A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT-PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV.

This review deals with the aetiology and the diagnosis of bovine viral abortion. While the abortion rates on beef and dairy cattle farms usually do not exceed 10%, significant economic losses because of abortion storms may be encountered. Determining the cause of abortions is usually a challenge, and it generally remains obscure in more than 50% of the necropsy submitted foetuses. Bovine viral diarrhoea virus and bovine herpesvirus-1 are the most common viruses causally associated with bovine abortions in farmed cattle globally. Rift Valley fever virus and bluetongue virus are important insect-transmitted abortogenic viruses. The geographic distribution of these two viruses is primarily dependent on the distribution of the insect vector, but direct transmission is possible. Recent global warming and subsequent insect vector expansion, coupled with the increase in international trade of animals and animal products, have been important factors in recent geographic advances of those two viruses. Bovine herpesviruses-4 and 5 in cattle, as well as other less frequent vector-borne viruses including epizootic haemorrhagic disease virus, Aino virus, Wesselsbron virus and lumpy skin disease virus, are discussed.

Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities.

Infectious salmon anemia virus (ISAV) is a very important fish virus in the Northern hemisphere and there is continued interest in understanding the mechanisms of its pathogenesis and persistence in fish. In this study, the permissive fish cell lines SHK-1, CHSE-214 and TO were used to determine whether ISAV-induced cytopathic effect (CPE) is due to apoptosis or necrosis. Characteristic apoptotic DNA fragmentation was observed only in ISAV-infected SHK-1 and CHSE-214 cells. Apoptosis in ISAV-infected SHK-1 cells was confirmed by fragment end-labelling assay, suggesting that CPE in these cells is associated with apoptosis. ISAV-infected TO cells did not undergo apoptosis, but showed leakage of high-mobility group 1 (HMGB1) protein from the nucleus, which is characteristic of cells undergoing necrosis; this suggests that CPE in these cells is associated with necrosis. ISAV-infected SHK-1 cells did not show leakage of HMGB1 protein. Infection with two different strains of ISAV showed that induction of apoptosis was correlated with the appearance of CPE in SHK-1 cells. ISAV-induced apoptosis was inhibited by a pan-caspase inhibitor, Z-VAD-fmk, indicating a caspase-activation pathway. The ISAV putative PB2 protein and proteins encoded by RNA segment 7 bound caspase-8 specifically in vitro, suggesting that these viral proteins may have a role in ISAV-induced apoptosis. These findings demonstrate for the first time that the mechanism of cell death during ISAV infection is dependent on the cell type, which may have implications for ISAV pathogenesis and persistence. INTRODUCTIONInfectious salmon anemia (ISA) virus (ISAV) is the causative agent of ISA, which is a highly infectious disease of farmed Atlantic salmon in the Northern hemisphere. This virus is a member of the family Orthomyxoviridae, genus Isavirus (Falk et al., 1997;Krossøy et al., 1999; www. ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm). The clinical disease in farmed Atlantic salmon is characterized by high mortality with exophthalmia, pale gills, ascites, haemorrhagic liver necrosis, renal interstitial haemorrhage and tubular nephrosis. ISAV is known to cause overt and fatal systemic infection in farmed Atlantic salmon and asymptomatic infection in feral fish, a situation analogous to that caused by avian influenza viruses in domestic poultry and feral birds (reviewed by Kibenge et al., 2004). ISAV may infect and replicate in sea trout, brown trout, rainbow trout, eels, herring and Arctic char, resulting in asymptomatic, probably lifelong carriers of the virus (reviewed by Kibenge et al., 2004). In Atlantic salmon, ISAV targets leukocytic cells and endothelial cells , or endothelial-associated cells (Falk et al., 2001) and macrophages (Moneke et al., 2003). Permissive fish cell lines for ISAV include salmon head kidney (SHK-1) cells ; TO cells (Wergeland & Jakobsen, 2001); and Atlantic salmon kidney (ASK-2) cells (Rolland et al., 2002), which are macrophage-like cell lines Wergeland & Jakobsen, 2001;Rolland et al., 2002) in which virus replicates with produc...

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R 2 = 0.974, p < 0.001). Compared to HN-negative clams (< 5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at ~10 to ~20% tetraploid cells, and the second at ~30 to ~80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at ~30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, ≥30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.