Strain McKrae Research Articles

Comparative Laboratory Diagnosis of Experimental Herpes Simplex Keratitis

We compared two commercially available tests, a direct immunofluorescence assay and an enzyme-linked immunosorbent assay (ELISA), to viral isolation in tissue culture for the laboratory diagnosis of untreated and partially treated experimental herpes simplex virus keratitis. New Zealand albino rabbits were inoculated bilaterally with herpes simplex virus-1 McKrae strain after corneal scarification. One eye of each rabbit was treated with a 1% trifluorothymidine solution daily, starting on the third day after inoculation. The direct immunofluorescence assay showed lower sensitivity for herpes simplex virus detection than viral isolation in tissue culture for both untreated and partially treated eyes. The Herpchek ELISA demonstrated similar sensitivity to tissue culture in detecting herpes simplex virus in untreated eyes. In the treated group, however, the Herpchek ELISA showed a higher percentage of eyes positive for herpes simplex virus than did viral isolation in tissue culture. After the initiation of antiviral therapy, eyes that no longer harbor infectious virus that can be isolated in tissue culture may remain herpes simplex virus antigen-positive and thus be more amenable to laboratory diagnosis using the rapid ELISA method.

Disinfection of Goldmann Tonometers Against Human Immunodeficiency Virus Type 1

Goldmann tonometer tips were inoculated with 5 X 10(5) IU of cell-free or cell-associated human immunodeficiency virus type 1 (lymphadenopathy virus type 1 isolate) or 10(4) plaque-forming units of herpes simplex virus type 1 (McKrae strain) or type 2 (Hicks strain). In an effort to mimic a "worst case" clinical scenario, each respective virus was allowed to air dry on the tonometer tip for 10 minutes. Inoculated tonometers were then (1) not treated, (2) wiped with a disposable (Kim-wipe) tissue or sterile gauze; (3) wiped with sterile gauze soaked with 3% hydrogen peroxide; or (4) wiped with a 70% isopropyl alcohol swab. The hydrogen peroxide treatment and the alcohol wipes both completely disinfected the tonometer tips for human immunodeficiency virus type 1 and herpes simplex virus types 1 and 2, whereas wiping with a sterile gauze or tissue was not effective. Wiping the Goldmann tonometer tip with an isopropyl alcohol swab and then allowing the alcohol to evaporate provides a ready and efficient means of inactivating these three enveloped viruses.

An improved model of recurrent herpetic eye disease in mice

Mice were passively immunized with serum containing antibodies to herpes simplex virus type 1 (HSV-1) before inoculation on the cornea with HSV-1 strain McKrae. After such immunization most mice survived and most had normal eyes. When primary infection had subsided, mice with normal eyes were selected and treated with cyclophosphamide, dexamethasone and UV irradiation of the inoculated eye or UV irradiation alone, to reactivate latent virus. After either treatment mice developed signs of recurrent infection (virus in eyewashings and recurrent corneal and/or lid disease). The incidence of such signs was 17/33 (52%) in mice receiving immunosuppressive drugs and UV irradiation and 19/32 (59%) in mice given UV irradiation alone. In mice treated with either stimulus dendritic or geographic ulceration of the cornea was seen. These closely resembled the herpetic lesions seen in humans. There was good correlation between the pattern and distribution of recurrent corneal disease and the distribution of cells containing virus antigens in corneal epithelial sheets. Again, as in humans, the induction of recurrent infection was found to correlate poorly with a rise in the level of serum neutralizing antibody. In mice treated with UV irradiation alone corneal ulcers healed and the eyes returned to normal. By contrast, in mice given immunosuppressive drugs and UV irradiation, the ulceration became more severe and the eyes became opaque and vascularized. The use of passive immunization has greatly improved our previously reported model of recurrent herpetic eye disease since it has increased the incidence of mice suitable for the induction of recurrent infection and has increased the incidence of such infection.

An in vitro study of ophthalmic antiviral agent toxicity on rabbit corneal epithelium

Using an in vitro system we measured the corneal epithelial cytotoxicity and the antiviral activity of the antiviral agents idoxuridine (IDU), trifluridine (TFT), ethyldeoxyuridine (EDU), and ( E)-5-(2-Bromovinyl)-2′-deoxyuridne (BVDU). Confluent rabbit corneal epithelial cell cultures were established, and the antiviral agents were added for 5, 30, or 60 min at a range of concentrations including that used clinically (IDU 0.1%, TFT 1.0%, BVDU 0.1%, EDU 2.0%). Twelve hour [ 3H]thymidine incorporation then was measured and expressed as % inhibition of control cultures. In separate experiments confluent corneal epithelial cell monolayers were inoculated with 10 4 plaque forming units (PFU) of HSV type 1 (McKrae strain) for 1 h, and IDU 0.1%, TFT 1.0%, and BVDU 0.1% were added to the culture for determination of PFU inhibition. Significant dose-, but not time-dependent, toxicity was observed at the clinical concentrations of IDU, TFT, and EDU. Toxicity was absent for BVDU. TFT and IDU were the most toxic, and EDU was of intermediate toxicity. IDU, TFT, and BVDU showed significant antiviral activity in this corneal epithelial cell culture system (TFT > BVDU >IDU). The results of this in vitro study paralleled the findings of previous in vivo corneal epithelial toxicity studies of IDU, TFT, and BVDU. Our data, however, suggest that EDU has a potential for clinical toxicity and further studies are recommended. Our model may be useful in the future toxicologic study of new antiviral agents.

Corneal nerves contain intra-axonal HSV-1 after virus reactivation by epinephrine iontophoresis

Experimental ocular models of herpes simplex virus type 1 (HSV-1) reactivation have been used to monitor viral shedding in the tear film and the appearance of corneal epithelial lesions, but the temporal correlation between reactivation and the presence of viral particles in the corneal nerves has not been made. Two New Zealand white rabbits were inoculated with 20 microliters of HSV-1 McKrae strain (5.0 x 10(6) PFU/ml) in each eye. Beginning on postinfection day 82, ocular iontophoresis (0.8 mAmps for 8 min) of 0.01% epinephrine was done once a day for 3 consecutive days to induce reactivation. Ten limbal nerves from four corneas processed for transmission electron microscopy contained 883 unmyelinated and 40 myelinated axons. Seven nerves were positive for virus. Viral particles were found only in unmyelinated axons, and in low frequency (24/883). Virus was not found in Schwann cells, perineurium, or adjacent stroma nor were virus particles seen exiting axons. No enveloped virions were found. Axons from six nerves of four control corneas from rabbits with latent, but not reactivated, HSV-1 did not contain virus particles. Induction by corneal iontophoresis of epinephrine suggests that HSV-1 is translocated from the ganglion to the cornea through axonal transport mechanisms. For the first time, evidence of anterograde, intra-axonal transport of HSV-1 particles in response to epinephrine reactivation is demonstrated.

Recovery of herpes simplex virus from the corneas of experimentally infected rabbits.

Rabbits were inoculated in the left cornea with one of three strains of herpes simplex virus (HSV) i.e. HSV-1 strain 17, HSV-1 strain McKrae, HSV-2 strain HG52, or with and HSV-1 McKrae/HSV-2 HG52 recombinant R40/2. Fifty-nine to 67 days after inoculation trigeminal ganglia and corneas were explanted and screened for release of infectious virus. Virus was isolated from all left trigeminal ganglia after organ culture irrespective of viral strain. Virus was isolated from three of 16 corneas of animals inoculated with HSV-1 strain McKrae and from one of four corneas from animals inoculated with the HSV-1/HSV-2 recombinant. The isolation of HSV from explanted corneas, after between 15 and 35 days in organ culture suggests that the cornea may be a site additional to dorsal root ganglia where latent HSV can reside in rabbits.

Recurrent herpes simplex virus type 1 corneal epithelial lesions after radial keratotomy in the rabbit.

The effects of radial keratotomy (RK) on herpes simplex virus type 1 ocular shedding and recurrent corneal epithelial lesions were assessed for 14 days after RK in rabbits latently infected with strain McKrae. Ten latently infected rabbits had RK performed on the right eye. The left eye was used as the control. Viral shedding was monitored by ocular tear-film swabs. The recurrent lesions were monitored by slit-lamp examination. In the RK-treated eyes, 15 (11%) of 140 swabs were positive, compared with six (4%) of 140 swabs in control eyes. Slit-lamp examination disclosed recurrent lesions on 18 (33%) of 55 days on which it was performed in the RK group vs three (5%) of 55 days for the control group. The results suggest that patients who have a history of ocular herpes simplex virus type 1 disease are at risk for recurrences after RK.

Strain specificity of spontaneous and adrenergically induced HSV-1 ocular reactivation in latently infected rabbits.

Spontaneous ocular shedding and adrenergic induction of ocular shedding were examined in rabbits infected with ten strains of herpes simplex virus type 1 (HSV-1): McKrae, KOS, F, Rodanus, 17 Syn+, RE, E-43, SC-16, MacIntyre, and CGA-3. All ocular inoculations were with 50 microliter of HSV-1 with titers between 1-10 X 10(6) PFU/ml. All corneas, except those that received the McKrae strain, were scarified. Acute ocular infection was determined by slit-lamp biomicroscopy. Dendritic keratitis or geographic ulcers developed in all eyes of all rabbits within 10 days after ocular inoculation. All eyes of all surviving rabbits were swabbed for 20 consecutive days during days 20-39 postinoculation (PI). On PI day 19, no active lesions were present as judged by slit-lamp biomicroscopy. Ocular tear film was collected on a Dacron-tipped swab and placed on primary rabbit kidney cell monolayers. The cell monolayers were monitored for cytopathic effects consistent with HSV-1 infection. Spontaneous HSV-1 shedding was detected in some eyes from all groups of latently infected rabbits, except those infected with CGA-3. Spontaneous shedding (positive swabs/total swabs) of the other nine strains ranged from 0.7% to 15.7%. After PI day 42, the rabbit eyes received 6-hydroxydopamine by iontophoresis, followed for 5 days by topical application of 2% epinephrine. This procedure results in induced HSV-1 ocular shedding for a duration of 3-5 days in rabbits infected with the McKrae strain. In rabbits latently infected with KOS, F, RE, MacIntyre, and CGA-3, no induced HSV-1 shedding was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of Efficacy of Adenosine-5′-Monophosphate Against HSV-1 Ocular Shedding in Rabbits

Adenosine-5'-monophosphate (AMP) was evaluated for efficacy in the prevention of spontaneous and induced herpes simplex virus type 1 (HSV-1) ocular shedding in latently infected rabbits with strain McKrae. Intraperitoneal injections (IP) of AMP (100 mg/kg) or NaCl (10 mg/kg) were given on postinoculation (PI) days 16-39. Spontaneous viral shedding was monitored by ocular tear film swabs on PI days 20-39. In the induced rabbits, one group received AMP (IP) and a second group received NaCl (IP) on PI days 66-77. In a third group, AMP (100 mg/kg) was given twice a day IP on PI days 66-77, and AMP was applied by iontophoresis to these eyes on PI days 68-74. In these three groups, ocular viral shedding was induced by ocular iontophoresis of 6-hydroxydopamine on PI day 70 followed by topical application of epinephrine for 5 days (PI days 70-74). HSV-1 ocular shedding was monitored on PI days 66-78. There were no significant differences in spontaneous or induced shedding patterns between the AMP (systemic or systemic plus ocular iontophoresis) and the NaCl groups. These results suggest that this dose of systemically administered AMP plus iontophoresis of AMP does not reduce ocular HSV-1 shedding in rabbits.

Adrenergically induced recurrent HSV-1 corneal epithelial lesions.

Herpes simplex virus type 1 (HSV-1) ocular shedding and recurrent HSV-1 corneal epithelial lesions were assessed after ocular iontophoresis of 0.1% 6-hydroxydopamine followed by topical ocular instillation of 0.1% Propine in ten rabbits latently infected with HSV-1 strain McKrae. Iontophoresis was performed once at 0.5 mAmp for five minutes and 0.1% Propine drops were instilled four times a day beginning three days after iontophoresis and continuing for five consecutive days. Over an eight day period beginning three days after iontophoresis, ocular tear film samples were collected on Dacron swabs with care taken to avoid contact with the corneal epithelium. The corneas were examined daily for the presence of epithelial lesions using a slit-lamp biomicroscope. Three types of lesions were observed: deep punctate lesions, dendritic lesions, and geographic epithelial defects. The ratio of positive HSV-1 eye swabs to total eye swabs was 36/157 (23%). The ratio of total positive days of corneal lesions to total days was 40/160 (25%). There were 23 deep punctate lesions, 13 dendritic lesions, and four geographic epithelial defects. There were 24/36 (67%) positive HSV-1 eye swabs associated with concurrent HSV-1 corneal epithelial lesions. There were 105/121 (87%) negative eye swabs with concurrent negative slit-lamp examinations. Chi square analysis showed significant (p less than 0.001) association of HSV-1 positive eye swabs and HSV-1 corneal lesions. These results suggest that adrenergic ocular treatment may induce both HSV-1 ocular shedding (reactivation) and HSV-1 corneal epithelial lesions (recurrence) in rabbits latently infected with HSV-1 strain McKrae.

Identifying HSV infected neurons after ocular inoculation.

ICR mice were inoculated intracamerally with McKrae strain herpes simplex virus (HSV) followed by intraperitoneal injection with 3H-thymidine. Infected mice were sacrificed after 3 or 4 days and the eyes, trigeminal ganglia (TG) and superior cervical ganglia (SCG) were embedded in glycol methacrylate, sectioned, and dipped for autoradiography. Light microscopy revealed silver grain labeling over neurons in the ipsilateral retina, TG and SCG of infected animals. No labeling of neurons was noted in the contralateral TG or SCG. Since the DNA of mature neurons does not replicate, we interpret these labeled neurons to represent cells with active replication of HSV. This technique allows the study of HSV infection of the nervous system with excellent tissue preservation. Furthermore, it may be used to distinguish those neurons with intrinsic viral synthesis from those harboring virus synthesized at a distant site with subsequent intracellular spread.

Quantitation and kinetics of induced HSV-1 ocular shedding.

Iontophoresis of 6-hydroxydopamine (6-HD) to the rabbit eye, followed by topical instillation of 2% epinephrine, induces ocular shedding of herpes simplex virus type 1 (HSV-1) reliably and with a high frequency in latently infected rabbits. Rabbit eyes inoculated with HSV-1 (McKrae strain) showed dendritic lesions indicative of acute HSV infection and subsequently shed virus spontaneously at least once during days 20 to 39 postinoculation (P.I.). Two iontophoretic conditions were employed. Group A (3 rabbits, 60.3 days P.I.) received iontophoresis of 1.0% 6-HD at 0.75 mAmp for 3 min. Group B (three rabbits, 67.3 days P.I.) received iontophoresis of 0.1% 6-HD at 0.5 mAmp for 8 min. Following iontophoresis, 2% epinephrine was instilled topically once on the day of iontophoresis and twice daily for four consecutive days. Tear film was collected on Dacron swabs and titered on African green monkey kidney cells by a plaque assay procedure. In group A, 100% (6/6) of the eyes shed virus, and the average duration of shedding was 4.0 days. The titers ranged from 2.0 to 7.7 X 10(4) plaque-forming units (PFU) per eye. The highest daily average titer, 9.89 X 10(3) PFU/eye, occurred on day 5 following iontophoresis. In Group B, 100% (6/6) of the eyes also shed virus, and the average duration of shedding was 5.3 days. The viral titer of the tear film ranged from 5.0 to 1.4 X 10(5) PFU/eye. The highest daily average titer, 4.68 X 10(3) PFU/eye, also occurred on day 5 following iontophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequential analysis of antibody responses in serum, aqueous humor and tear film during latent and induced recurrent HSV infections.

To study HSV specific antibody responses during latent HSV infection and induced HSV ocular shedding, rabbits were infected binocularly with McKrae strain HSV-1. The titer and class of anti-HSV antibody response in serum, aqueous humor and eye washes were determined sequentially during latent HSV infection and following intentional reactivation. In all instances the only antibody class detected was IgG. The highest anti-HSV titers were in the serum and aqueous humor, whereas a significantly lower level of anti-HSV IgG was found in eye washes. Anti-HSV IgG antibody titers were consistently 100-fold higher in serum than in the aqueous humor, which suggests an absence of local ocular antibody synthesis. Neither anti-HSV IgM nor secretory IgA antibodies were detected in any samples.

Use of recombinant interferon to prevent recurrent herpesvirus shedding.

Iontophoresis with 6-hydroxydopamine was performed in rabbits previously infected with herpes simplex virus, McKrae strain. Viral shedding into the tear film was significantly decreased by the use of recombinant alpha interferon subtype D given as one drop qid. Interferon was noted in the tear film of rabbits 16-18 hours after the last placement of interferon drops into the inferior cul-de-sac.

Acyclovir and vidarabine monophosphate: Comparison of lontophoretic and intravenous administration for the treatment of HSV-1 stromal keratitis

We determined the therapeutic efficacy of iontophoretic application of acyclovir and vidarabine monophosphate (ara-AMP) for the treatment of herpes simplex virus (HSV) type 1-induced stromal keratitis in rabbits. The therapeutic efficacy of intravenous administration of acyclovir was assessed in the same model. Stromal keratitis was produced by intrastromal injection of 10 μl purified HSV-1, McKrae strain. Treatment began the first day after intrastromal injection. Iontophoresis (0.5 mAmp for four minutes) of 3.4 percent (0.1 M) ara-AMP and 5.0 percent (0.22 M) acyclovir was performed once daily for five consecutive days in two treatment groups. Intravenous administration of 50 mg acyclovir/kg was performed twice daily for eight consecutive days. Intravenous administration of NaCl (0.14 M) and ocular iontophoresis of NaCl (0.14 M) were performed as controls in the two treatment groups. At least two scorers performed a single masked evaluation of the disease severity (lesion scoring) of the conjunctiva, corneal epithelium, stroma, and iris by slit lamp examination. The eyes were scored daily for 12 consecutive days, and then every other day up to day 22. Iontophoresis of acyclovir or ara-AMP significantly reduced the course of the disease compared with iontophoresis of NaCl. Intravenous administration of acyclovir significantly reduced the disease compared with intravenous NaCl. This suggests that iontophoresis of acyclovir or ara-AMP either alone or in combination with intravenous administration of acyclovir may be of value in the treatment of HSV-1 stromal keratitis.

Initial herpes simplex virus type 1 infection prevents ganglionic superinfection by other strains.

The ganglia of rabbits infected with a relatively benign strain of herpesvirus (E-43) and challenged with either of two virulent neurotrophic strains (MP or McKrae) were found to be colonized only by the initial benign infecting strain. Primary infection with the E-43 strain resulted in milder disease when the animals were infected with MP or McKrae strains and also prevented colonization of the ganglion by these strains. Neutralization with anti-glycoprotein C, plaque morphology, cytopathic effects, reconstruction experiments, and restriction endonuclease analysis indicated that the virus recovered from the ganglion was the initial infecting E-43 strain; no traces of the challenging MP and McKrae strains were found. The challenging McKrae strain was shed for several weeks in a few animals, but the virus isolated from the trigeminal ganglia of these animals was the primary infecting E-43 strain. These results suggest that initial infection with a relatively benign strain of herpesvirus may prevent superinfection of the ganglion (but not necessarily the end organ) by highly virulent herpes simplex virus strains and could have significant implications in the consideration of immunization against this disease in humans.

Failure of Systemically Administered Adenine Arabinoside to Affect Humoral and Cell-Mediated Immunity

The effect of a high dosage (250 mg/kg of body weight) of adenine arabinoside or ara-A (9-beta-D-arabinofuranosyladenine) on humoral immunity was studied in New Zealand white rabbits infected with the McKrae strain of herpes simplex virus. The rabbits were treated daily for 14 days with subcutaneous injections of ara-A. The primary and secondary humoral responses, as measured by neutralizing antibody titers, developed similarly in control and treated groups. Similar drug treatment was used on guinea pigs before or after sensitization with BCG vaccine. Subsequent challenge of the sensitized animals with Old tuberculin solution indicated that ara-A treatment had no effect on the induction or previously established cell-mediated immunity. The lack of immunosuppressive activity of ara-A at dosage levels higher than those used in primates makes this drug a potentially effective agent in the systemic treatment of herpetic infections.