ITR) to that of authentic m8, was determined by long-read next-generation sequencing (NGS). The ITR contains x 18 to 32 of the 70 and x 30 to 45 of 54 base pair tandem repeats, and the number of tandem repeats was different between the ITR left and right. Since the virus recovered from pLC16m8.8S-BAC was expected to retain the identical viral genome to that of m8, including the ITR, a reference-based alignment following a short-read NGS was performed to validate the sequence of the recovered virus. Based on the pattern of coverage depth in the ITR, no remarkable differences were observed between the virus and m8, and the other region was confirmed to be identical as well. In summary, this new system can recover the virus, which is geno- and phenotypically indistinguishable from authentic m8.

Application of next-generation sequencing to detect acyclovir-resistant herpes simplex virus type 1 variants at low frequency in thymidine kinase gene of the isolates recovered from patients with hematopoietic stem cell transplantation.

Ion Torrent next-generation sequencing (NGS) technology was applied to study the mode of emergence of acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) in patients with hematopoietic stem cell transplantation (HSCT) by quantitatively detecting mutations in the viral thymidine kinase (vTK) gene in the HSV-1 isolates recovered from HSCT patients. All of the mutations detected with the Sanger sequencing method in the vTK genes of HSV-1 isolates were also detected with the NGS assay. Furthermore, different mutations, which conferred ACV resistance and were not detected with the Sanger sequencing method, were also detected in a quantitative manner by using the NGS assay. The approach described here is applicable to studying the emergence process of vTK gene mutation-associated ACVr HSV-1 more in detail than the Sanger method. The NGS assay makes it possible to make a diagnosis of vTK gene mutation-associated ACVr HSV-1 infections at the early stage, which the ratio of ACVr HSV-1 is much lower than that of ACV-sensitive (ACVs) HSV-1.

Association of the Emergence of Acyclovir-Resistant Herpes Simplex Virus Type 1 With Prognosis in Hematopoietic Stem Cell Transplantation Patients.

Background: Antiviral-resistant herpes simplex virus type 1 (HSV-1) has been recognized as an emerging clinical problem among patients undergoing hematopoietic stem cell transplantation (HSCT). Methods: A prospective observational study was conducted at a hematological center over a 2-year period. Oropharyngeal swab samples were serially collected each week from 1 week before and up to 100 days after HSCT and were tested for virus isolation. The HSV-1 isolates were tested for sensitivity to acyclovir (ACV). The prognosis of patients with ACV-resistant (ACVr) HSV-1 and the genetic background of the ACVr HSV-1 isolates were assessed. Results: Herpes simplex virus type 1 was isolated in 39 of 268 (15%) HSCT patients within 100 days after transplantation. Acyclovir-resistant HSV-1 emerged in 11 of these 39 patients (28%). The 100-day death rates of HSCT patients without HSV-1 shedding, those with only ACV-sensitive HSV-1 shedding, and those with ACVr HSV-1 shedding were 31%, 39%, and 64%, respectively. Patients with HSV-1, including ACVr HSV-1, shedding showed a significantly higher mortality rate. Relapsed malignancies were a significant risk factor for the emergence of ACVr HSV-1. Acyclovir resistance was attributable to viral thymidine kinase and DNA polymerase mutations in 6 and 5 patients, respectively. Conclusions: Herpes simplex virus type 1, including ACVr HSV-1, shedding was associated with poorer outcome in HSCT patients, even if HSV disease did not always occur. Patients with relapsed malignancies were at especially high risk for the emergence of ACVr HSV-1.

Association between sensitivity of viral thymidine kinase-associated acyclovir-resistant herpes simplex virus type 1 and virulence.

BACKGROUND: Acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) infections are concern in immunocompromised patients. Most clinical ACVr HSV-1 isolates have mutations in the viral thymidine kinase (vTK) genes. The vTK-associated ACVr HSV-1 shows reduced virulence, but the association between the level of resistance and the virulence of the vTK-associated ACVr HSV-1 is still unclear. METHODS: The virulence in mice of 5 vTK-associated ACVr HSV-1 clones with a variety of ACV sensitivities, when inoculated through intracerebral and corneal routes, was evaluated in comparison with ACV-sensitive (ACVs) parent HSV-1 TAS. RESULTS: Although all the 5 ACVr HSV-1 clones and ACVs HSV-1 TAS showed a similar single-step growth capacity in vitro, the virulence of ACVr HSV-1 clones significantly decreased. A 50% lethal dose (LD50) of each clone was closely correlated with 50% inhibitory concentrations (IC50), demonstrating that the higher the ACV-sensitvity, the the higher the virulence among the ACVr clones. One of the ACVr HSV-1 clones with a relatively low IC50 value maintained similar virulence to that of the parent TAS. The infection in mice with ACVr HSV-1 due to a single amino acid substitution in vTK induced local diseases, keratitis and dermatitis, while vTK-deficient clone did not. CONCLUSIONS: A statistically significant correlation between the virulence and susceptibility to ACV among ACVr HSV-1 clones was demonstrated.