Modulation of cellular tropism and innate antiviral response by viral glycans.

Arthropod-borne viruses (arboviruses) are a significant cause of human and animal disease worldwide. Multiple interactions between virus and the host innate immune system ultimately determine the pathogenesis and clinical outcome of the infection. Evidence is rapidly emerging that suggests viral glycans play a key role in viral pathogenesis by regulating host cell tropism and interactions with the host innate immune response. Glycan-mediated interactions are especially important for arboviruses which must adapt to variable glycosylation systems and cellular receptors within both vertebrate and invertebrate hosts. This review focuses on emerging evidence which supports a crucial role for viral glycans in mediating host cell tropism and regulating the innate antiviral response.

Ross River virus envelope glycans contribute to type I interferon production in myeloid dendritic cells.

Alphaviruses are mosquito-transmitted viruses that cause significant human disease, and understanding how these pathogens successfully transition from the mosquito vector to the vertebrate host is an important area of research. Previous studies demonstrated that mosquito and mammalian-cell-derived alphaviruses differentially induce type I interferons (alpha/beta interferon [IFN-alpha/beta]) in myeloid dendritic cells (mDCs), where the mosquito cell-derived virus is a poor inducer of IFN-alpha/beta compared to the mammalian-cell-derived virus. Furthermore, the reduced IFN-alpha/beta induction by the mosquito cell-derived virus is attributed to differential N-linked glycosylation. To further evaluate the role of viral envelope glycans in regulating the IFN-alpha/beta response, studies were performed to assess whether the mosquito cell-derived virus actively inhibits IFN-alpha/beta induction or is simply a poor inducer of IFN-alpha/beta. Coinfection studies using mammalian- and mosquito cell-derived Ross River virus (mam-RRV and mos-RRV, respectively) indicated that mos-RRV was unable to suppress IFN-alpha/beta induction by mam-RRV in mDC cultures. Additionally, a panel of mutant viruses lacking either individual or multiple N-linked glycosylation sites was used to demonstrate that N-linked glycans were essential for high-level IFN-alpha/beta induction by the mammalian-cell-derived virus. These results suggest that the failure of the mosquito cell-derived virus to induce IFN-alpha/beta is due to a lack of complex carbohydrates on the virion rather than the active suppression of the DC antiviral response.

In vivo experimentation with simian herpesviruses: assessment of biosafety and molecular contamination.

In vivo studies with highly pathogenic viruses prompt concerns regarding the persistence of infectious virus in pathology specimens. Although formalin fixation of tissues may inactivate infectious virus, fixation may also degrade viral nucleic acid and antigens, thereby limiting detection of virus in tissues by polymerase chain reaction (PCR) amplification or immunohistochemistry (IHC). We sought to: 1) assess the rate of inactivation of infectious virus in tissue specimens during formalin fixation, 2) assess IHC recognition of viral antigens and PCR detection of viral DNA after long-term (14 d) formalin fixation, and 3) investigate microtome contamination by DNA carry-over to subsequently sectioned tissues. Infectious baboon herpesvirus HVP2 could be recovered from fresh tissues of infected mice but not those fixed in formalin for >/=24 h. The intensity of IHC staining of viral antigen was unaffected by the duration of formalin fixation. PCR detection of viral DNA was negatively impacted by formalin fixation and/or heat inherent to paraffin processing; however, amplification of very short DNA sequences using real-time PCR was not affected. Lastly, microtome contamination by viral DNA was demonstrated by PCR screening of uninoculated control tissues that were sectioned after sectioning infected tissues. In summary, infectious virus is inactivated after only 24 h of formalin fixation whereas IHC staining remains sensitive in tissues fixed for up to 14 d. Formalin fixation does degrade DNA, but viral DNA can be detected by PCR amplification of very short DNA sequences. In addition, viral DNA can contaminate a microtome knife such that subsequently sectioned uninoculated control tissues exhibit false positive PCR amplification.

A naturally occurring fatal case of Herpesvirus papio 2 pneumonia in an infant baboon (Papio hamadryas anubis).

Here we describe the unusual finding of herpesvirus pneumonia in a 7-d-old infant baboon (Papio hamadryas anubis). This animal had been separated from its dam the morning of its birth and was being hand-reared for inclusion in a specific pathogen-free colony. The baboon was presented for anorexia and depression of 2 d duration. Physical examination revealed a slightly decreased body temperature, lethargy, and dyspnea. The baboon was placed on a warm-water blanket and was given amoxicillin-clavulanate orally and fluids subcutaneously. The animal's clinical condition continued to deteriorate despite tube feeding, subcutaneous fluid administration, and antibiotic therapy, and it died 2 d later. Gross necropsy revealed a thin carcass and severe bilateral diffuse pulmonary consolidation. Histopathology of the lung revealed severe diffuse necrotizing pneumonia. Numerous epithelial and endothelial cells contained prominent intranuclear herpetic inclusion bodies. Virus isolated from lung tissue in cell culture was suspected to be Herpesvirus papio 2 (HVP2) in light of the viral cytopathic effect. Real-time polymerase chain reaction (PCR) analysis and DNA sequencing of PCR products both confirmed that the virus was HVP2. This case is interesting because the age at onset suggests perinatal transmission at or immediately after birth, and the disease course suggests inoculation of the virus into the respiratory tract.

Neuropathogenesis of herpesvirus papio 2 in mice parallels infection with Cercopithecine herpesvirus 1 (B virus) in humans.

Cercopithecine herpesvirus 1 (monkey B virus; BV) produces extremely severe and usually fatal infections when transmitted from macaque monkeys to humans. Cercopithecine herpesvirus 16 (herpesvirus papio 2; HVP2) is very closely related to BV, yet cases of human HVP2 infection are unknown. However, following intramuscular inoculation of mice, HVP2 rapidly invades the peripheral nervous system and ascends the central nervous system (CNS) resulting in death, very much like human BV infections. In this study, the neurovirulence of HVP2 in mice was further evaluated as a potential model system for human BV infections. HVP2 was consistently neurovirulent when administered by epidermal scarification, intracranial inoculation and an eye splash. Quantitative real-time PCR, histopathology and immunohistochemistry were used to follow the temporal spread of virus following skin scarification and to compare the pathogenesis of neurovirulent and apathogenic isolates of HVP2. Apathogenic isolates were found to be capable of reaching the CNS but were extremely inefficient at replicating within the CNS. It is concluded that neurovirulent strains of HVP2 exhibit a pathogenesis in mice that parallels that observed in human BV infections and that this model system may prove useful in dissecting the viral determinants underlying the extreme severity of zoonotic BV infections.

Experimental infection of baboons (Papio cynocephalus anubis) with apathogenic and neurovirulent subtypes of herpesvirus papio 2.

Cercopithecine herpesvirus 16 (Herpesvirus papio 2; HVP2) is an alpha-herpesvirus of baboons (Papio spp.) that generally causes minimal to inapparent disease in the natural host species. HVP2 is very closely related genetically and antigenically to Cercopithecine herpesvirus 1 (monkey B virus; BV) of macaques, which is well known for its extreme lethality in nonmacaque species including humans. Preliminary evidence suggests that a mouse model of HVP2 would be an excellent tool for studying zoonotic BV infections. Although the pathogenicity of different BV isolates in mice spans the full range of severity from apathogenic to extremely neurovirulent, testing of multiple HVP2 isolates revealed only two distinct phenotypes in mice regardless of route of inoculation: apathogenic (HVP2ap) and highly neurovirulent (HVP2nv). For the HVP2nv mouse model to truly reflect BV infection in both its natural host and the differential pathogenicity of BV in aberrant host species, HVP2nv should not produce severe disease in its natural host. To test this, juvenile baboons were inoculated with doses of 10(6) or 10(4) plaque-forming units of HVP2ap or HVP2nv by using an oral subdermal inoculation route. Parameters followed included the appearance of lesions, shedding of infectious virus, general health, and the immune response to the infection. Regardless of the inoculum dose used, no differences were noted between the two HVP2 subtypes in baboons in any of the parameters measured. These findings further support the use of the HVP2nv mouse system as a model to elucidate and study the viral determinants associated with cross-species BV neurovirulence.

Alternate circulation of recent equine-2 influenza viruses (H3N8) from two distinct lineages in the United States.

Phylogenetic and antigenic analyses indicate that recent circulating equine-2 influenza viruses in the United States have been alternating between two genetic and antigenic distinct lineages since 1996. The evolution rates for these two lineages, the Kentucky and the Florida lineage, are very similar. For the earlier isolates in the Kentucky lineage, there are multiple and sequential nonsynonymous substitutions at antigenic sites B and D. However, there are no changes at any of these antigenic sites for KY98 and OK00. In the Florida lineage, except for NY99 with one amino acid substitution at antigenic site B, viruses in this lineage do not have nonsynonymous substitutions at any of the antigenic sites. The lack of amino acid substitutions at these antigenic sites suggests a mechanism other than immune selection is responsible for the maintenance of these viral lineages. Serological analysis indicates that these two lineages are antigenic distinct, and the pattern of reactivity of horse sera towards these two lineages alternates in consecutive years, parallel to the "switching" of virus lineage seen in the phylogenetic tree. This alternate circulation may play a role in the maintenance of these two lineages of equine-2 influenza virus.

Pathogenicity of different baboon herpesvirus papio 2 isolates is characterized by either extreme neurovirulence or complete apathogenicity.

In comparisons of the pathogenicity of simian alphaherpesviruses in mice, two isolates of the baboon virus HVP2 were nearly as lethal as monkey B virus, a biological safety level 4 agent (J. W. Ritchey, K. A. Ealey, M. Payton, and R. Eberle, J. Comp. Pathol. 127:150-161, 2002). To confirm these results, mice were inoculated intramuscularly with 10(5) PFU of HVP2 isolates obtained from different baboon subspecies and primate centers. Some of the HVP2 isolates (6 of 13) caused paralysis and death in the mice, while 7 of 13 HVP2 isolates produced no clinical signs of disease. The apathogenic HVP2 isolates (HVP2ap) induced only low levels of serum antiviral immunoglobulin G relative to levels observed in sera from mice infected with the neurovirulent isolates of HVP2 (HVP2nv). Histological examination of tissues from mice inoculated with HVP2nv isolates showed extensive neural tissue destruction, while mice infected with HVP2ap isolates showed no lesions. Tissue samples collected at 48-h intervals postinfection suggested that HVP2ap isolates failed to replicate at the site of inoculation. There was no significant difference in the in vitro replication, plaque size, or cytopathic effect morphology of HVP2ap versus HVP2nv isolates. While HVP2 isolates replicated better in Vero monkey kidney cells than in murine L cells, plaquing efficiency of individual isolates did not correlate with the dichotomous pathogenic properties seen in mice. Phylogenetic analyses of both coding and intergenic regions (US4-6) of the HVP2 genome separated isolates into two distinct clades that correlated with the two in vivo virulence phenotypes. Taken together, these results demonstrate that two subtypes of HVP2 exist that are very closely related but differ dramatically in their ability to cause disease in a murine model.