Shimon Perk

West Nile virus infection in crocodiles.

To the Editor: Recently West Nile virus (WNV) infection has been reported in three alligators (Alligator sp.) from central Florida (1) and one captive crocodile monitor (Varanus salvadori) with neurologic signs from the District of Columbia and Maryland area (2). These first reports of the virus in American reptiles highlight the possible role of this group of vertebrates in the WNV life cycle. To our knowledge, WNV in a reptile was reported only once before in a serosurvey conducted in Israel from 1965 to 1966, in which 22 reptiles and 96 amphibians were tested for hemagglutination-inhibiting antibodies against several viruses, including WNV; one turtle (Clemmys caspica) was seropositive (3). Experimental infection of the lake frog (Rana ridibunda) with a Russian strain of WNV resulted in high levels of viremia (4). At present, the role of reptiles and amphibians in the life cycle and epidemiology of WNV is not known. We report, for the first time, WNV infection in crocodiles (Crocodylus niloticus). To assess the potential role of crocodiles in the life cycle of WNV in Israel, serum specimens were collected from 20 healthy crocodiles on a commercial farm in the Negev Desert, in southern Israel (31°14′N, 34°19′E). The crocodiles came from two separate breeding farms (32°03′N, 35°26′E and 30°18′N, 35°07′E) in the Syrian-African Rift Valley, which is on the main route of bird migration from Africa to Europe. Five males and 15 females, 1–2.5 years of age, were examined. Blood was withdrawn from the crocodiles’ ventral caudal vein, separated by centrifugation, and kept at –20°C until analyzed. Neutralizing antibody titers were determined against WN-goose-98 (5) and attempts to isolate the virus were performed by using Vero cell culture (6) and by using direct reverse transcription–polymerase chain reaction (RT-PCR) on the serum specimens. To eliminate the possibilities of nonspecific reaction, all serum samples were concurrently tested for the only other flavivirus known to be present in Israel; Israeli turkey meningo-encephalitis virus (ITV) (7). Because ITV does not produce cytopathic effects (CPE) in Vero cells, virus neutralization was conducted on BHK cells for both WNV and ITV by using WN-goose-98 and ITV (vaccine strain). In this case, the virus stocks (10-4.2 50% tissue culture infective dose) were diluted 1:400, and virus neutralization titers were checked 3 days later. Viral RNA was extracted from serum samples with the QIAamp RNA blood kit (QIAGEN, Valencia, CA) , according to the manufacturer’s protocol and resuspended in 30 μl of RNase-free water. The primer pair WN240-Kun848 (respective genome positions 5′: 848 and 1,645) was used to synthesize an 800-bp product in the E gene region (8,9). The resulting DNA fragment was visualized on 1.5% agarose gel stained with ethidium bromide. The seroprevalence rate in the first set of virus neutralization assays in Vero cells was 14/20 (70%, with titers ranging from 1:20 to 1:320 [3x1:20, 3x1:40, 3x1:80, 2x1:160, 3x1:320]). No differences were discernible in either the seroprevalence rate or in the average titers of crocodiles from two different breeding farms. In BHK cells, a similar seroprevalence rate was observed, with titers ranging from 1:40 to 1:1,280 (3x1:40, 2x1:80, 1x1:160, 4x1:320, 3x1:640, 1x1:1280). All serum samples, except one, were <1:10 against ITV virus, which had a titer of 1:640 against WNV and 1:10 against ITV. Viremia was not detected in any of the 20 samples in Vero cell culture or by RT-PCR. These results demonstrate a high rate of infection with WNV in crocodiles in Israel. The crocodiles may have been exposed to the virus during the summer at their present location, since no difference in prevalence was seen between the two groups (which differed only in the farm of origin) and since the younger crocodiles had been hatched in the spring of 2002. Furthermore, a cross-reaction with the other prevalent flavivirus in Israel, ITV, was ruled out. Preliminary results from an equine seroprevalence study (involving 800 horses over a 3-year period) of virus neutralization antibodies to WNV collected during fall 2002, indicate that most horses sampled in Israel’s Arava Valley (a desert in the Syrian-African Rift near the Jordanian and Egyptian borders) and the Gulf of Aqaba/Eilat (30°59′N, 35°18′E to 29°34′N, 34°57′E) (85%, 79/90) were positive (A. Steinman and S. Tal, unpub. data,). WNV was also isolated from mosquitoes in the same region (10). The seroprevalence of WNV antibodies among horses and local birds from the Negev Desert is not known nor is the time when the horses acquired WNV infection. However, the isolation of WNV from mosquitoes (10) and the presence of antibodies to WNV in young crocodiles demonstrate arboviral activity in this region in the summer of 2002, although clinical cases were few. That virus was not isolated from crocodiles in late November (past outbreaks of WNV in Israel mainly occurred between August and October) (6,11). WNV has been endemic in Israel since the early 1950s (12). More recently, in the summer of 2000, an extensive outbreak occurred, affecting hundreds of people (11), dozens of horses (6), and several flocks of geese (5). However, no deaths of crocodiles were reported. This contrasts with the report from Florida (1), where WNV was isolated from dead alligators, and where hundreds of cases of sudden death had been reported in previous years; these deaths are now suspected to result, at least in part, from WNV disease. The role of various reptile species in the epidemiology of other arboviruses such as western equine encephalitis, eastern equine ecephalitis, and Venezuelan equine encephalitis is well documented (13–15). At present, the role of reptiles and amphibians in the life cycle and epidemiology of WNV is not known, and further research is necessary.

Genetic characterization of avian influenza viruses isolated in Israel during 2000-2006.

Our aim was to establish the phylogenetic and genetic relationships among avian influenza viruses (AIV) recently isolated from poultry in Israel. During this study we analyzed complete nucleotide sequences of two envelope (hemagglutinin and neuraminidase) and six internal genes (polymerase B1, polymerase B2, polymerase A, nucleoprotein, nonstructural, and matrix) of 29 selected H9N2 and six internal genes of five H5N1 viruses isolated in Israel during 2000–2006. Comparative genetic and phylogenetic analyses of these sequences revealed that the local H5N1 viruses are closely related to H5N1 viruses isolated in European, Asian, and Middle Eastern countries in 2005–2006. The H9N2 Israeli isolates, together with viruses isolated in Jordan and Saudi Arabia formed a single group. Our data support the claim that during recent years a new endemic focus of H9N2 has been formed in the Middle East. The introduction of H5N1 and co-circulation of these two subtypes of AIV in this region may augment the risk of potentially pandemic strains emergence.

H9N2 Influenza Viruses from Israeli Poultry: A Five-Year Outbreak

Abstract Since 2000, hundreds of H9N2 viruses have been isolated from all types of domestic birds. Although H9N2 is a low-pathogenicity virus, disease has been observed in all types of poultry in the field. Clinical signs ranged from very mild disease to high morbidity and mortality when the virus was associated with a secondary pathogen. Because of the wide range of the virus and the great losses it caused, initially a local vaccination program was implemented, but mass vaccination was quickly authorized. A local strain, isolated in 2002 was selected and is currently in use as an inactivated vaccine. An intensive operation is in progress to characterize the isolates. Several genes (hemagglutinin [HA], neuraminidase, nonstructural protein, nucleoprotein, and matrix) were sequenced, revealing three main groups: the first group included two isolates from 2000, the second group included isolates from 2001 to the beginning of 2003, and the third group included all isolates from 2003 to date. The differences between the second and third groups, in a part of the HA gene, ranged from 3.49% to 6.97% (average 4.57%) of the nucleotides. Similar differences were recorded in the other tested genes. These data could indicate the probable introduction of distinct progenitor viruses into the Israeli poultry population. Furthermore, sequencing of the HA protein of some Israeli isolates revealed the presence of L216 in the binding site; this finding was typical of the H9N2 viruses isolated from humans, which raises the possibility of an influence on host specificity and virulence.

Characterization of Israeli avian metapneumovirus strains in turkeys and chickens

Although the disease associated with the avian metapneumovirus (aMPV) has been described in Israel for more than 20 years and one Israeli isolate was characterized as subtype A, this is the first study investigating the strains circulating in Israel (period 2002 to 2004). Viral RNA was detected by reverse transcription-polymerase chain reaction in 44% of the examined flocks, and one-half of the virus isolation attempts were successful. Characterization showed that 34 non-vaccinated turkey flocks had aMPV subtype B, three had aMPV subtype A and three remained undetermined. Sequence analysis of part of the attachment protein G gene (1161 base pairs) showed a relative homogeneity within the Israeli subtype B group, although they were different from the European and vaccine strains. Seven out of 10 vaccinated flocks were reverse transcription-polymerase chain reaction-positive for aMPV, and sequence analysis of 239 base pairs of the G gene revealed that field strains and not vaccine strains were recovered from these flocks. Overall, the virological examination confirmed the high prevalence of aMPV in Israel and revealed a co-circulation of two subtypes, A and B, with aMPV subtype B being the dominant subtype.

Modulation of the severity of highly pathogenic H5N1 influenza in chickens previously inoculated with Israeli H9N2 influenza viruses.

The continued evolution of H9N2 and H5N1 viruses and their spread and re-emergence across Eurasia raise concern that prior H9N2 virus infection may limit the detection of subsequent H5N1 infection in gallinaceous poultry by attenuating the severity of disease. We show that H9N2 viruses isolated from Israeli turkeys during 2000-2004 were antigenically and genetically distinguishable. These three H9N2 viruses caused no overt signs of disease in chickens. The 2004 isolate replicated and spread most efficiently, and chickens previously inoculated with this H9N2 virus showed 90%-100% survival after inoculation 1 to 35 days later with lethal H5N1 virus. Chickens that survived did not show signs of disease but did shed lethal H5N1 virus from the cloaca. The modulation of survivability was time-dependent; the effect was maximal 5 days after H9N2 inoculation. These findings suggest that co-circulation of H9N2 viruses can contribute to the spread of lethal H5N1 viruses.

Longitudinal Survey of Avian Metapneumoviruses in Poultry in Israel: Infiltration of Field Strains into Vaccinated Flocks

Abstract Avian metapneumovirus (aMPV) is a respiratory pathogen and includes four subtypes, which are differentially distributed worldwide. In Israel, two aMPV subtypes, A and B, are cocirculating in an area with a relatively high density of poultry houses. We performed a longitudinal survey in commercial flocks of turkeys and chickens (six and two flocks, respectively) with differing vaccination programs. This study revealed that all monitored flocks were infected by aMPV during the sampling period, as detected either by serology or by molecular methods. Moreover, to identify the subtypes infecting the birds and to distinguish between field and vaccine strains, we sequenced fragments of the attachment glycoprotein gene. This analysis revealed that both vaccine and field strains of both aMPV-A and -B could be recovered from the birds. In four out of six turkey flocks, aMPV field strains were recovered, emphasizing the broad distribution of this highly contagious pathogen. Importantly, in two out of four vaccinated turkey flocks, homologous field subtypes of aMPV-A or -B were detected even after boost administration, suggesting failure of the vaccine to protect the flocks from secondary infection. Overall, the results of this longitudinal study call for a more efficient vaccination program against aMPV, which is critical for an area of intensive commercial poultry farming.

Avian influenza virus H9N2 survival at different temperatures and pHs.

Abstract The H9N2 avian influenza virus (AIV) subtype has become endemic in Israel since its introduction in 2000. The disease has been economically damaging to the commercial poultry industry, in part because of the synergistic pathology of coinfection with other viral and/or bacterial pathogens. Avian influenza virus viability in the environment depends on the cumulative effects of chemical and physical factors, such as humidity, temperature, pH, salinity, and organic compounds, as well as differences in the virus itself. We sought to analyze the viability of AIV H9N2 strains at three temperatures (37, 20, and 4 C) and at 2 pHs (5.0 and 7.0). Our findings indicated that at 37 C AIV H9N2 isolate 1525 (subgroup IV) survived for a period of time 18 times shorter at 20 C, and 70 times shorter period at 4 C, as measured by a decrease in titer. In addition, the virus was sensitive to a lower pH (pH 5.0) with no detectable virus after 1 wk incubation at 20 C as compared to virus at pH 7.0, which was viable for at least 3 wk at that temperature. The temperature sensitivity of the virus corresponds to the occurrence of H9N2 outbreaks during the winter, and lower pH can greatly affect the viability of the virus.

Bagaza virus and Israel turkey meningoencephalomyelitis virus are a single virus species

Bagaza virus (BAGV) and Israel turkey meningoencephalomyelitis virus (ITV) are classified in the genus Flavivirus of the family Flaviviridae. Serologically, they are closely related, belonging to the Ntaya serocomplex. Nucleotide sequences available to date consist of several complete sequences of BAGV isolates, but only partial sequences of ITV isolates. Sequence comparisons of partial envelope (E) and NS5 regions reveal a close genetic relationship between these viruses. Despite this, BAGV and ITV are considered as separate virus species in the database of the International Committee on Taxonomy of Viruses. In this work, complete nucleotide sequences for five ITV isolates are provided, thereby permitting a phylogenetic comparison with other complete sequences of flaviviruses in the Ntaya serogroup. We conclude that BAGV and ITV are the same virus species and propose that both viruses be designated by a new unified name: Avian meningoencephalomyelitis virus.

Molecular characterization of the glycoprotein genes of H5N1 influenza A viruses isolated in Israel and the Gaza Strip during 2006 outbreaks

Highly pathogenic H5N1 avian influenza A viruses (AIV) have caused outbreaks among domestic poultry and wild aquatic birds in many Asian, European, and African countries since 1997. In March 2006 an avian H5N1 influenza A virus was isolated from poultry in Israel. In the present study we molecularly characterized the hemagglutinin (HA) and neuraminidase (NA) genes of eleven H5N1 viruses isolated from domestic poultry in Israel and Gaza in March–April 2006. Phylogenetic analysis of the HA and NA genes showed that the Israeli and Gazian viruses were closely related to viruses isolated in Egypt in 2006.