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Generalized parapoxvirus infection associated with increased antibody titres for varicella zoster virus and measles

European Journal of Dermatology. Volume 19, Number 4, 375-9, July-August 2009, Clinical report

DOI : 10.1684/ejd.2009.0700

Summary

Author(s) : Clara Larcher, Elfriede Daniel, Elisabetta Pagani, Klaus Maier, Valentina Pasquetto, Maria Francesca Mellina-Bares, Edna Nemati, Edoardo Egarter-Vigl, Pierfrancesco Zampieri, Hartwig P Huemer , Laboratory of Microbiology & Virology, Azienda Sanitaria dell’Alto Adige, Bolzano, Italy, Dept. Dermatology, Regional Hospital Franz Tappeiner, Merano, Italy, Dept. Histopathology, Central Hospital Bolzano, Bolzano, Italy, Dept. Physiology & Medical Physics, Innsbruck Medical University, Innsbruck, Austria, Dept. Hygiene, Microbiology & Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str.3, R.301, A-6020 Innsbruck, Austria.

Summary : Human parapoxvirus infections are rare, self-limiting, zoonotic diseases. A 35-year-old veterinarian presented with a generalized rash of large umbilicated vesicles that appeared after antibiotic treatment for erysipelas on the forearm. The erysipelas arose from an erupted pustular thumb lesion that appeared after examining a sheep. An outbreak of chickenpox in the village suggested parapoxvirus or varicella zoster virus (VZV) was the most likely agent. No poxvirus was detected by electron microscopy or in cell cultures from lesion material. PCR revealed parapoxvirus DNA with a sequence similar to orf-viruses from Finland. Orf-virus immunofluorescence showed a titre increase, supporting the parapoxvirus diagnosis. VZV was not detected by PCR, but varicella antibodies increased three-fold in serum samples drawn two weeks apart. In addition, the patient had high antibody titres for measles and reported recent contact with individuals exposed to an outbreak of measles in nearby Austria. To explain the unusually generalized symptoms in this young and healthy patient, these findings could be variously interpreted as: i) a booster by community VZV infections\; ii) a subclinical VZV (re)infection that was superinfected by the parapoxvirus\; iii) an orf-virus mediated immune stimulation\; iv) a post-infectious syndrome\; or v) a temporary immunosuppression by subclinical measles.

Keywords : antibody titre, measles, orf virus, parapoxvirus, varicella, VZV

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ARTICLE

Auteur(s) : Clara Larcher¹, Elfriede Daniel², Elisabetta Pagani¹, Klaus Maier³, Valentina Pasquetto¹, Maria Francesca Mellina-Bares², Edna Nemati⁴, Edoardo Egarter-Vigl³, Pierfrancesco Zampieri², Hartwig P Huemer⁵

¹Laboratory of Microbiology & Virology, Azienda Sanitaria dell’Alto Adige, Bolzano, Italy
²Dept. Dermatology, Regional Hospital Franz Tappeiner, Merano, Italy
³Dept. Histopathology, Central Hospital Bolzano, Bolzano, Italy
⁴Dept. Physiology & Medical Physics, Innsbruck Medical University, Innsbruck, Austria
⁵Dept. Hygiene, Microbiology & Social Medicine, Innsbruck Medical University, Fritz-Pregl-Str.3, R.301, A-6020 Innsbruck, Austria

accepté le 11 Mars 2009

Orf-virus (Parapoxvirus ovis) causes contagious ecthyma in small ruminants worldwide. Humans may become infected by contact with small skin lesions. Therefore, most primary lesions in humans are found on the hands, presenting as painless pustules that develop a central necrosis. Although the orf-virus infection is self-limiting in immunocompetent hosts, a differential diagnosis is important because the skin lesions can resemble potentially life-threatening zoonotic infections, including tularemia, cutaneous anthrax, or erysipeloid [1].

Many studies have reported a broad spectrum of sequelae associated with human orf-virus infection. The most commonly reported symptoms are fever, lymphangitis, lymphadenopathy, and secondary bacterial infection [2]. Others have reported erythema multiforme, papulovesicular (widespread blistering) eruptions (rare), bullous pemphigoid-like eruptions several weeks after an orf infection, and mucous membrane pemphigoid lesions [3-5]. Orf-virus can infect animals repeatedly and an orf-virus infection does not fully protect against a secondary infection. Thus, it can severely compromise the immune system of the host [6]. Parapoxviruses have the most impressive spectrum of putative immunoregulatory proteins, including an interferon resistance protein, a viral orthologue of mammalian IL-10, and inhibitors of the cytokines GM-CSF and IL-2. Orf-virus also encodes a virulence protein homologous to mammalian vascular endothelial growth factor. Orf-virus influences several different pathways in the host immune and inflammatory responses; thus, its mechanism of action is very complex and not fully understood [7].

Due to the lack of a reporting system, the frequency of occurrence of zoonotic poxvirus infections in humans is not well established. Many cases are not submitted to a hospital or laboratory examination due to the benign and self limiting nature of the skin lesions. The abandonment of smallpox vaccinations in1977 may have rendered the population more vulnerable to poxviruses and may have contributed to the numerous reports of cowpox infections in young Europeans. There is some serological cross-reactivity between the different genera of poxviruses. However, it seems rather unlikely that the smallpox vaccine, an orthopoxvirus, might have a cross-protective effect against parapoxviruses. A previous parapoxvirus infection does not even fully protect against a second infection [6]. Jenner noted this based on empirical evidence in the 18^th century and warned against the use of “spurious pox” (i.e. parapoxvirus) vaccinations, instead of the cowpoxvirus.

Case report

A 35-year-old man, a veterinarian by profession, presented at the dermatology outpatient clinic with a pruritic rash that had developed within the previous five days. He reported that, for the past two weeks, he had experienced headache, ulcerous lesions on the lips, and within the past day, joint pain in the right wrist and the left great toe. Upon clinical examination, umbilicated vesicles of approximately 7 mm diameter were observed on the face, neck, head, arms, and symmetrically on both knees (figure 1). The lymph nodes of the right elbow were palpable. There was some swelling of the eye lids, but no fever or signs of meningitis.

Two and a half weeks previously, the patient had examined an anorectic sheep and sustained a slight abrasion on the right hand from the teeth of the sheep. The sheep showed no lesions on the mouth or teats; however, some animals in that area had had “scabby mouth” disease. Three days later, a pustular lesion and associated perifocal swelling had appeared on the patient’s thumb, supporting the diagnosis of orf-virus. The patient treated this primary lesion with birch tar and ichthyol (ammonium bituminosulfonate) as a self medication. The patient then removed the crust, resulting in some bleeding and followed by a skin redness of the forearm that extended up to the elbow. Lymphangitis developed and was regarded as bacterial superinfection; in contrast to lymphadenopathy, lymphangitis is not a typical symptom of orf infection. The patient was treated with amoxicillin-clavulanic acid for six days for suspected erysipelas; after 3 days of treatment with no response, the initial standard dose was increased to 6 g/d.

During that time, there had been an outbreak of chickenpox in the patient’s village, but he reported that he had had chickenpox during childhood. Furthermore, the skin lesions were three-times larger than those usually associated with VZV. In addition, the patient reported that he had had contact with individuals who had been exposed to a measles outbreak in nearby Austria [8]; measles had also been observed in the north of Italy [9]. Unfortunately, serological data for the exposed contacts were not available.

The patient’s past medical history indicated no evidence of an atopic skin diathesis and listed no known causes of immunosuppression, including lymphoma, tumors, HIV, or other immune disorders. There was no evidence of drug-induced immunosuppression, a condition that has previously been suggested to play a role in orthopoxvirus infections [10]. The patient had not been vaccinated against measles, varicella, or smallpox viruses.

Two skin biopsies were performed for microbiological diagnosis and histopathology. One swab was performed for bacterial culture, to exclude anthrax. Two serum samples were drawn within two weeks for serological examination.

Laboratory findings

Clostridium perfringens was cultured from a swab that the patient had taken himself from the primary pustule, but was regarded as a colonizer. Swabs from the generalized rash excluded anthrax but remained otherwise uninformative. Attempts to grow parapoxvirus in cell culture were unsuccessful. Vesicle homogenates were generated in a microtube cell grinder and spotted onto slides. Samples were analysed by electron microscopy using a negative stain technique with phosphotungstic acid as the contrasting agent. Analysis of the electron micrographs showed no parapoxvirus particles.

PCR was performed to detect parapoxvirus DNA in the skin biopsy. The biopsy DNA was digested with proteinase K, extracted with the Qiagen DNA Mini Kit, and amplified with the Platinum Blue PCR Supermix (Invitrogen) according to the semi-nested amplification protocol described by Inoshima et al. [11]. The PCR products were analysed with an ABI-3130 DNA sequencer and reference sequences were aligned with Proseq2.91. Homology trees were generated by the neighbour-joining method using Jukes and Cantor’s calculation of genetic distance (figure 2). Interestingly, the DNA sequencing analysis showed that the sequence from the parapoxvirus (IT08PradStJ) detected in the patient from the region of Prad, Stilfser Joch in the far north of Italy was most similar to that of the orf-viruses found in Finland. Contamination of the PCR can be excluded because the strain used as a positive control originated from Germany (RVB065-Burghessler) and had a clearly different DNA sequence (figure 2) The B2L gene sequences of strains IT08PradStJ and RVB065-Burghessler were deposited in EMBL/Genbank under the submission numbers FM178392 and FM178391, respectively. A nested PCR for VZV was performed using an established protocol [12]. No VZV-specific amplification products were obtained from biopsy material.

Sera were drawn at three and five weeks after the initial incident and were analysed by two different methods. Antibodies against ortho- and parapoxvirus were detected by indirect immunofluorescence performed on RK13 rabbit kidney cells that had been infected with the vaccinia virus strain WR or the orf-virus control strain (figure 3). A FITC-labelled anti-human IgG Fab-antibody (Sigma Co.) was used as secondary antibody. Only a weak antibody response (most likely a cross-reaction) against orthopoxvirus was detected (< 1:50); however, antibodies against orf-virus were detected and serum sample titres were 1:400 at 3 weeks and 1:800 at 5 weeks after the initial incident. This titre was considerably higher when compared to other reported Orf-virus infections. Most previous studies found antibody responses to be rather modest, due to immuno-regulatory phenomena, especially in the natural hosts; this might explain the propensity for this virus to re-infect the same host.

Antibodies against VZV, measles, HSV1/2, and EBV were detected with a commercial EIA system (Enzygnost, Dade-Behring). Interestingly, there was a three-fold increase in specific IgG antibodies to VZV in the two serum samples. Titres against other herpes viruses, including EBV and HSV, were unremarkable. However, there was an unusually high EIA IgG titre against measles virus observed in both samples. Specific IgM antibodies were not found.

Histopathology of a vesicle/skin biopsy showed an inflammatory infiltrate that was restricted to the cutis. Deeper parts of the dermis were not affected. The infiltrate was mixed lympho-granulocytic with a perivascular location, consistent with allergic vasculitis (figure 4).

Discussion

Support for a diagnosis of varicella (with superinfection by orf-virus)

To support the diagnosis of varicella, we found rising antibody titres, a possibility of exposure in the village, and itching lesions. In contrast, orf-pustules are rarely reported to be pruritic [1]. On the other hand, the lesions were too large to be chickenpox and we did not detect VZV by PCR. This suggests rapid clearance of the virus by a pre-existing immune response. A primary infection seems unlikely due to the case history of childhood chickenpox and the presence of specific IgG and absence of IgM antibodies in the first serum sample. Re-infections with varicella occur rather frequently [13]. The increase in antibody titres in immune subjects after re-exposure to VZV is an established phenomenon [14]. Adult immigrants whose primary varicella infections occurred in Africa or Asia showed asymptomatic superinfections with different strains and establishment of co-latency. One third experienced reactivation of a strain molecularly distinct from those normally found in Africa or Asia [15]. Bacterial superinfections are frequent in chickenpox, but a parapoxvirus superinfection has not been reported to date. Although poxviruses have a tendency to infect pre-injured skin, as in eczema vaccinatum, it is not certain whether damage to the skin microvascular system by VZV would also favour the extravasation and haematogenous spread of orf-virus.

Support for an additional recent case of measles

We observed unusually high antibody titres against the measles virus in both serum samples. This indicated a recent boost in the specific IgG-titres and possibly temporary immunosuppression by measles virus. Secondary infections and subclinical measles infections are not uncommon. A secondary immune response against measles in a vaccinated population, presumably due to field exposure, was observed in approximately 5% of the population in Luxemburg. The waning of the immune response years after vaccination is an important factor in subclinical measles infections [16]. In another study, subclinical measles occurred in a surprisingly high percentage of vaccinated individuals. The frequency was inversely related to pre-exposure antibody titres and the subclinical cases had an average of 45-fold increases in titres that remained elevated for at least six months [17]. This would fit the time frame of a potential measles exposure in our patient. Though he had not been vaccinated, our patient could have contracted a wild type measles virus infection during childhood, and that might explain a high titre in response to a subclinical infection contracted by possible exposure to measles in Austria within 6 months of the present study.

Support for an immunological origin of the pustules

Firstly, the histopathological findings were compatible with allergic vasculitis rather than VZV, comparable to inflammation following insect stings. We detected orf-virus only by PCR and not by electron microscopy or cell culture; thus, we presume that either there were very small amounts of virus present, or they were sequestered in inactivated immune-complexes. This phenomenon would be consistent with the findings described for HSV-associated erythema multiforme, where no intact virus could be detected in the skin lesions, though viral DNA material was transported to the skin [18]. In support of an immune-complex sequestration, the joint symptoms reported by our patient were compatible with reactive arthritis and resolved rather quickly.

Suggestions of the involvement of an immune mediated post-infectious syndrome

To explain the unusual lesions, it is possible that the parapoxvirus infection was associated with a post-infectious syndrome known as Sweet’s syndrome (SS). Also called acute, febrile, neutrophilic dermatosis, SS is an inflammatory disorder that can affect many organ systems [19]. Our patient showed some, but not all of the symptoms of SS. SS is characterized by pyrexia (not present in our patient), painful erythematous plaques, and aseptic neutrophilic infiltrations that spare the epidermis. Signs of vasculitis are usually absent in SS. In addition, unlike SS, the lesions of our patient were not painful, but rather itching and the mixed lymphocytic infiltrates were mostly perivascular. Common complications of SS include arthralgias/arthritis and conjunctivitis; both were present in our patient. However, our patient also had oral ulcers, which seem to be uncommon in classical SS. Streptococcal infections of the upper respiratory tract have been especially implicated in initiating SS. Our patient most likely had a streptococcal skin infection (lymphangitis, erysipelas) and was exposed to measles and VZV. One third of patients with SS experience a recurrence of SS. Although our patient had no history of previous symptoms of SS, we could not rule out a genetic predisposition. In addition, cytokines play an important etiological role in SS [19] and parapoxviruses have an important influence on various cytokine pathways [7]. This is also reflected in the proliferative character of orf lesions compared to the more ulcerative lesions caused by orthopoxviruses.

Possible immunoregulatory contributions of orf-virus

The orf-virus has a complex involvement with different pathways of the immune and inflammatory responses; thus, it is not exclusively immunosuppressive [7]. Orf-virus has been shown as a potent immune stimulator in heterologous hosts. In mice, inactivated viral particles provided protection against unrelated viral agents, including hepatitis B and HSV [20]. Accordingly, the orf-virus infection per se could have mitigated the outcome of a concomitant VZV infection in our patient, suppressing it to a subclinical level. Orf-virus also mediated immune stimulation in pigs. This provided evidence for a viral component functioning as a superantigen capable of stimulating immune cells independent of their specific antigen receptors [21].

Similar mechanisms, albeit less potent, are observed in normal immune responses. Many antigens have been shown to activate “bystander cells”. A tetanus toxoid boost was associated with an increase in anti-tetanus IgG and increases in other IgG against unrelated viral antigens. Thus, repeated, non-specific polyclonal activation of memory B cells might offer a natural means for maintaining serological memory for long periods of time [22]. Therefore, one could speculate that the strong stimulation of the immune system by the orf-virus on its own could increase antibody titres against unrelated antigens (like VZV) even in the absence of the antigen.

Acknowledgements

The work was supported by the Austrian Federal Health Ministery (BMGF) as a contribution to the Austrian smallpox emergency preparedness plan. (Pockenalarmplan). There is no conflict of interest of any of the authors.

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