During late summer 2001 in Austria, a series of deaths in several species of birds occurred, similar to the beginning of the West Nile virus (WNV) epidemic in the United States. We necropsied the dead birds and examined them by various methods; pathologic and immunohistologic investigations suggested a WNV infection. Subsequently, the virus was isolated, identified, partially sequenced, and subjected to phylogenetic analysis. The isolates exhibited 97% identity to Usutu virus (USUV), a mosquito-borne Flavivirus of the Japanese encephalitis virus group; USUV has never previously been observed outside Africa nor associated with fatal disease in animals or humans. If established in central Europe, this virus may have considerable effects on avian populations; whether USUV has the potential to cause severe human disease is unknown.

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.

NS1 protein of influenza virus is a virulence factor that counteracts Type I interferon (IFN)- Key words: conditionally-replicating-virus; STAT1; SCIDKnowledge of the pathogenesis of viral diseases and the ability to manipulate specific regions of viral genomes permit the construction of conditionally replicating viruses that are attenuated in normal cells but retain their ability to lyse tumor cells. [1][2][3] We show that the NS1 protein of influenza A virus is a virulence factor that counteracts the interferon (IFN)-mediated antiviral cellular response. 4 As a consequence, an influenza virus that lacks a functional NS1 protein due to an almost total deletion of the NS1 open reading frame (delNS1 virus) fails to replicate in normal cells and is apathogenic for wild-type mice. 5 The delNS1 virus, however, grows to titers similar to wild-type virus in dsRNA-activated kinase (PKR) knockout mice. 6 The conditionally replicating phenotype of the delNS1 virus in PKR-defective systems could be exploited for virally-induced oncolysis in tumors expressing oncogenic ras. 7 This observation is based on the fact that oncogenic ras inhibits PKR activation. 8 For this reason, melanoma cells became permissive for productive delNS1 virus replication upon transfection and expression of oncogenic N-ras. Moreover, delNS1 virus treatment of subcutaneous N-ras-expressing melanomas in SCID mice showed that this virus has tumor-ablative potentials in vivo.The delNS1 virus was also shown to replicate effectively in STAT1 knockout mice. 9 STAT1 exists in 2 isoforms, a 91-kD protein (STAT1␣) and a 84-kD splice variant (STAT1␤). 10 Upon activation by Type I IFN signaling STAT1 forms a heterodimer with STAT2 and becomes an essential part of the IFN-receptor induced transcription complex ISGF3. A reduced expression of STAT1 is associated with IFN resistance. 11 Alterations in the IFN-dependent signal cascades, including changes in STAT1 and Type I IFN-receptor molecules, have been described to occur frequently in malignantly transformed cells. For example, several melanoma and lymphoma cell lines contain no or reduced levels of STAT1. 12,13 In addition, leukemia cell lines were shown to be defective in IFN genes. 14 We hypothesized, therefore, that IFN resistance is a common tumor characteristic, which may allow delNS1 virus-mediated oncolysis.We analyzed the growth of delNS1 virus in IFN-resistant tumor cell lines of various histological origins. We compared the oncolytic effect of the delNS1 virus with a second virus, in which the NS1 was only deleted to the N-terminal 99 amino acids (NS1-99). In mice, the latter virus has intermediate attenuation properties ranging between delNS1 and wild-type viruses. 15 The NS1-99 virus gave us the possibility to investigate whether the attenuation level of NS1-deletion viruses influences the efficiency of oncolysis in vivo. The in vitro growth of the NS1-deletion mutants inversely correlated with the IFN resistance of the assayed cell lines. Both delNS1 and NS1-99 viruses induced a tumor-ablative ef...

The correlation between parvovirus infections and lesions in the central nervous system other than cerebellar hypoplasia was studied in 100 cats. The animals were necropsied with a history of various diseases, one third showing typical clinical and pathomorphological signs of panleukopenia. In 18 cats polyclonal antiserum against canine parvovirus consistently labeled neurons mainly in diencephalic regions, whereas the cerebellar cortex remained negative in all cases. In situ hybridization with digoxigenin-labeled minus-sense RNA probes, hybridizing with monomer-replicative form DNA or mRNA, revealed positive signals in nuclei of several neurons of the brain, again excluding the cerebellum. PCR applied to formalin-fixed and paraffin-embedded brain tissue and intestinal tissues of the diseased cats and subsequent DNA sequence analysis yielded canine parvovirus type 2 (CPV-2)-like sequences in the central nervous system. Two aspects of these findings are intriguing: (i) parvoviruses appear to be capable of replicating in neurons, cells that are considered to be terminally differentiated and (ii) CPV-like viruses of the old antigenic type CPV-2 appear to be able to infect cats.Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are considered to be host range variants among the feline parvovirus subgroup in the genus Parvovirus (12). Whereas FPV is known to have infected cats for many decades, CPV emerged suddenly in the mid-1970s and spread throughout the world in 1978. It was a new pathogen for the dog, and retrospective studies revealed the first signs of its appearance in 1976. The original virus from 1978, designated CPV type 2 (CPV-2) to separate it from a nonrelated parvovirus isolated in 1973 from skin tissue of a dog (2), was replaced throughout the world between 1979 and 1985 by two different but closely related antigenic variants: CPV type 2a (CPV-2a) and CPV-2b (10). Besides the antigenic differences between CPV-2 and the antigenic types CPV-2a and -2b, they show distinct biological properties. Whereas CPV-2 is only known to infect and replicate in dogs, CPV-2a and -2b can infect, replicate, and cause disease in cats (14). Experimental infections of cats with CPV-2 consistently failed to demonstrate virus replication (14,15).Parvovirus replication is restricted to the nucleus and is dependent on certain helper functions from the host cell. This is due to the single-stranded DNA genome of the virus that needs to be completed to a double-stranded intermediate to start transcription and translation of the viral genome and proteins, respectively. The DNA polymerase responsible for the synthesis of the complementary strand is a cellular polymerase that is only expressed in mammalian cells during the S phase of the cell cycle (1).Replication of FPVs in dogs and cats is predominantly seen in some highly mitotically active tissues, such as the lymphoid tissue, including lymph nodes, spleen, and thymus, as well as bone marrow and the epithelium of the gastrointestinal tract. Infection of the central ner...