Detection of pathogenic viruses in the urban wastewater in Kuwait-implications for monitoring viral disease outbreaks.

Effective surveillance for epidemic-prone viral diseases is essential for emergency preparedness to respond to threats and occurrences of pandemics. While it is difficult and expensive to conduct health facility-based surveillance, there is a growing interest in conducting sewage-based epidemiological studies to monitor the health of the urban population because of the relative ease of sample collection and the availability of advanced molecular techniques for the detection of pathogens in the sewage. Sewage samples offer unique means to study the aggregate health of the population as opposed to the monitoring of the health of any individual by traditional methods. We worked together with the Ministry of Public Works in Kuwait and developed a platform for the collection and testing of sewage samples from different regions of Kuwait for studying population health. In this report, we describe the results of a cross-sectional study conducted between 16 and 23 September 2019 in an attempt to detect influenza, Noro, Rota, hepatitis A, and hepatitis E viruses in urban sewage samples collected in Kuwait. All five targeted viruses were detected in the samples collected from urban wastewater in Kuwait using reverse-transcriptase quantitative PCR (RT-qPCR). We recently checked for the presence of SARS-CoV-2 in the stored cDNA samples and confirmed the absence of SARS-CoV-2 in them. This is the first report that demonstrates the preparedness in Kuwait for using sewage samples for the detection and monitoring of many pathogenic viruses which may greatly increase the capacity of the country to deal with a viral disease outbreak in the future.

Advances in the Development of Therapeutics for Cytomegalovirus Infections.

The development of therapeutics for cytomegalovirus (CMV) infections, while progressing, has not matched the pace of new treatments of human immunodeficiency virus (HIV) infections; nevertheless, recent developments in the treatment of CMV infections have resulted in improved human health and perhaps will encourage the development of new therapeutic approaches. First, the deployment of ganciclovir and valganciclovir for both the prevention and treatment of CMV infections and disease in transplant recipients has been further improved with the licensure of the efficacious and less toxic letermovir. Regardless, late-onset CMV disease, specifically pneumonia, remains problematic. Second, the treatment of congenital CMV infections with valganciclovir has beneficially improved both hearing and neurologic outcomes, both fundamental advances for these children. In these pediatric studies, viral load was decreased but not eliminated. Thus, an important lesson learned from studies in both populations is the need for new antiviral agents and the necessity for combination therapies as has been shown to be beneficial in the treatment of HIV infections, among others. The development of monoclonal antibodies, sirtuins, and cyclopropovir may provide new treatment options.

Filociclovir Is a Potent In Vitro and In Vivo Inhibitor of Human Adenoviruses.

Human adenovirus (HAdV) infection is common in the general population and can cause a range of clinical manifestations, among which pneumonia and keratoconjunctivitis are the most common. Although HAdV infections are mostly self-limiting, infections in immunocompromised individuals can be severe. No antiviral drug has been approved for treating adenoviruses. Filociclovir (FCV) is a nucleoside analogue which has successfully completed phase I human clinical safety studies and is now being developed for treatment of human cytomegalovirus (HCMV)-related disease in immunocompromised patients. In this report, we show that FCV is a potent broad-spectrum inhibitor of HAdV types 4 to 8, with 50% effective concentrations (EC50s) ranging between 1.24 and 3.6 µM and a 50% cytotoxic concentration (CC50) of 100 to 150 µM in human foreskin fibroblasts (HFFs). We also show that the prophylactic oral administration of FCV (10 mg/kg of body weight) 1 day prior to virus challenge and then daily for 14 days to immunosuppressed Syrian hamsters infected intravenously with HAdV6 was sufficient to prevent morbidity and mortality. FCV also mitigated tissue damage and inhibited virus replication in the liver. The 10-mg/kg dose had similar effects even when the treatment was started on day 4 after virus challenge. Furthermore, FCV administered at the same dose after intranasal challenge with HAdV6 partially mitigated body weight loss but significantly reduced pathology and virus replication in the lung. These findings suggest that FCV could potentially be developed as a pan-adenoviral inhibitor.

Activation of 6-Alkoxy-Substituted Methylenecyclopropane Nucleoside Analogs Requires Enzymatic Modification by Adenosine Deaminase-Like Protein 1.

To determine the mechanism of action of third-generation methylenecyclopropane nucleoside analogs (MCPNAs), DNA sequencing of herpes simplex virus 1 (HSV-1) isolates resistant to third-generation MCPNAs resulted in the discovery of G841S and N815S mutations in HSV-1 UL30. Purified HSV-1 UL30 or human cytomegalovirus (HCMV) UL54 was then subjected to increasing concentrations of MBX-2168-triphosphate (TP), with results demonstrating a 50% inhibitory concentration (IC50) of ∼200 µM, indicating that MBX-2168-TP does not inhibit the viral DNA polymerase. Further metabolic studies showed the removal of a moiety on the guanine ring of MBX-2168. Therefore, we hypothesized that enzymatic removal of a moiety at the 6-position of the guanine ring of third-generation MCPNAs is an essential step in activation. To test this hypothesis, pentostatin (deoxycoformycin [dCF]), an adenosine deaminase-like protein 1 (ADAL-1) inhibitor, was coincubated with MBX-2168. The results showed that dCF antagonized the effect of MBX-2168, with a >40-fold increase in the 50% effective concentration (EC50) at 50 µM dCF (EC50 of 63.1 ± 8.7 µM), compared with MBX-2168 alone (EC50 of 0.2 ± 0.1 µM). Purified ADAL-1 demonstrated time-dependent removal of the moiety on the guanine ring of MBX-2168-monophosphate (MP), with a Km of 17.5 ± 2.4 µM and a Vmax of 0.12 ± 0.04 nmol min-1 Finally, synguanol-TP demonstrated concentration-dependent inhibition of HSV-1 UL30 and HCMV UL54, with IC50s of 0.33 ± 0.16 and 0.38 ± 0.11 µM, respectively. We conclude that ADAL-1 is the enzyme responsible for removing the moiety from the guanine ring of MBX-2168-MP prior to conversion to a TP, the active compound that inhibits the viral DNA polymerase.

Enantiomeric 4'-Truncated 3-deaza-1',6'-isoneplanocins: Synthesis and antiviral properties including Ebola.

Enantiomeric 3-deaza-1',6'-isoneplanocins (C-3 unsubstituted 7a/7b and C-3 with a bromine 8a/8b) lacking the 4'-hydroxymethyl as mechanistically designed anti-viral targets have been prepared by utilizing the Ullmann reaction. Anti-Ebola properties were found for the D-like 7a and 8a and L-like 8b. All four products showed effects against human cytomegalovirus while D-like 7a/8a affected measles; 7a was effective versus norovirus and 8a inhibited Pichinde. Both 7a and 8a produced SAHase inhibitory effects. However, the anti-EBOV activity of 7a and 8a cannot be readily correlated with this observation due with their contrasting IC50 values (8a > 7a). It is to be noted that 7b showed no effects on this enzyme and 8b was minimally inhibitory. These results offer preliminary insight into the differing mechanisms of action of D- and L- like structures and enlighten structural features to guide additional antiviral agent pursuit in the isoneplanocin series.

Ecosystem Interactions Underlie the Spread of Avian Influenza A Viruses with Pandemic Potential.

Despite evidence for avian influenza A virus (AIV) transmission between wild and domestic ecosystems, the roles of bird migration and poultry trade in the spread of viruses remain enigmatic. In this study, we integrate ecosystem interactions into a phylogeographic model to assess the contribution of wild and domestic hosts to AIV distribution and persistence. Analysis of globally sampled AIV datasets shows frequent two-way transmission between wild and domestic ecosystems. In general, viral flow from domestic to wild bird populations was restricted to within a geographic region. In contrast, spillover from wild to domestic populations occurred both within and between regions. Wild birds mediated long-distance dispersal at intercontinental scales whereas viral spread among poultry populations was a major driver of regional spread. Viral spread between poultry flocks frequently originated from persistent lineages circulating in regions of intensive poultry production. Our analysis of long-term surveillance data demonstrates that meaningful insights can be inferred from integrating ecosystem into phylogeographic reconstructions that may be consequential for pandemic preparedness and livestock protection.

Reassortment of Influenza A Viruses in Wild Birds in Alaska before H5 Clade 2.3.4.4 Outbreaks.

Sampling of mallards in Alaska during September 2014-April 2015 identified low pathogenic avian influenza A virus (subtypes H5N2 and H1N1) that shared ancestry with highly pathogenic reassortant H5N2 and H5N1 viruses. Molecular dating indicated reassortment soon after interhemispheric movement of H5N8 clade 2.3.4.4, suggesting genetic exchange in Alaska or surrounds before outbreaks.

Genetic characterization of H5N2 influenza viruses isolated from wild birds in Japan suggests multiple reassortment.

Low-pathogenic avian influenza viruses (LPAIVs) of the H5 subtype can mutate to highly pathogenic forms, potentially destabilizing the poultry industry. Wild migratory birds are considered a natural reservoir of LPAIVs capable of dispersing both high- and low-pathogenic forms of the virus. Therefore, surveillance and characterization of AIV in wild birds are essential. Here, we report on the isolation and genetic characterization of 10 AIVs of the H5N2 subtype obtained through surveillance in Hokkaido, Japan, during 2009 and 2011. Full-genome sequencing revealed that the H5 and N2 genes of these isolates are all closely related to each other, belonging to the Eurasian avian-like lineage, but they are unrelated to H5 highly pathogenic strains of clade 2.3.4.4. The internal genes of the isolates were found to be diverse, consistent with our hypothesis that these H5N2 strains have undergone multiple reassortment events. Even though all of the H5N2 isolates were characterized as LPAIV based on the amino acid sequences at the HA cleavage site, this analysis demonstrates a diverse pool of precursors that may seed future outbreaks in poultry and possible human transmissions, suggesting the need for high-quality surveillance.

Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan.

This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses.

Autophagic clearance of Sin Nombre hantavirus glycoprotein Gn promotes virus replication in cells.

Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.

Middle eastern college of obstetricians and gynecologists (MCOG) practice guidelines: Role of prediction models in management of trial of labor after cesarean section. Practice guideline no. 05-O-22✰,✰✰,☠,☠☠.

Cesarean delivery rates have been steadily rising since the beginning of the 21st century. The growing incidence is even more prominent in developing countries owing to lack of evidence-based guidance and audit, and the expansion of private practice. The uprise in Cesarean delivery rate has been associated with considerable financial burden and has increased the risk otherwise uncommon serious complications such as placenta accreta disorders and uterine rupture. In addition to primary prevention of Cesarean delivery, trial of labor after cesarean section is one of the most successful strategies to reduce Cesarean deliveries and minimize risks associated with higher order Cesarean deliveries. This guideline appraises patient selection strategies and use of prediction model to promote counseling and enhance safety in women considering vaginal birth after Cesarean.

Characterization of the Interaction between hantavirus nucleocapsid protein (N) and ribosomal protein S19 (RPS19).

Hantaviruses, members of the Bunyaviridae family, are negative-stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of the viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and the 5' UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.

Filociclovir is a potent inhibitor of human adenovirus F41.

Clusters of acute non HepA-E hepatitis cases in previously healthy children have been reported globally. At least, 1010 cases were identified in 35 countries, 5% of those cases required liver transplantation and 2% died. The exact cause is not yet known, but there is circumstantial evidence suggesting that human adenovirus F41 (HAdV-F41) might be playing a role. No antiviral drug has been approved for treating human adenovirus infections. Furthermore, HAdV-F41 is notoriously difficult to grow in cell culture, which hindered studying the efficacy of an antiviral compound against this virus. Here, we show that filociclovir (FCV), a nucleoside analog, is a potent inhibitor of HAdV-F41 in cell culture using 2 approaches, namely immunostaining of infected cells and virus yield reduction assay. The activity of FCV was compared to 3 other known antivirals: cidofovir (CDV), ganciclovir (GCV) and valganciclovir (VGCV). Among the 4 compounds examined in this study, FCV was the most potent, with an EC50 of 3.5 µM. These compounds can be ranked by potency as follows: FCV > CDV > GCV ≥ VGCV. In addition, FCV was 10-fold more potent than CDV in a virus yield reduction assay. This report provides timely and valuable methodologies to the research community for testing antivirals against HAdV-F41. Our findings also support the continued development of FCV for various therapeutic applications, including pediatric hepatitis, if a causal relationship is firmly established in the future.

Delineation of the preferences and requirements of the human immunodeficiency virus type 1 dimerization initiation signal by using an in vivo cell-based selection approach.

HIV-1 packages two copies of RNA into one particle, and the dimerization initiation signal (DIS) in the viral RNA plays an important role in selecting the copackaged RNA partner. We analyzed the DIS sequences of the circulating HIV-1 isolates in the GenBank database and observed that, in addition to the prevalent GCGCGC, GTGCAC, and GTGCGC sequences, there are many other minor variants. To better understand the requirements for the DIS to carry out its function, we generated a plasmid library containing a subtype B HIV-1 genome with a randomized DIS, infected cells with viruses derived from the library, and monitored the emergence of variants at different time points until 100 days postinfection. We observed rapid loss of viral diversity and found that the selected variants contained palindromes in the DIS. The "wild-type" GCGCGC-containing virus was a major variant, whereas GTGCAC- and GTGCGC-containing viruses were present at low frequencies. Additionally, other 6-nucleotide (nt) palindromic sequences were selected; a major category of the selected variants contained two GC dyads in the center of the palindrome, flanked by a non-GC dyad. Surprisingly, variants with GC-rich 4-nt palindromes were sustained throughout the selection period at significant frequencies ( approximately 12 to 38%); of these, variants containing the CGCGC sequence were observed frequently, suggesting that this sequence has a selection advantage. These results revealed that multiple sequences can fulfill the function of the HIV-1 DIS. A common feature of the selected DIS sequence is a 4- or 6-nt GC-rich palindrome, although not all sequences with these characteristics were selected, suggesting the presence of other unidentified interactions.

Recent advances in hantavirus molecular biology and disease.

Hantaviruses are emerging zoonotic pathogens that belong to the Bunyaviridae family. They have been classified as category A pathogens by CDC (centers for disease control and prevention). Hantaviruses pose a serious threat to human health because their infection causes two highly fatal diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). These pathogens are transmitted to humans through aerosolized excreta of their infected rodent hosts. Hantaviruses have a tripartite-segmented negative-sense RNA genome. The three genomic RNA segments, S, M, and L, encode a nucleocapsid protein (N), a precursor glycoprotein that is processed into two envelope glycoproteins (Gn and Gc) and the viral RNA-dependent RNA polymerase (RdRp), respectively. N protein is the major structural component of the virus, its main function is to protect and encapsidate the three genomic RNAs forming three viral ribonucleocapsids. Recent studies have proposed that N in conjunction with RdRp plays important roles in the transcription and replication of viral genome. In addition, N preferentially facilitates the translation of viral mRNA in cells. Glycoproteins, Gn and Gc, play major roles in viral attachment and entry to the host cells, virulence, and assembly and packaging of new virions in infected cells. RdRp functions as RNA replicase and transcriptase to replicate and transcribe the viral RNA and is also thought to have endonuclease activity. Currently, no antiviral therapy or vaccine is available for the treatment of hantavirus-associated diseases. Understanding the molecular details of hantavirus life cycle will help in the identification of targets for antiviral therapeutics and in the design of potential antiviral drug for the treatment of HFRS and HCPS. Due to the alarming fatality of hantavirus diseases, development of an effective vaccine against hantaviruses is a necessity.

Filociclovir Is an Active Antiviral Agent against Ocular Adenovirus Isolates In Vitro and in the Ad5/NZW Rabbit Ocular Model.

Presently, there is no FDA- or EMA-approved antiviral for the treatment of human adenovirus (HAdV) ocular infections. This study determined the antiviral activity of filociclovir (FCV) against ocular HAdV isolates in vitro and in the Ad5/NZW rabbit ocular model. The 50% effective concentrations (EC50) of FCV and cidofovir (CDV) were determined for several ocular HAdV types using standard plaque reduction assays. Rabbits were topically inoculated in both eyes with HAdV5. On day 1, the rabbits were divided into four topical treatment groups: (1) 0.5% FCV 4x/day × 10 d; (2) 0.1% FCV 4x/day × 10 d; (3) 0.5% CDV 2x/day × 7 d; (4) vehicle 4x/day × 10 d. Eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. The resulting viral eye titers were determined using standard plaque assays. The mean in vitro EC50 for FCV against tested HAdV types ranged from 0.50 to 4.68 µM, whereas those treated with CDV ranged from 0.49 to 30.3 µM. In vivo, compared to vehicle, 0.5% FCV, 0.1% FCV, and 0.5% CDV produced lower eye titers, fewer numbers of positive eye cultures, and shorter durations of eye infection. FCV demonstrated anti-adenovirus activity in vitro and in vivo.

The discovery and development of filociclovir for the prevention and treatment of human cytomegalovirus-related disease.

Human cytomegalovirus (HCMV) infections are widespread among the human population. Infection is persistent and mostly asymptomatic, except in immunocompromised individuals, particularly transplant patients, where significant morbidity and mortality can occur. Currently approved drugs for treating HCMV-related disease [including ganciclovir (GCV), valganciclovir (VGCV), cidofovir (CDV) and foscarnet (FOS)] all target the viral DNA polymerase and suffer from dose-limiting toxicity and resistance issues. The most recently approved drug, letermovir (LMV), was approved only for prophylaxis in adult HCMV-seropositive stem cell transplant recipients. Although LMV is highly potent, high-grade resistance mutations in the terminase gene were shown to readily emerge in vitro and in treated patients. Therefore, there is a need for new drugs that can be used for combinatorial therapeutic and/or prophylactic regimens to counteract the emergence of resistant mutants. Filociclovir (FCV), also known as cyclopropavir or MBX-400, is a methylenecyclopropane nucleoside analog, which has successfully completed Phase I safety studies, and is now entering Phase II clinical efficacy studies for the treatment of HCMV-related disease in transplant patients. FCV is 10-fold more active than GCV against HCMV in vitro, and has activity against all human herpesviruses except HSV-1 and HSV-2. Recently, FCV was also shown to be highly potent against human adenoviruses. This activity spectrum suggests that FCV could be used to treat/prevent infection with several viruses that pose significant risk to transplant patients. The active triphosphate form of FCV (FCV-TP) reaches higher peak levels than GCV-TP in HCMV-infected cells, and exhibits about 10-fold higher affinity to HCMV DNA polymerase UL54. Furthermore, FCV was shown to retain activity against a panel of GCV-resistant HCMV isolates, suggesting that it could be a useful alternative therapy for treating patients infected with some GCV-resistant HCMV strains. This review summarizes the early discovery work of FCV and highlights the recent advances in the continued development of this clinical candidate.

Viral contamination in biologic manufacture and implications for emerging therapies.

Recombinant protein therapeutics, vaccines, and plasma products have a long record of safety. However, the use of cell culture to produce recombinant proteins is still susceptible to contamination with viruses. These contaminations cost millions of dollars to recover from, can lead to patients not receiving therapies, and are very rare, which makes learning from past events difficult. A consortium of biotech companies, together with the Massachusetts Institute of Technology, has convened to collect data on these events. This industry-wide study provides insights into the most common viral contaminants, the source of those contaminants, the cell lines affected, corrective actions, as well as the impact of such events. These results have implications for the safe and effective production of not just current products, but also emerging cell and gene therapies which have shown much therapeutic promise.

Penciclovir is a potent inhibitor of feline herpesvirus-1 with susceptibility determined at the level of virus-encoded thymidine kinase.

Feline herpesvirus-1 (FHV-1) is the causative agent of a severe ocular disease in cats for which a safe potent antiviral chemotherapeutic agent is highly demanded. The sensitivity of FHV-1 to inhibition by three anti-herpetic nucleoside analogues [acyclovir (ACV), penciclovir (PCV) and cidofovir (CDV)] was tested by means of yield reduction assay. ACV showed very poor ability to inhibit FHV-1 replication. At low multiplicity of infection (MOI), both PCV and CDV were nearly equally effective with IC50 values ranging between 6 and 8 microg/ml. However, when the MOI was raised to 3PFU/cell, the activity of CDV was markedly reduced (IC50 25 microg/ml), while that of PCV remained relatively low (IC50 10 microg/ml). Although FHV-1 is normally insensitive to ACV, it exhibited >1000-fold increase in sensitivity when the thymidine kinase (TK) encoded by herpes simplex virus-1 (HSV-1) was supplied in trans. Furthermore, three PCV-resistant FHV-1 variants selected in vitro were shown to carry mutations in the TK gene. Taken together, these data provided direct evidence that PCV is a potent selective inhibitor of FHV-1 and that the virus-encoded TK is an important determinant of the virus susceptibility to nucleoside analogues.

Development of a quantitative real-time TaqMan PCR assay for testing the susceptibility of feline herpesvirus-1 to antiviral compounds.

Feline herpesvirus-1 (FHV-1) is considered as the most common viral infection of domestic cats worldwide. It causes a disease characterized by upper respiratory and ocular clinical signs. Several attempts are currently underway to develop antiviral chemotherapy for treating FHV-1 infections. The availability of a rapid quantitative method for detecting FHV-1 would greatly facilitate prompt therapy, and hence enhance the success of any antiviral regime. In this study, a TaqMan real-time PCR assay was established for measuring FHV-1 DNA levels in culture supernatants. This assay was shown to be highly specific, reproducible and allows quantitation over a range of 2 to 2 x 10(8) copies per reaction. The assay was then applied to measure the reduction of FHV-1 DNA levels in the presence of increasing concentrations of acyclovir (ACV), penciclovir (PCV) and cidofovir (CDV). The 50% inhibitory concentrations (IC(50s)) obtained with the B927 laboratory strain of FHV-1 were 15.8 microM for ACV, 7.93 microM for CDV and 1.2 microM for PCV. The assay described here is sensitive, time-saving and does not involve prior titration of virus stocks or monitoring virus-induced cytopathic effects. Therefore, it is suitable for routine anti-FHV-1 drug susceptibility testing in veterinary clinics.